Designing Allosteric Control into Enzymes by Chemical Rescue of Structure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deckert, Katelyn; Budiardjo, S. Jimmy; Brunner, Luke C.

2012-08-07

Ligand-dependent activity has been engineered into enzymes for purposes ranging from controlling cell morphology to reprogramming cellular signaling pathways. Where these successes have typically fused a naturally allosteric domain to the enzyme of interest, here we instead demonstrate an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. We present two examples, W33G in a {beta}-glycosidase enzyme ({beta}-gly) and W492Gmore » in a {beta}-glucuronidase enzyme ({beta}-gluc), in which we engineer indole-dependent activity into enzymes by removing a buried tryptophan side chain that serves as a buttress for the active site architecture. In both cases, we observe a loss of function, and in both cases we find that the subsequent addition of indole can be used to restore activity. Through a detailed analysis of {beta}-gly W33G kinetics, we demonstrate that this rescued enzyme is fully functionally equivalent to the corresponding wild-type enzyme. We then present the apo and indole-bound crystal structures of {beta}-gly W33G, which together establish the structural basis for enzyme inactivation and rescue. Finally, we use this designed switch to modulate {beta}-glycosidase activity in living cells using indole. Disruption and recovery of protein structure may represent a general technique for introducing allosteric control into enzymes, and thus may serve as a starting point for building a variety of bioswitches and sensors.« less

Chemical Modification in the Design of Immobilized Enzyme Biocatalysts: Drawbacks and Opportunities.

PubMed

Rueda, Nazzoly; Dos Santos, Jose C S; Ortiz, Claudia; Torres, Rodrigo; Barbosa, Oveimar; Rodrigues, Rafael C; Berenguer-Murcia, Ángel; Fernandez-Lafuente, Roberto

2016-06-01

Chemical modification of enzymes and immobilization used to be considered as separate ways to improve enzyme properties. This review shows how the coupled use of both tools may greatly improve the final biocatalyst performance. Chemical modification of a previously immobilized enzyme is far simpler and easier to control than the modification of the free enzyme. Moreover, if protein modification is performed to improve its immobilization (enriching the enzyme in reactive groups), the final features of the immobilized enzyme may be greatly improved. Chemical modification may be directed to improve enzyme stability, but also to improve selectivity, specificity, activity, and even cell penetrability. Coupling of immobilization and chemical modification with site-directed mutagenesis is a powerful instrument to obtain fully controlled modification. Some new ideas such as photoreceptive enzyme modifiers that change their physical properties under UV exposition are discussed. © 2016 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nelson, Cassandra E.; Rogowski, Artur; Morland, Carl

Degradation of polysaccharides forms an essential arc in the carbon cycle, provides a percentage of our daily caloric intake, and is a major driver in the renewable chemical industry. Microorganisms proficient at degrading insoluble polysaccharides possess large numbers of carbohydrate active enzymes, many of which have been categorized as functionally redundant. Here we present data that suggests that carbohydrate active enzymes that have overlapping enzymatic activities can have unique, non-overlapping biological functions in the cell. Our comprehensive study to understand cellodextrin utilization in the soil saprophyte Cellvibrio japonicus found that only one of four predicted β-glucosidases is required in amore » physiological context. Gene deletion analysis indicated that only the cel3B gene product is essential for efficient cellodextrin utilization in C. japonicus and is constitutively expressed at high levels. Interestingly, expression of individual β-glucosidases in Escherichia coli K-12 enabled this non-cellulolytic bacterium to be fully capable of using cellobiose as a sole carbon source. Furthermore, enzyme kinetic studies indicated that the Cel3A enzyme is significantly more active than the Cel3B enzyme on the oligosaccharides but not disaccharides. Finally, our approach for parsing related carbohydrate active enzymes to determine actual physiological roles in the cell can be applied to other polysaccharide-degradation systems.« less

Characterization of Human Aspartoacylase: the brain enzyme responsible for Canavan disease†

PubMed Central

Le Coq, Johanne; An, Hyun-Joo; Lebrilla, Carlito; Viola, Ronald E.

2008-01-01

Aspartoacylase catalyzes the deacetylation of N-acetylaspartic acid (NAA) to produce acetate and L-aspartate, and is the only brain enzyme that has been shown to effectively metabolize NAA. Although the exact role of this enzymatic reaction has not yet been completely elucidated, the metabolism of NAA appears to be necessary in the formation of myelin lipids and defects in this enzyme lead to Canavan disease, a fatal neurological disorder. The low catalytic activity and inherent instability observed with the Escherichia coli-expressed form of aspartoacylase suggested the need for a suitable eukaryotic expression system that would be capable of producing a fully functional, mature enzyme. Human aspartoacylase has now been successfully expressed in Pichia pastoris. While the expression yields are lower than in E. coli, the purified enzyme is significantly more stable. This enzyme form has the same substrate specificity, but is 150-fold more active than the E. coli-expressed enzyme. The molecular weight of the purified enzyme, measured by mass spectrometry, is higher than predicted, suggesting the presence of some posttranslational modifications. Deglycosylation of aspartoacylase or mutation at the glycosylation site causes decreased enzyme stability and diminished catalytic activity. A carbohydrate component has been removed and characterized by mass spectrometry. In addition to this carbohydrate moiety, the enzyme has also been shown to contain one zinc atom per subunit. Chelation studies to remove the zinc results in a reversible loss of catalytic activity, thus establishing aspartoacylase as a zinc metalloenzyme. PMID:16669630

Characterization of human aspartoacylase: the brain enzyme responsible for Canavan disease.

PubMed

Le Coq, Johanne; An, Hyun-Joo; Lebrilla, Carlito; Viola, Ronald E

2006-05-09

Aspartoacylase catalyzes the deacetylation of N-acetylaspartic acid (NAA) to produce acetate and L-aspartate and is the only brain enzyme that has been shown to effectively metabolize NAA. Although the exact role of this enzymatic reaction has not yet been completely elucidated, the metabolism of NAA appears to be necessary in the formation of myelin lipids, and defects in this enzyme lead to Canavan disease, a fatal neurological disorder. The low catalytic activity and inherent instability observed with the Escherichia coli-expressed form of aspartoacylase suggested the need for a suitable eukaryotic expression system that would be capable of producing a fully functional, mature enzyme. Human aspartoacylase has now been successfully expressed in Pichia pastoris. While the expression yields are lower than in E. coli, the purified enzyme is significantly more stable. This enzyme form has the same substrate specificity but is 150-fold more active than the E. coli-expressed enzyme. The molecular weight of the purified enzyme, measured by mass spectrometry, is higher than predicted, suggesting the presence of some post-translational modifications. Deglycosylation of aspartoacylase or mutation at the glycosylation site causes decreased enzyme stability and diminished catalytic activity. A carbohydrate component has been removed and characterized by mass spectrometry. In addition to this carbohydrate moiety, the enzyme has also been shown to contain one zinc atom per subunit. Chelation studies to remove the zinc result in a reversible loss of catalytic activity, thus establishing aspartoacylase as a zinc metalloenzyme.

Allosteric substrate switching in a voltage-sensing lipid phosphatase.

PubMed

Grimm, Sasha S; Isacoff, Ehud Y

2016-04-01

Allostery provides a critical control over enzyme activity, biasing the catalytic site between inactive and active states. We found that the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP), which modifies phosphoinositide signaling lipids (PIPs), has not one but two sequential active states with distinct substrate specificities, whose occupancy is allosterically controlled by sequential conformations of the voltage-sensing domain (VSD). Using fast fluorescence resonance energy transfer (FRET) reporters of PIPs to monitor enzyme activity and voltage-clamp fluorometry to monitor conformational changes in the VSD, we found that Ci-VSP switches from inactive to a PIP3-preferring active state when the VSD undergoes an initial voltage-sensing motion and then into a second PIP2-preferring active state when the VSD activates fully. This two-step allosteric control over a dual-specificity enzyme enables voltage to shape PIP concentrations in time, and provides a mechanism for the complex modulation of PIP-regulated ion channels, transporters, cell motility, endocytosis and exocytosis.

Allosteric substrate switching in a voltage sensing lipid phosphatase

PubMed Central

Grimm, Sasha S.; Isacoff, Ehud Y.

2016-01-01

Allostery provides a critical control over enzyme activity, biasing the catalytic site between inactive and active states. We find the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP), which modifies phosphoinositide signaling lipids (PIPs), to have not one but two sequential active states with distinct substrate specificities, whose occupancy is allosterically controlled by sequential conformations of the voltage sensing domain (VSD). Using fast FRET reporters of PIPs to monitor enzyme activity and voltage clamp fluorometry to monitor conformational changes in the VSD, we find that Ci-VSP switches from inactive to a PIP3-preferring active state when the VSD undergoes an initial voltage sensing motion and then into a second PIP2-preferring active state when the VSD activates fully. This novel 2-step allosteric control over a dual specificity enzyme enables voltage to shape PIP concentrations in time, and provides a mechanism for the complex modulation of PIP-regulated ion channels, transporters, cell motility and endo/exocytosis. PMID:26878552

Engineered Mononuclear Variants in Bacillus cereus Metallo-β-lactamase BcII Are Inactive†

PubMed Central

Abriata, Luciano A.; González, Lisandro J.; Llarrull, Leticia I.; Tomatis, Pablo E.; Myers, William K.; Costello, Alison L.; Tierney, David L.; Vila, Alejandro J.

2008-01-01

Metallo-β-lactamases (MβLs) are zinc enzymes able to hydrolyze almost all β-lactam antibiotics, rendering them inactive, at the same time endowing bacteria high levels of resistance. The design of inhibitors active against all classes of MβLs has been hampered by their structural diversity and by the heterogeneity in metal content in enzymes from different sources. BcII is the metallo-β-lactamase from Bacillus cereus, which is found in both the mononuclear and dinuclear forms. Despite extensive studies, there is still controversy about the nature of the active BcII species. Here we have designed two mutant enzymes in which each one of the metal binding sites was selectively removed. Both mutants were almost inactive, despite preserving most of the structural features of each metal site. These results reveal that neither site isolated in the MβL scaffold is sufficient to render a fully active enzyme. This suggests that only the dinuclear species is active or that the mononuclear variants can be active only if aided by other residues that would be metal ligands in the dinuclear species. PMID:18652482

PlantCAZyme: a database for plant carbohydrate-active enzymes

PubMed Central

Ekstrom, Alexander; Taujale, Rahil; McGinn, Nathan; Yin, Yanbin

2014-01-01

PlantCAZyme is a database built upon dbCAN (database for automated carbohydrate active enzyme annotation), aiming to provide pre-computed sequence and annotation data of carbohydrate active enzymes (CAZymes) to plant carbohydrate and bioenergy research communities. The current version contains data of 43 790 CAZymes of 159 protein families from 35 plants (including angiosperms, gymnosperms, lycophyte and bryophyte mosses) and chlorophyte algae with fully sequenced genomes. Useful features of the database include: (i) a BLAST server and a HMMER server that allow users to search against our pre-computed sequence data for annotation purpose, (ii) a download page to allow batch downloading data of a specific CAZyme family or species and (iii) protein browse pages to provide an easy access to the most comprehensive sequence and annotation data. Database URL: http://cys.bios.niu.edu/plantcazyme/ PMID:25125445

Nanoarmored Enzymes for Organic Enzymology: Synthesis and Characterization of Poly(2-Alkyloxazoline)-Enzyme Conjugates.

PubMed

Leurs, Melanie; Tiller, Joerg C

2017-01-01

The properties of enzymes can be altered significantly by modification with polymers. Numerous different methods are known to obtain such polymer-enzyme conjugates (PECs). However, there is no universal method to render enzymes into PECs that are fully soluble in organic solvents. Here, we present a method, which achieves such high degree of modification of proteins that the majority of modified enzymes will be soluble in organic solvents. This is achieved by preparing poly(2-alkyloxazoline)s (POx) with an NH 2 end group and coupling this functional polymer via pyromellitic acid dianhydride onto the amino groups of the respective protein. The resulting PECs are capable of serving as surfactants for unmodified proteins, rendering the whole mixture organosoluble. Depending on the nature of the POx and the molecular weight and the nature of the enzyme, the PECs are soluble in chloroform or even toluene. Another advantage of this method is that the poly(2-alkyloxazoline) can be activated with the coupling agent and used for the enzyme conjugation without further purification. The POx-enzyme conjugates generated by this modification strategy show modulated catalytic activity in both, aqueous and organic, systems. © 2017 Elsevier Inc. All rights reserved.

A fully human anti-Ep-CAM scFv-beta-glucuronidase fusion protein for selective chemotherapy with a glucuronide prodrug

PubMed Central

de Graaf, M; Boven, E; Oosterhoff, D; van der Meulen-Muileman, I H; Huls, G A; Gerritsen, W R; Haisma, H J; Pinedo, H M

2002-01-01

Monoclonal antibodies against tumour-associated antigens could be useful to deliver enzymes selectively to the site of a tumour for activation of a non-toxic prodrug. A completely human fusion protein may be advantageous for repeated administration, as host immune responses may be avoided. We have constructed a fusion protein consisting of a human single chain Fv antibody, C28, against the epithelial cell adhesion molecule and the human enzyme β-glucuronidase. The sequences encoding C28 and human enzyme β-glucuronidase were joined by a sequence encoding a flexible linker, and were preceded by the IgGκ signal sequence for secretion of the fusion protein. A CHO cell line was engineered to secrete C28-β-glucuronidase fusion protein. Antibody specificity and enzyme activity were retained in the secreted fusion protein that had an apparent molecular mass of 100 kDa under denaturing conditions. The fusion protein was able to convert a non-toxic prodrug of doxorubicin, N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-β-glucuronyl carbamate to doxorubicin, resulting in cytotoxicity. A bystander effect was demonstrated, as doxorubicin was detected in all cells after N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-β-glucuronyl carbamate administration when only 10% of the cells expressed the fusion protein. This is the first fully human and functional fusion protein consisting of an scFv against epithelial cell adhesion molecule and human enzyme β-glucuronidase for future use in tumour-specific activation of a non-toxic glucuronide prodrug. British Journal of Cancer (2002) 86, 811–818. DOI: 10.1038/sj/bjc/6600143 www.bjcancer.com © 2002 Cancer Research UK PMID:11875747

Observing single FoF1-ATP synthase at work using an improved fluorescent protein mNeonGreen as FRET donor

NASA Astrophysics Data System (ADS)

Heitkamp, Thomas; Deckers-Hebestreit, Gabriele; Börsch, Michael

2016-02-01

Adenosine triphosphate (ATP) is the universal chemical energy currency for cellular activities provided mainly by the membrane enzyme FoF1-ATP synthase in bacteria, chloroplasts and mitochondria. Synthesis of ATP is accompanied by subunit rotation within the enzyme. Over the past 15 years we have developed a variety of single-molecule FRET (smFRET) experiments to monitor catalytic action of individual bacterial enzymes in vitro. By specifically labeling rotating and static subunits within a single enzyme we were able to observe three-stepped rotation in the F1 motor, ten-stepped rotation in the Fo motor and transient elastic deformation of the connected rotor subunits. However, the spatial and temporal resolution of motor activities measured by smFRET were limited by the photophysics of the FRET fluorophores. Here we evaluate the novel FRET donor mNeonGreen as a fusion to FoF1-ATP synthase and compare it to the previously used fluorophore EGFP. Topics of this manuscript are the biochemical purification procedures and the activity measurements of the fully functional mutant enzyme.

Relevance of substrates and products on the desorption of lipases physically adsorbed on hydrophobic supports.

PubMed

Virgen-Ortíz, Jose J; Tacias-Pascacio, Veymar G; Hirata, Daniela B; Torrestiana-Sanchez, Beatriz; Rosales-Quintero, Arnulfo; Fernandez-Lafuente, Roberto

2017-01-01

Lipase B from Candida antarctica (CALB) has been physically immobilized on octyl-agarose via interfacial activation. The incubation of the enzyme in 80% ethanol at pH 5 and 25°C has not significant effect on enzyme activity. Moreover, the hydrolysis of 100mM tributyrin catalyzed by this biocatalyst exhibited a quite linear reaction course. However, a new cycle of tributyrin hydrolysis showed a drastic drop in the activity. SDS-PAGE gels of the supernatant and the biocatalyst showed a significant enzyme desorption after the reaction. Similar results could be appreciated using triacetin or sunflower oil, while using 300mM methyl phenyl acetate, butyl butyrate or ethyl butyrate most enzyme molecules remained immobilized. The results show that the detergent properties of some reaction products increase the enzyme release from the hydrophobic support, and this problem increased if the concentration of the reactants increased. Using 500mM tributyrin, even in fully aqueous medium, some enzyme desorption from the support may be observed. Thus, the results show a limitation of this kind of biocatalysts that should be considered in the selection of an industrial lipase biocatalyst. Copyright © 2016 Elsevier Inc. All rights reserved.

Thai Fruits Exhibit Antioxidant Activity and Induction of Antioxidant Enzymes in HEK-293 Cells.

PubMed

Anantachoke, Natthinee; Lomarat, Pattamapan; Praserttirachai, Wasin; Khammanit, Ruksinee; Mangmool, Supachoke

2016-01-01

The cellular antioxidant enzymes play the important role of protecting the cells and organisms from the oxidative damage. Natural antioxidants contained in fruits have attracted considerable interest because of their presumed safety and potential nutritional value. Even though antioxidant activities of many fruits have been reported, the effects of phytochemicals contained in fruits on the induction of antioxidant enzymes in the cells have not been fully defined. In this study, we showed that extracts from Antidesma ghaesembilla , Averrhoa bilimbi , Malpighia glabra , Mangifera indica, Sandoricum koetjape , Syzygium malaccense, and Ziziphus jujuba inhibited H 2 O 2 -induced intracellular reactive oxygen species production in HEK-293 cells. Additionally, these Thai fruit extracts increased the mRNA and protein expressions of antioxidant enzymes, catalase, glutathione peroxidase-1, and manganese superoxide dismutase. The consumption of Thai fruits rich in phenolic compounds may reduce the risk of oxidative stress.

Thai Fruits Exhibit Antioxidant Activity and Induction of Antioxidant Enzymes in HEK-293 Cells

PubMed Central

Anantachoke, Natthinee; Lomarat, Pattamapan; Praserttirachai, Wasin; Khammanit, Ruksinee

2016-01-01

The cellular antioxidant enzymes play the important role of protecting the cells and organisms from the oxidative damage. Natural antioxidants contained in fruits have attracted considerable interest because of their presumed safety and potential nutritional value. Even though antioxidant activities of many fruits have been reported, the effects of phytochemicals contained in fruits on the induction of antioxidant enzymes in the cells have not been fully defined. In this study, we showed that extracts from Antidesma ghaesembilla, Averrhoa bilimbi, Malpighia glabra, Mangifera indica, Sandoricum koetjape, Syzygium malaccense, and Ziziphus jujuba inhibited H2O2-induced intracellular reactive oxygen species production in HEK-293 cells. Additionally, these Thai fruit extracts increased the mRNA and protein expressions of antioxidant enzymes, catalase, glutathione peroxidase-1, and manganese superoxide dismutase. The consumption of Thai fruits rich in phenolic compounds may reduce the risk of oxidative stress. PMID:28074103

Prompt and easy activation by specific thioredoxins of calvin cycle enzymes of Arabidopsis thaliana associated in the GAPDH/CP12/PRK supramolecular complex.

PubMed

Marri, Lucia; Zaffagnini, Mirko; Collin, Valérie; Issakidis-Bourguet, Emmanuelle; Lemaire, Stéphane D; Pupillo, Paolo; Sparla, Francesca; Miginiac-Maslow, Myroslawa; Trost, Paolo

2009-03-01

The Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) can form under oxidizing conditions a supramolecular complex with the regulatory protein CP12. Both GAPDH and PRK activities are inhibited within the complex, but they can be fully restored by reduced thioredoxins (TRXs). We have investigated the interactions of eight different chloroplast thioredoxin isoforms (TRX f1, m1, m2, m3, m4, y1, y2, x) with GAPDH (A(4), B(4), and B(8) isoforms), PRK and CP12 (isoform 2), all from Arabidopsis thaliana. In the complex, both A(4)-GAPDH and PRK were promptly activated by TRX f1, or more slowly by TRXs m1 and m2, but all other TRXs were ineffective. Free PRK was regulated by TRX f1, m1, or m2, while B(4)- and B(8)-GAPDH were absolutely specific for TRX f1. Interestingly, reductive activation of PRK caged in the complex was much faster than reductive activation of free oxidized PRK, and activation of A(4)-GAPDH in the complex was much faster (and less demanding in terms of reducing potential) than activation of free oxidized B(4)- or B(8)-GAPDH. It is proposed that CP12-assembled supramolecular complex may represent a reservoir of inhibited enzymes ready to be released in fully active conformation following reduction and dissociation of the complex by TRXs upon the shift from dark to low light. On the contrary, autonomous redox-modulation of GAPDH (B-containing isoforms) would be more suited to conditions of very active photosynthesis.

Genetics of carbon catabolite repression in Saccharomycess cerevisiae: genes involved in the derepression process.

PubMed

Zimmermann, F K; Kaufmann, I; Rasenberger, H; Haubetamann, P

1977-02-28

A recessive mutant cat1-1, wild type CAT1, was isolated in Saccharomyces cerevisiae. It did not grow on glycerol nor ferment maltose even with fully constitutive, glucose resistant maltase synthesis. It prevented derepression of isocitrate lyase, fructose-1,6-diphosphatase and maltase in a constitutive but glucose sensitive maltase mutant. Derepression of malate dehydrogenase was retarded and slowed down. Sucrose fermentation and invertase synthesis was not affected. Respiration was normal. From this mutant, two reverse mutants were isolated. One was recessive, acted as a suppressor of cat1-1 and was called cat2-1, wild type CAT2; the other was dominant and allelic to CAT1 and designated CAT1-2d and cat2-1 caused an earlier derepression of enzymes studied but did not affect the repressed nor the fully derepressed enzyme levels. CAT1-2d and cat2-1 did not show any additive effects. It is proposed that carbon catabolite repression acts in two ways. The direct way represses synthesis of sensitive enzymes, during growth on repressing carbon sources whereas the other way regulates the derepression process. After alleviation of carbon catabolite repression, gene CAT1 becomes active and prevents the activity of CAT2 which functions as a repressor of sensitive enzyme synthesis. The CAT2 gene product has to be eliminated before derepression can actually occur. The time required for this causes a delay in derepression after the depletion of a repressible carbon source. cat1-1 cannot block CAT2 activity and therefore, derepression is blocked. cat2-1 is inactive and derepression can start after carbon catabolite repression has ceased. CAT1-2d permanently active as a repressor of CAT2 and eliminates the delay in derepression.

SlyD-dependent nickel delivery limits maturation of [NiFe]-hydrogenases in late-stationary phase Escherichia coli cells.

PubMed

Pinske, Constanze; Sargent, Frank; Sawers, R Gary

2015-04-01

Fermentatively growing Escherichia coli cells have three active [NiFe]-hydrogenases (Hyd), two of which, Hyd-1 and Hyd-2, contribute to H2 oxidation while Hyd-3 couples formate oxidation to H2 evolution. Biosynthesis of all Hyd involves the insertion of a Fe(CN)2CO group and a subsequent insertion of nickel ions through the HypA/HybF, HypB and SlyD proteins. With high nickel concentrations the presence of none of these proteins is required, but under normal growth conditions and during late stationary growth SlyD is important for hydrogenase activities. The slyD mutation reduced H2 production during exponential phase growth by about 50%. Assaying stationary phase grown cells for the coupling of Hyd activity to the respiratory chain or formate-dependent H2 evolution showed that SlyD is essential for both H2 evolution and H2 oxidation. Although introduction of plasmid-coded slyD resulted in an overall decrease of Hyd-2 polypeptides in slyD and hypA slyD mutants, processing and dye-reducing activity of the Hyd-2 enzyme was nevertheless restored. Similarly, introduction of the slyD plasmid restored only some H2 evolution in the slyD mutant while Hyd-3 polypeptides and dye-reducing activity were fully restored. Taken together, these results indicate an essential role for SlyD in the generation of the fully cofactor-equipped hydrogenase large subunits in the stationary phase where the level of each Hyd enzyme is finely tuned by SlyD for optimal enzyme activity.

Putrescine Aminopropyltransferase Is Responsible for Biosynthesis of Spermidine, Spermine, and Multiple Uncommon Polyamines in Osmotic Stress-Tolerant Alfalfa.

PubMed Central

Bagga, S.; Rochford, J.; Klaene, Z.; Kuehn, G. D.; Phillips, G. C.

1997-01-01

The biosynthesis of polyamines from the diamine putrescine is not fully understood in higher plants. A putrescine aminopropyltransferase (PAPT) enzyme activity was characterized in alfalfa (Medicago sativa L.). This enzyme activity was highly specific for putrescine as the initial substrate and did not recognize another common diamine, 1,3-diaminopropane, or higher-molecular-weight polyamines such as spermidine and spermine as alternative initial substrates. The enzyme activity was inhibited by a general inhibitor of aminopropyltransferases, 5[prime]-methylthioadenosine, and by a specific inhibitor of PAPTs, cyclohexylammonium sulfate. The initial substrate specificity and inhibition characteristics of the enzyme activity suggested that it is a classical example of a PAPT. However, this enzyme activity yielded multiple polyamine products, which is uncharacteristic of PAPTs. The major reaction product of PAPT activity in alfalfa was spermidine. The next most abundant products of the enzyme reaction using putrescine as the initial substrate included the tetramines spermine and thermospermine. These two tetramines were distinguished by thin-layer chromatography to be distinct reaction products exhibiting differential rates of formation. In addition, the uncommon polyamines homocaldopentamine and homocaldohexamine were tentatively identified as minor enzymatic reaction products but only in extracts prepared from osmotic stresstolerant alfalfa cultivars. PAPT activity from alfalfa was highest in meristematic shoot tip and floral bud tissues and was not detected in older, nonmeristematic tissues. Product inhibition of the enzyme activity was observed after spermidine was added into the in vitro assay for alfalfa PAPT activity. A biosynthetic pathway is proposed that accounts for the characteristics of this PAPT activity and accommodates a novel scheme by which certain uncommon polyamines are produced in plants. PMID:12223719

Systems analysis in Cellvibrio japonicus resolves predicted redundancy of β-glucosidases and determines essential physiological functions: Functional analysis of C. japonicus β-glucosidases

DOE PAGES

Nelson, Cassandra E.; Rogowski, Artur; Morland, Carl; ...

2017-02-28

Degradation of polysaccharides forms an essential arc in the carbon cycle, provides a percentage of our daily caloric intake, and is a major driver in the renewable chemical industry. Microorganisms proficient at degrading insoluble polysaccharides possess large numbers of carbohydrate active enzymes, many of which have been categorized as functionally redundant. Here we present data that suggests that carbohydrate active enzymes that have overlapping enzymatic activities can have unique, non-overlapping biological functions in the cell. Our comprehensive study to understand cellodextrin utilization in the soil saprophyte Cellvibrio japonicus found that only one of four predicted β-glucosidases is required in amore » physiological context. Gene deletion analysis indicated that only the cel3B gene product is essential for efficient cellodextrin utilization in C. japonicus and is constitutively expressed at high levels. Interestingly, expression of individual β-glucosidases in Escherichia coli K-12 enabled this non-cellulolytic bacterium to be fully capable of using cellobiose as a sole carbon source. Furthermore, enzyme kinetic studies indicated that the Cel3A enzyme is significantly more active than the Cel3B enzyme on the oligosaccharides but not disaccharides. Finally, our approach for parsing related carbohydrate active enzymes to determine actual physiological roles in the cell can be applied to other polysaccharide-degradation systems.« less

Using soil enzymes to explain observed differences in the response of soil decomposition to nitrogen fertilization

NASA Astrophysics Data System (ADS)

Stone, M.; Weiss, M.; Goodale, C. L.

2010-12-01

Soil microbes produce extracellular enzymes that degrade a variety of carbon-rich polymers contained within soil organic matter (SOM). These enzymes are key regulators of the terrestrial carbon cycle. However, basic information about the kinetics of extracellular enzymes and key environmental variables that regulate their catalytic ability is lacking. This study aims to clarify the mechanisms by which microbial carbon-degrading enzymes drive different responses to nitrogen (N) fertilization in soil decomposition at two sites with long-term N fertilization experiments, the Bear Brook (BB) forest in Maine and Fernow Forest (FF) in West Virginia. We examined a suite of cellulolytic and lignolytic enzymes that break down common SOM constituents. We hypothesized that enzymes derived from the site with a higher mean annual temperature (FF) would be more heat-tolerant, and retain their catalytic efficiency (Km) as temperature rises, relative to enzymes from the colder environment (BB). We further hypothesized that cellulolytic enzyme activity would be unaffected by N, while oxidative enzyme activity would be suppressed in N-fertilized soils. To test these hypotheses and examine the interactive effects of temperature and N, we measured enzyme activity in unfertilized and N-fertilized soils under a range of laboratory temperature manipulations. Preliminary results show a significant decrease in cellulolytic enzyme efficiency with temperature at the colder site (BB), as well as a significant increase in efficiency due to N-fertilization for two cellulolytic enzymes. Oxidative enzyme activity shows a marginally significant reduction due to N-fertilization at BB. These results suggest that soil warming may produce a negative feedback on carbon turnover in certain climates, while N-fertilization may alter the relative decomposition rates of different soil organic matter constituents. FF activity will be analyzed in a similar manner and the two sites will be compared in order to fully assess our hypotheses.

Structure of choline oxidase in complex with the reaction product glycine betaine.

PubMed

Salvi, Francesca; Wang, Yuan-Fang; Weber, Irene T; Gadda, Giovanni

2014-02-01

Choline oxidase from Arthrobacter globiformis, which is involved in the biosynthesis of glycine betaine from choline, has been extensively characterized in its mechanistic and structural properties. Despite the knowledge gained on the enzyme, the details of substrate access to the active site are not fully understood. The `loop-and-lid' mechanism described for the glucose-methanol-choline enzyme superfamily has not been confirmed for choline oxidase. Instead, a hydrophobic cluster on the solvent-accessible surface of the enzyme has been proposed by molecular dynamics to control substrate access to the active site. Here, the crystal structure of the enzyme was solved in complex with glycine betaine at pH 6.0 at 1.95 Å resolution, allowing a structural description of the ligand-enzyme interactions in the active site. This structure is the first of choline oxidase in complex with a physiologically relevant ligand. The protein structures with and without ligand are virtually identical, with the exception of a loop at the dimer interface, which assumes two distinct conformations. The different conformations of loop 250-255 define different accessibilities of the proposed active-site entrance delimited by the hydrophobic cluster on the other subunit of the dimer, suggesting a role in regulating substrate access to the active site.

Printing of polymer microcapsules for enzyme immobilization on paper substrate.

PubMed

Savolainen, Anne; Zhang, Yufen; Rochefort, Dominic; Holopainen, Ulla; Erho, Tomi; Virtanen, Jouko; Smolander, Maria

2011-06-13

Poly(ethyleneimine) (PEI) microcapsules containing laccase from Trametes hirsuta (ThL) and Trametes versicolor (TvL) were printed onto paper substrate by three different methods: screen printing, rod coating, and flexo printing. Microcapsules were fabricated via interfacial polycondensation of PEI with the cross-linker sebacoyl chloride, incorporated into an ink, and printed or coated on the paper substrate. The same ink components were used for three printing methods, and it was found that laccase microcapsules were compatible with the ink. Enzymatic activity of microencapsulated TvL was maintained constant in polymer-based ink for at least eight weeks. Thick layers with high enzymatic activity were obtained when laccase-containing microcapsules were screen printed on paper substrate. Flexo printed bioactive paper showed very low activity, since by using this printing method the paper surface was not fully covered by enzyme microcapsules. Finally, screen printing provided a bioactive paper with high water-resistance and the highest enzyme lifetime.

NisT, the transporter of the lantibiotic nisin, can transport fully modified, dehydrated, and unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides.

PubMed

Kuipers, Anneke; de Boef, Esther; Rink, Rick; Fekken, Susan; Kluskens, Leon D; Driessen, Arnold J M; Leenhouts, Kees; Kuipers, Oscar P; Moll, Gert N

2004-05-21

Lantibiotics are lanthionine-containing peptide antibiotics. Nisin, encoded by nisA, is a pentacyclic lantibiotic produced by some Lactococcus lactis strains. Its thioether rings are posttranslationally introduced by a membrane-bound enzyme complex. This complex is composed of three enzymes: NisB, which dehydrates serines and threonines; NisC, which couples these dehydrated residues to cysteines, thus forming thioether rings; and the transporter NisT. We followed the activity of various combinations of the nisin enzymes by measuring export of secreted peptides using antibodies against the leader peptide and mass spectroscopy for detection. L. lactis expressing the nisABTC genes efficiently produced fully posttranslationally modified prenisin. Strikingly, L. lactis expressing the nisBT genes could produce dehydrated prenisin without thioether rings and a dehydrated form of a non-lantibiotic peptide. In the absence of the biosynthetic NisBC enzymes, the NisT transporter was capable of excreting unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides. Our data show that NisT specifies a broad spectrum (poly)peptide transporter that can function either in conjunction with or independently from the biosynthetic genes. NisT secretes both unmodified and partially or fully posttranslationally modified forms of prenisin and non-lantibiotic peptides. These results open the way for efficient production of a wide range of peptides with increased stability or novel bioactivities.

Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes.

PubMed

Fujishima, Kosuke; Wang, Kendrick M; Palmer, Jesse A; Abe, Nozomi; Nakahigashi, Kenji; Endy, Drew; Rothschild, Lynn J

2018-01-29

Amino acid biosynthesis pathways observed in nature typically require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine: serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase (CysK/CysM). To solve this chicken-and-egg problem, we substituted alternate amino acids in CysE, CysK and CysM for cysteine and methionine, which are the only two sulfur-containing proteinogenic amino acids. Using a cysteine-dependent auxotrophic E. coli strain, CysE function was rescued by cysteine-free and methionine-deficient enzymes, and CysM function was rescued by cysteine-free enzymes. CysK function, however, was not rescued in either case. Enzymatic assays showed that the enzymes responsible for rescuing the function in CysE and CysM also retained their activities in vitro. Additionally, substitution of the two highly conserved methionines in CysM decreased but did not eliminate overall activity. Engineering amino acid biosynthetic enzymes to lack the so-produced amino acids can provide insights into, and perhaps eventually fully recapitulate via a synthetic approach, the biogenesis of biotic amino acids.

Purification and substrate specificity of Staphylococcus hyicus lipase.

PubMed

van Oort, M G; Deveer, A M; Dijkman, R; Tjeenk, M L; Verheij, H M; de Haas, G H; Wenzig, E; Götz, F

1989-11-28

The Staphylococcus hyicus lipase gene has been cloned and expressed in Staphylococcus carnosus. From the latter organism the enzyme was secreted into the medium as a protein with an apparent molecular mass of 86 kDa. This protein was purified, and the amino-terminal sequence showed that the primary gene product was indeed cleaved at the proposed signal peptide cleavage site. The protein was purified from large-scale preparations after tryptic digestion. This limited proteolysis reduced the molecular mass to 46 kDa and increased the specific activity about 3-fold. Although the enzyme had a low specific activity in the absence of divalent cations, the activity increased about 40-fold in the presence of Sr2+ or Ca2+ ions. The purified lipase has a broad substrate specificity. The acyl chains were removed from the primary and secondary positions of natural neutral glycerides and from a variety of synthetic glyceride analogues. Thus triglycerides were fully hydrolyzed to free fatty acid and glycerol. The enzyme hydrolyzed naturally occurring phosphatidylcholines, their synthetic short-chain analogues, and lysophospholipids to free fatty acids and water-soluble products. The enzyme had a 2-fold higher activity on micelles of short-chain D-lecithins than on micelles composed of the L-isomers. Thus the enzyme from S. hyicus has lipase activity and also high phospholipase A and lysophospholipase activity.

Human tyrosinase produced in insect cells: a landmark for the screening of new drugs addressing its activity.

PubMed

Fogal, Stefano; Carotti, Marcello; Giaretta, Laura; Lanciai, Federico; Nogara, Leonardo; Bubacco, Luigi; Bergantino, Elisabetta

2015-01-01

Human tyrosinase is the first enzyme of the multistep process of melanogenesis. It catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine and the following oxidation of o-diphenol to the corresponding quinone, L-dopaquinone. In spite of its biomedical relevance, its reactivity is far from being fully understood, mostly because of the lack of a suitable expression system. Indeed, until now, studies on substrates and inhibitors of tyrosinases have been performed in vitro almost exclusively using mushroom or bacterial enzymes. We report on the production of a recombinant human tyrosinase in insect cells (Sf9 line). Engineering the protein, improving cell culture conditions, and setting a suitable purification protocol optimized product yield. The obtained active enzyme was truthfully characterized with a number of substrate and inhibitor molecules. These results were compared to those gained from a parallel analysis of the bacterial (Streptomyces antibioticus) enzyme and those acquired from the literature for mushroom tyrosinase, showing that the reactivity of the human enzyme appears unique and pointing out the great bias introduced when using non-human tyrosinases to measure the inhibitory efficacy of new molecules. The described enzyme is therefore an indispensable paradigm in testing pharmaceutical or cosmetic agents addressing tyrosinase activity.

Ascorbic acid metabolism during bilberry (Vaccinium myrtillus L.) fruit development.

PubMed

Cocetta, Giacomo; Karppinen, Katja; Suokas, Marko; Hohtola, Anja; Häggman, Hely; Spinardi, Anna; Mignani, Ilaria; Jaakola, Laura

2012-07-15

Bilberry (Vaccinium myrtillus L.) possesses a high antioxidant capacity in berries due to the presence of anthocyanins and ascorbic acid (AsA). Accumulation of AsA and the expression of the genes encoding the enzymes of the main AsA biosynthetic route and of the ascorbate-glutathione cycle, as well as the activities of the enzymes involved in AsA oxidation and recycling were investigated for the first time during the development and ripening of bilberry fruit. The results showed that the AsA level remained relatively stable during fruit maturation. The expression of the genes encoding the key enzymes in the AsA main biosynthetic route showed consistent trends with each other as well as with AsA levels, especially during the first stages of fruit ripening. The expression of genes and activities of the enzyme involved in the AsA oxidation and recycling route showed more prominent developmental stage-dependent changes during the ripening process. Different patterns of activity were found among the studied enzymes and the results were, for some enzymes, in accordance with AsA levels. In fully ripe berries, both AsA content and gene expression were significantly higher in skin than in pulp. Copyright © 2012 Elsevier GmbH. All rights reserved.

The Megavirus Chilensis Cu,Zn-Superoxide Dismutase: the First Viral Structure of a Typical Cellular Copper Chaperone-Independent Hyperstable Dimeric Enzyme

PubMed Central

Lartigue, Audrey; Burlat, Bénédicte; Coutard, Bruno; Chaspoul, Florence; Claverie, Jean-Michel

2014-01-01

ABSTRACT Giant viruses able to replicate in Acanthamoeba castellanii penetrate their host through phagocytosis. After capsid opening, a fusion between the internal membranes of the virion and the phagocytic vacuole triggers the transfer in the cytoplasm of the viral DNA together with the DNA repair enzymes and the transcription machinery present in the particles. In addition, the proteome analysis of purified mimivirus virions revealed the presence of many enzymes meant to resist oxidative stress and conserved in the Mimiviridae. Megavirus chilensis encodes a predicted copper, zinc superoxide dismutase (Cu,Zn-SOD), an enzyme known to detoxify reactive oxygen species released in the course of host defense reactions. While it was thought that the metal ions are required for the formation of the active-site lid and dimer stabilization, megavirus chilensis SOD forms a very stable metal-free dimer. We used electron paramagnetic resonance (EPR) analysis and activity measurements to show that the supplementation of the bacterial culture with copper and zinc during the recombinant expression of Mg277 is sufficient to restore a fully active holoenzyme. These results demonstrate that the viral enzyme's activation is independent of a chaperone both for disulfide bridge formation and for copper incorporation and suggest that its assembly may not be as regulated as that of its cellular counterparts. A SOD protein is encoded by a variety of DNA viruses but is absent from mimivirus. As in poxviruses, the enzyme might be dispensable when the virus infects Acanthamoeba cells but may allow megavirus chilensis to infect a broad range of eukaryotic hosts. IMPORTANCE Mimiviridae are giant viruses encoding more than 1,000 proteins. The virion particles are loaded with proteins used by the virus to resist the vacuole's oxidative stress. The megavirus chilensis virion contains a predicted copper, zinc superoxide dismutase (Cu,Zn-SOD). The corresponding gene is present in some megavirus chilensis relatives but is absent from mimivirus. This first crystallographic structure of a viral Cu,Zn-SOD highlights the features that it has in common with and its differences from cellular SODs. It corresponds to a very stable dimer of the apo form of the enzyme. We demonstrate that upon supplementation of the growth medium with Cu and Zn, the recombinant protein is fully active, suggesting that the virus's SOD activation is independent of a copper chaperone for SOD generally used by eukaryotic SODs. PMID:25355875

Watch Out for the "Living Dead": Cell-Free Enzymes and Their Fate.

PubMed

Baltar, Federico

2017-01-01

Microbes are the engines driving biogeochemical cycles. Microbial extracellular enzymatic activities (EEAs) are the "gatekeepers" of the carbon cycle. The total EEA is the sum of cell-bound (i.e., cell-attached), and dissolved (i.e., cell-free) enzyme activities. Cell-free enzymes make up a substantial proportion (up to 100%) of the total marine EEA. Although we are learning more about how microbial diversity and function (including total EEA) will be affected by environmental changes, little is known about what factors control the importance of the abundant cell-free enzymes. Since cell-attached EEAs are linked to the cell, their fate will likely be linked to the factors controlling the cell's fate. In contrast, cell-free enzymes belong to a kind of "living dead" realm because they are not attached to a living cell but still are able to perform their function away from the cell; and as such, the factors controlling their activity and fate might differ from those affecting cell-attached enzymes. This article aims to place cell-free EEA into the wider context of hydrolysis of organic matter, deal with recent studies assessing what controls the production, activity and lifetime of cell-free EEA, and what their fate might be in response to environmental stressors. This perspective article advocates the need to go "beyond the living things," studying the response of cells/organisms to different stressors, but also to study cell-free enzymes, in order to fully constrain the future and evolution of marine biogeochemical cycles.

Small-Angle Neutron Scattering Reveals pH-Dependent Conformational Changes in Trichoderma reesei Cellobiohydrolase I: Implications for Enzymatic Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pingali, Sai Venkatesh; O'Neill, Hugh Michael; McGaughey, Joseph

2011-01-01

Cellobiohydrolase I (Cel7A) of the fungus Trichoderma reesei (now classified as an anamorph of Hypocrea jecorina) hydrolyzes crystalline cellulose to soluble sugars, making it of key interest for producing fermentable sugars from biomass for biofuel production. The activity of the enzyme is pH-dependent, with its highest activity occurring at pH 4 5. To probe the response of the solution structure of Cel7A to changes in pH, we measured small angle neutron scattering of it in a series of solutions having pH values of 7.0, 6.0, 5.3, and 4.2. As the pH decreases from 7.0 to 5.3, the enzyme structure remainsmore » well defined, possessing a spatial differentiation between the cellulose binding domain and the catalytic core that only changes subtly. At pH 4.2, the solution conformation of the enzyme changes to a structure that is intermediate between a properly folded enzyme and a denatured, unfolded state, yet the secondary structure of the enzyme is essentially unaltered. The results indicate that at the pH of optimal activity, the catalytic core of the enzyme adopts a structure in which the compact packing typical of a fully folded polypeptide chain is disrupted and suggest that the increased range of structures afforded by this disordered state plays an important role in the increased activity of Cel7A through conformational selection.« less

The Algicidal Fungus Trametes versicolor F21a Eliminating Blue Algae via Genes Encoding Degradation Enzymes and Metabolic Pathways Revealed by Transcriptomic Analysis.

PubMed

Dai, Wei; Chen, Xiaolin; Wang, Xuewen; Xu, Zimu; Gao, Xueyan; Jiang, Chaosheng; Deng, Ruining; Han, Guomin

2018-01-01

The molecular mechanism underlying the elimination of algal cells by fungal mycelia has not been fully understood. Here, we applied transcriptomic analysis to investigate the gene expression and regulation at time courses of Trametes versicolor F21a during the algicidal process. The obtained results showed that a total of 193, 332, 545, and 742 differentially expressed genes were identified at 0, 6, 12, and 30 h during the algicidal process, respectively. The gene ontology terms were enriched into glucan 1,4-α-glucosidase activity, hydrolase activity, lipase activity, and endopeptidase activity. The KEGG pathways were enriched in degradation and metabolism pathways including Glycolysis/Gluconeogenesis, Pyruvate metabolism, the Biosynthesis of amino acids, etc. The total expression levels of all Carbohydrate-Active enZYmes (CAZyme) genes for the saccharide metabolism were increased by two folds relative to the control. AA5, GH18, GH5, GH79, GH128, and PL8 were the top six significantly up-regulated modules among 43 detected CAZyme modules. Four available homologous decomposition enzymes of other species could partially inhibit the growth of algal cells. The facts suggest that the algicidal mode of T. versicolor F21a might be associated with decomposition enzymes and several metabolic pathways. The obtained results provide a new candidate way to control algal bloom by application of decomposition enzymes in the future.

Analysis of galactosemia-linked mutations of GALT enzyme using a computational biology approach.

PubMed

Facchiano, A; Marabotti, A

2010-02-01

We describe the prediction of the structural and functional effects of mutations on the enzyme galactose-1-phosphate uridyltransferase related to the genetic disease galactosemia, using a fully computational approach. One hundred and seven single-point mutants were simulated starting from the structural model of the enzyme obtained by homology modeling methods. Several bioinformatics programs were then applied to each resulting mutant protein to analyze the effect of the mutations. The mutations have a direct effect on the active site, or on the dimer assembly and stability, or on the monomer stability. We describe how mutations may exert their effect at a molecular level by altering H-bonds, salt bridges, secondary structure or surface features. The alteration of protein stability, at level of monomer and/or dimer, is the main effect observed. We found an agreement between our results and the functional experimental data available in literature for some mutants. The data and analyses for all the mutants are fully available in the web-accessible database hosted at http://bioinformatica.isa.cnr.it/GALT.

Strategies for microbial synthesis of high-value phytochemicals

NASA Astrophysics Data System (ADS)

Li, Sijin; Li, Yanran; Smolke, Christina D.

2018-03-01

Phytochemicals are of great pharmaceutical and agricultural importance, but often exhibit low abundance in nature. Recent demonstrations of industrial-scale production of phytochemicals in yeast have shown that microbial production of these high-value chemicals is a promising alternative to sourcing these molecules from native plant hosts. However, a number of challenges remain in the broader application of this approach, including the limited knowledge of plant secondary metabolism and the inefficient reconstitution of plant metabolic pathways in microbial hosts. In this Review, we discuss recent strategies to achieve microbial biosynthesis of complex phytochemicals, including strategies to: (1) reconstruct plant biosynthetic pathways that have not been fully elucidated by mining enzymes from native and non-native hosts or by enzyme engineering; (2) enhance plant enzyme activity, specifically cytochrome P450 activity, by improving efficiency, selectivity, expression or electron transfer; and (3) enhance overall reaction efficiency of multi-enzyme pathways by dynamic control, compartmentalization or optimization with the host's metabolism. We also highlight remaining challenges to — and future opportunities of — this approach.

In Vitro Assembly of Catalase*

PubMed Central

Baureder, Michael; Barane, Elisabeth; Hederstedt, Lars

2014-01-01

Most aerobic organisms contain catalase, which functions to decompose hydrogen peroxide. Typical catalases are structurally complex homo-tetrameric enzymes with one heme prosthetic group buried in each subunit. It is not known how catalase in the cell is assembled from its constituents. The bacterium Enterococcus faecalis cannot synthesize heme but can acquire it from the environment to form a cytoplasmic catalase. We have in E. faecalis monitored production of the enzyme polypeptide (KatA) depending on the availability of heme and used our findings to devise a procedure for the purification of preparative amounts of in vivo-synthesized apocatalase. We show that fully active catalase can be obtained in vitro by incubating isolated apoprotein with hemin. We have characterized features of the assembly process and describe a temperature-trapped hemylated intermediate of the enzyme maturation process. Hemylation of apocatalase does not require auxiliary cell components, but rapid assembly of active enzyme seemingly is assisted in the cell. Our findings provide insight about catalase assembly and offer new experimental possibilities for detailed studies of this process. PMID:25148685

Expression and Characterization of Hyperthermostable Exo-polygalacturonase TtGH28 from Thermotoga thermophilus.

PubMed

Wagschal, Kurt; Rose Stoller, J; Chan, Victor J; Lee, Charles C; Grigorescu, Arabela A; Jordan, Douglas B

2016-07-01

D-galacturonic acid is a potential platform chemical comprising the principal component of pectin in the citrus processing waste stream. Several enzyme activities are required for the enzymatic production of galacturonic acid from pectin, including exo- and endo-polygalacturonases. The gene TtGH28 encoding a putative GH28 polygalacturonase from Pseudothermotoga thermarum DSM 5069 (Theth_0397, NCBI# AEH50492.1) was synthesized, expressed in Escherichia coli, and characterized. Alignment of the amino acid sequence of gene product TtGH28 with other GH28 proteins whose structures and details of their catalytic mechanism have been elucidated shows that three catalytic Asp residues and several other key active site residues are strictly conserved. Purified TtGH28 was dimeric and hyperthermostable, with K t (0.5)  = 86.3 °C. Kinetic parameters for activity on digalacturonic acid, trigalacturonic acid, and polygalacturonic acid were obtained. No substrate inhibition was observed for polygalacturonate, while the K si values for the oligogalacturonides were in the low mM range, and K i for product galacturonic acid was in the low μM range. Kinetic modeling of the progress of reaction showed that the enzyme is both fully exo- and fully non-processional.

Furin is a chemokine-modifying enzyme: in vitro and in vivo processing of CXCL10 generates a C-terminally truncated chemokine retaining full activity.

PubMed

Hensbergen, Paul J; Verzijl, Dennis; Balog, Crina I A; Dijkman, Remco; van der Schors, Roel C; van der Raaij-Helmer, Elizabeth M H; van der Plas, Mariena J A; Leurs, Rob; Deelder, André M; Smit, Martine J; Tensen, Cornelis P

2004-04-02

Chemokines comprise a class of structurally related proteins that are involved in many aspects of leukocyte migration under basal and inflammatory conditions. In addition to the large number of genes, limited processing of these proteins by a variety of enzymes enhances the complexity of the total spectrum of chemokine variants. We have recently shown that the native chemokine CXCL10 is processed at the C terminus, thereby shedding the last four amino acids. The present study was performed to elucidate the mechanism in vivo and in vitro and to study the biological activity of this novel isoform of CXCL10. Using a combination of protein purification and mass spectrometric techniques, we show that the production of C-terminally truncated CXCL10 by primary keratinocytes is inhibited in vivo by a specific inhibitor of pro-protein convertases (e.g. furin) but not by inhibition of matrix metalloproteinases. Moreover, CXCL10 is processed by furin in vitro, which is abrogated by a mutation in the furin recognition site. Using GTPgammaS binding, Ca(2+) mobilization, and chemotaxis assays, we demonstrate that the C-terminally truncated CXCL10 variant is a potent ligand for CXCR3. Moreover, the inverse agonist activity on the virally encoded receptor ORF74 and the direct antibacterial activity of CXCL10 are fully retained. Hence, we have identified furin as a novel chemokine-modifying enzyme in vitro and most probably also in vivo, generating a C-terminally truncated CXCL10, which fully retains its (inverse) agonistic properties.

Catalytic and immunochemical properties of arylsulphatase A from urine, modified by potassium ferrate.

PubMed

Laidler, P M; Steczko, J

1986-01-01

Arylsulphatase A (EC 3.1.6.1.) from urine was inactivated with potassium ferrate, a strong oxidizing agent. The inhibition could be prevented by competitive inhibitors, tetraborate and orthophosphate. Tetraborate which was shown to be a powerful competitive inhibitor (determined Ki = 4 X 10(-5) M) gave more efficient protection. The partially inactivated enzyme exhibited a Km value similar to that of the unmodified arylsulphatase A, and its Vmax decreased in proportion to the loss of enzymatic activity. The partially modified enzyme did not lose its ability to catalyse hydrolysis of p-nitrocatechol sulphate according to the "anomalous kinetics" exhibited towards this substrate and characteristic for arylsulphatase A. The immunochemical properties of arylsulphatase A either fully or partially inactivated were similar to those of the native enzyme. The results allow to conclude that ferrate reacts with arylsulphatase A in its active site. Thus ferrate seems to be a very sensitive probe for amino acid residues essential for catalytic activity of arylsulphatase A.

Watch Out for the “Living Dead”: Cell-Free Enzymes and Their Fate

PubMed Central

Baltar, Federico

2018-01-01

Microbes are the engines driving biogeochemical cycles. Microbial extracellular enzymatic activities (EEAs) are the “gatekeepers” of the carbon cycle. The total EEA is the sum of cell-bound (i.e., cell-attached), and dissolved (i.e., cell-free) enzyme activities. Cell-free enzymes make up a substantial proportion (up to 100%) of the total marine EEA. Although we are learning more about how microbial diversity and function (including total EEA) will be affected by environmental changes, little is known about what factors control the importance of the abundant cell-free enzymes. Since cell-attached EEAs are linked to the cell, their fate will likely be linked to the factors controlling the cell’s fate. In contrast, cell-free enzymes belong to a kind of “living dead” realm because they are not attached to a living cell but still are able to perform their function away from the cell; and as such, the factors controlling their activity and fate might differ from those affecting cell-attached enzymes. This article aims to place cell-free EEA into the wider context of hydrolysis of organic matter, deal with recent studies assessing what controls the production, activity and lifetime of cell-free EEA, and what their fate might be in response to environmental stressors. This perspective article advocates the need to go “beyond the living things,” studying the response of cells/organisms to different stressors, but also to study cell-free enzymes, in order to fully constrain the future and evolution of marine biogeochemical cycles. PMID:29354095

Pre-Steady State Studies of Phosphite Dehydrogenase Demonstrate that Hydride Transfer is Fully Rate-Limiting†

PubMed Central

Fogle, Emily J.

2008-01-01

Phosphite dehydrogenase (PTDH)1 is a unique NAD-dependent enzyme that catalyzes the oxidation of inorganic phosphite to phosphate. The enzyme has great potential for cofactor regeneration and mechanistic studies have provided some insight into the residues that are important for catalysis. In this investigation, pre-steady state studies were performed on the His6-tagged wild type (WT) enzyme, several active site mutants, a thermostable mutant (12X-PTDH), and a thermostable mutant with dual cofactor specificity (NADP-12X-PTDH). Stopped-flow kinetic experiments indicate that slow steps after hydride transfer do not significantly limit the rate of reaction for WT, the active site mutants, or the thermostable mutant. Pre-steady state kinetic isotope effects (KIEs) and single turn-over experiments further confirm that slow steps after the chemical step do not significantly limit the rate of reaction for any of these proteins. Collectively, these results suggest that the hydride transfer step is fully rate determining in PTDH and that the observed KIE on kcat is the intrinsic effect in WT PTDH and the mutants examined. In contrast, a slow step after catalysis may partially limit the rate of phosphite oxidation by NADP-12X-PTDH with NADP as cofactor. Finally, site directed mutagenesis of Asp79 indicates that this residue is important in orienting Arg237 for proper interaction with phosphite. PMID:17949110

Remote Control by Inter-Enzyme Allostery: A Novel Paradigm for Regulation of the Shikimate Pathway.

PubMed

Munack, Steffi; Roderer, Kathrin; Ökvist, Mats; Kamarauskaite, Jurate; Sasso, Severin; van Eerde, André; Kast, Peter; Krengel, Ute

2016-03-27

DAHP synthase and chorismate mutase catalyze key steps in the shikimate biosynthetic pathway en route to aromatic amino acids. In Mycobacterium tuberculosis, chorismate mutase (MtCM; Rv0948c), located at the branch point toward phenylalanine and tyrosine, has poor activity on its own. However, it is efficiently activated by the first enzyme of the pathway, DAHP synthase (MtDS; Rv2178c), through formation of a non-covalent MtCM-MtDS complex. Here, we show how MtDS serves as an allosteric platform for feedback regulation of both enzymes, using X-ray crystallography, small-angle X-ray scattering, size-exclusion chromatography, and multi-angle light scattering. Crystal structures of the fully inhibited MtDS and the allosterically down-regulated MtCM-MtDS complex, solved at 2.8 and 2.7Å, respectively, reveal how effector binding at the internal MtDS subunit interfaces regulates the activity of MtDS and MtCM. While binding of all three metabolic end products to MtDS shuts down the entire pathway, the binding of phenylalanine jointly with tyrosine releases MtCM from the MtCM-MtDS complex, hence suppressing MtCM activation by 'inter-enzyme allostery'. This elegant regulatory principle, invoking a transient allosteric enzyme interaction, seems to be driven by dynamics and is likely a general strategy used by nature. Copyright © 2016 Elsevier Ltd. All rights reserved.

Opposing effects of two osmolytes--trehalose and glycerol--on thermal inactivation of rabbit muscle 6-phosphofructo-1-kinase.

PubMed

Faber-Barata, Joana; Sola-Penna, Mauro

2005-01-01

Trehalose and glycerol are known as good stabilizers of function and structure of several macromolecules against stress conditions. We previously reported that they have comparable effectiveness on protecting two yeast cytosolic enzymes against thermal inactivation. However, enzyme protection has always been associated to a decrease in catalytic activity at the stabilizing conditions i.e., the presence of the protective molecule. In the present study we tested trehalose and glycerol on thermal protection of the mammalian cytosolic enzyme phosphofructokinase. Here we found that trehalose was able to protect phosphofructokinase against thermal inactivation as well as to promote an activation of its catalytic activity. The enzyme incubated in the presence of 1 M trehalose did not present any significant inactivation within 2 h of incubation at 50 degrees C, contrasting to control experiments where the enzyme was fully inactivated during the same period exhibiting a t0.5 for thermal inactivation of 56+/-5 min. On the other hand, enzyme incubated in the presence of 37.5% (v/v) glycerol was not protected against incubation at 50 degrees C. Indeed, when phosphofructokinase was incubated for 45 min at 50 degrees C in the presence of lower concentrations of glycerol (7.5-25%, v/v), the remaining activity was 2-4 times lower than control. These data show that the compatibility of effects previously shown for trehalose and glycerol with some yeast cytosolic enzymes can not be extended to all globular enzyme system. In the case of phosphofructokinase, we believe that its property of shifting between several different complex oligomers configurations can be influenced by the physicochemical properties of the stabilizing molecules.

Eimeria tenella enolase and pyruvate kinase: a likely role in glycolysis and in others functions.

PubMed

Labbé, Marie; Péroval, Marylène; Bourdieu, Christiane; Girard-Misguich, Fabienne; Péry, Pierre

2006-12-01

Two cDNA codings for glycolytic enzymes were cloned from a cDNA library constructed from the schizont stage of the avian parasite Eimeria tenella. Enolase and pyruvate kinase cDNA were fully sequenced and compared with sequences of enzymes from other organisms. Although these enzymes were already detected in the sporozoite stage, their expression was enhanced during the first schizogony in accordance with the anaerobic conditions of this part of the life cycle of the parasite. Under activating conditions, microscopic observations suggest that these glycolytic enzymes were relocalised inside sporozoites and moreover were in part secreted. The enzymes were also localised at the apex of the first generation of merozoites. Enolase was partly observed inside the nucleus of sporozoites and schizonts. Taken together, these results suggest that glycolytic enzymes not only have a function in glycolysis during anaerobic intracellular stages but may also participate in the invasion process and, for enolase, in the control of gene regulation.

Cytochrome P450 monooxygenases: perspectives for synthetic application.

PubMed

Urlacher, Vlada B; Eiben, Sabine

2006-07-01

Cytochrome P450 monooxygenases are versatile biocatalysts that introduce oxygen into a vast range of molecules. These enzymes catalyze diverse reactions in a regio- and stereoselective manner, and their properties have been used for drug development, bioremediation and the synthesis of fine chemicals and other useful compounds. However, the potential of P450 monooxygenases has not been fully exploited; there are some drawbacks limiting the broader implementation of these catalysts for commercial needs. Protein engineering has produced P450 enzymes with widely altered substrate specificities, substantially increased activity and higher stability. Furthermore, electrochemical and enzymatic approaches for the replacement or regeneration of NAD(P)H have been developed, enabling the more cost-effective use of P450 enzymes. In this review, we focus on the aspects relevant to the synthetic applications of P450 enzymes and their optimization for commercial needs.

Soy Pulp Extract Inhibits Angiotensin I-Converting Enzyme (ACE) Activity In Vitro: Evidence for Its Potential Hypertension-Improving Action.

PubMed

Nishibori, Naoyoshi; Kishibuchi, Reina; Morita, Kyoji

2017-05-04

Soy pulp, called "okara" in Japanese, is known as a by-product of the production of bean curd (tofu), and expected to contain a variety of biologically active substances derived from soybean. However, the biological activities of okara ingredients have not yet been fully understood, and the effectiveness of okara as a functional food seems necessary to be further evaluated. Then the effect of okara extract on angiotensin I-converting enzyme (ACE) activity was examined in vitro, and the extract was shown to cause the inhibition of ACE activity in a manner depending on its concentration. Kinetic analysis indicated that this enzyme inhibition was accompanied by an increase in the Km value without any change in Vmax. Further studies suggested that putative inhibitory substances contained in the extract might be heat stable and dialyzable, and recovered mostly in the peptide fraction obtained by a spin-column separation and a high performance liquid chromatography (HPLC) fractionation. Therefore, the extract was speculated to contain small-size peptides responsible for the inhibitory effect of okara extract on ACE activity, and could be expected to improve the hypertensive conditions by reducing the production of hypertensive peptide.

The Algicidal Fungus Trametes versicolor F21a Eliminating Blue Algae via Genes Encoding Degradation Enzymes and Metabolic Pathways Revealed by Transcriptomic Analysis

PubMed Central

Dai, Wei; Chen, Xiaolin; Wang, Xuewen; Xu, Zimu; Gao, Xueyan; Jiang, Chaosheng; Deng, Ruining; Han, Guomin

2018-01-01

The molecular mechanism underlying the elimination of algal cells by fungal mycelia has not been fully understood. Here, we applied transcriptomic analysis to investigate the gene expression and regulation at time courses of Trametes versicolor F21a during the algicidal process. The obtained results showed that a total of 193, 332, 545, and 742 differentially expressed genes were identified at 0, 6, 12, and 30 h during the algicidal process, respectively. The gene ontology terms were enriched into glucan 1,4-α-glucosidase activity, hydrolase activity, lipase activity, and endopeptidase activity. The KEGG pathways were enriched in degradation and metabolism pathways including Glycolysis/Gluconeogenesis, Pyruvate metabolism, the Biosynthesis of amino acids, etc. The total expression levels of all Carbohydrate-Active enZYmes (CAZyme) genes for the saccharide metabolism were increased by two folds relative to the control. AA5, GH18, GH5, GH79, GH128, and PL8 were the top six significantly up-regulated modules among 43 detected CAZyme modules. Four available homologous decomposition enzymes of other species could partially inhibit the growth of algal cells. The facts suggest that the algicidal mode of T. versicolor F21a might be associated with decomposition enzymes and several metabolic pathways. The obtained results provide a new candidate way to control algal bloom by application of decomposition enzymes in the future. PMID:29755442

Cloning, expression, purification and characterization of lipase from Bacillus licheniformis, isolated from hot spring of Himachal Pradesh, India.

PubMed

Kaur, Gagandeep; Singh, Amninder; Sharma, Rohit; Sharma, Vinay; Verma, Swati; Sharma, Pushpender K

2016-06-01

In the present investigation, a gene encoding extracellular lipase was cloned from a Bacillus licheniformis. The recombinant protein containing His-tag was expressed as inclusion bodies in Esherichia coli BL21DE3 cells, using pET-23a as expression vector. Expressed protein purified from the inclusion bodies demonstrated ~22 kDa protein band on 12 % SDS-PAGE. It exhibited specific activity of 0.49 U mg -1 and % yield of 8.58. Interestingly, the lipase displayed activity at wide range of pH and temperature, i.e., 9.0-14.0 pH and 30-80 °C, respectively. It further demonstrated ~100 % enzyme activity in presence of various organic solvents. Enzyme activity was strongly inhibited in the presence of β-ME. Additionally, the serine and histidine modifiers also inhibited the enzyme activities strongly at all concentrations that suggest their role in the catalytic center. Enzyme could retain its activity in presence of various detergents (Triton X-100, Tween 20, Tween 40, SDS). Sequence and structural analysis employing in silico tools revealed that the lipase contained two highly conserved sequences consisting of ITITGCGNDL and NLYNP, arranged as parallel β-sheet in the core of the 3D structure. The function of these conserve sequences have not fully understood.

Characterization of oxidative phosphorylation enzymes in Euglena gracilis and its white mutant strain W(gm)ZOflL.

PubMed

Krnáčová, Katarína; Rýdlová, Ivana; Vinarčíková, Michaela; Krajčovič, Juraj; Vesteg, Matej; Horváth, Anton

2015-03-12

The enzymes involved in Euglena oxidative phosphorylation (OXPHOS) were characterized in this study. We have demonstrated that Euglena gracilis strain Z and its stable bleached non-photosynthetic mutant strain WgmZOflL both possess fully functional OXPHOS apparatus as well as pathways requiring terminal alternative oxidase(s) and alternative mitochondrial NADH-dehydrogenase(s). Light (or dark) and plastid (non)functionality seem to have little effect on oxygen consumption, the activities of the enzymes involved in OXPHOS and the action of respiration inhibitors in Euglena. This study also demonstrates biochemical properties of complex III (cytochrome c reductase) in Euglena. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Complementary DNA cloning, functional expression and characterization of a novel cytochrome P450, CYP2D50, from equine liver.

PubMed

DiMaio Knych, H K; Stanley, S D

2008-10-01

Members of the CYP2D family constitute only about 2-4% of total hepatic CYP450s, however, they are responsible for the metabolism of 20-25% of commonly prescribed therapeutic compounds. CYP2D enzymes have been identified in a number of different species. However, vast differences in the metabolic activity of these enzymes have been well documented. In the horse, the presence of a member of the CYP2D family has been suggested from studies with equine liver microsomes, however its presence has not been definitively proven. In this study a cDNA encoding a novel CYP2D enzyme (CYP2D50) was cloned from equine liver and expressed in a baculovirus expression system. The nucleotide sequence of CYP2D50 was highly homologous to that of human CYP2D6 and therefore the activity of the enzyme was characterized using dextromethorphan and debrisoquine, two isoform selective substrates for the human orthologue. CYP2D50 displayed optimal catalytic activity with dextromethorphan using molar ratios of CYP2D50 to NADPH CYP450 reductase of 1:15. Although CYP2D50 and CYP2D6 shared significant sequence homology, there were striking differences in the catalytic activity between the two enzymes. CYP2D50 dextromethorphan-O-demethylase activity was nearly 180-fold slower than the human counterpart, CYP2D6. Similarly, rates of formation of 4-hydroxydebrisoquine activity were 50-fold slower for CYP2D50 compared to CYP2D6. The results of this study demonstrate substantial interspecies variability in metabolism of substrates by CYP2D orthologues in the horse and human and support the need to fully characterize this enzyme system in equids.

NAD+ glycohydrolase, an ecto-enzyme of calf spleen cells.

PubMed Central

Muller, H M; Muller, C D; Schuber, F

1983-01-01

By using a sensitive fluorimetric assay of NAD+ glycohydrolase (EC 3.2.2.6), we showed that calf spleen cells are able to hydrolyse 1,N6-etheno-NAD+ given in the medium. The observed rates of substrate hydrolysis and product accumulation in the medium are equivalent. Moreover, the splenocytes are able to cleave the nicotinamide-ribose bond of a water-soluble polymer of NAD+, and their NAD+ glycohydrolase activity is fully inhibited by a high-molecular-weight Blue Dextran. Selective permeation of the cellular membrane digitonin revealed an intracellular pool of NAD+ glycohydrolase, which accounts for 25% of the total activity. We conclude that NAD+ glycohydrolase associated with the splenocytes has the characteristics of an ecto-enzyme. PMID:6192807

NREL Explains the Higher Cellulolytic Activity of a Vital Microorganism

DOE Office of Scientific and Technical Information (OSTI.GOV)

The discovery of a new mode of action by C. thermocellum to convert biomass to biofuels is significant because the bacterium is already recognized as one of the most effective in the biosphere. Researchers found that, in addition to using common cellulase degradation mechanisms attached to cells, C. thermocellum also uses a new category of cell-free scaffolded enzymes. The new discovery will influence the strategies used to improve the cellulolytic activity of biomass degrading microbes going forward. Better understanding of this bacterium could lead to cheaper production of ethanol and drop-in fuels. Also, this discovery demonstrates that nature's biomass conversionmore » behaviors are not fully understood and remain as opportunities for future microbial/enzyme engineering efforts.« less

Effect of decabromodiphenyl ether (BDE-209) on a soil-biota system: Role of earthworms and ryegrass.

PubMed

Feng, Mingbao; He, Qun; Shi, Jiaqi; Qin, Li; Zhang, Xuesheng; Sun, Ping; Wang, Zunyao

2016-06-01

In the present study, the toxic effect of decabromodiphenyl ether (BDE-209), an important brominated fire retardant, on soil was evaluated by amending with different concentrations (0 mg/kg, 1 mg/kg, 10 mg/kg, and 500 mg/kg dry wt) for 40 d. The activities of 3 soil enzymes (urease, catalase, and alkaline phosphatase) were measured as the principal assessment endpoints. Meanwhile, the effects of natural environmental factors, such as light conditions and soil biota, on BDE-209 intoxication were studied. For the latter, 30 earthworms (Metaphire guillelmi) with fully matured clitella or ryegrass (Lolium perenne) with fully matured leaves were exposed in soil amended with BDE-209. The activities of the soil enzymes were adversely affected by BDE-209, especially for the high-concentration treatments, with greater adverse effects in the dark than in the light. The presence of earthworms reduced toxicity to BDE-209, whereas ryegrass did not. The calculated integrated biomarker response index, which provides a general indicator of the health status of test species by combining different biomarker signals, further validated these findings. Moreover, the antioxidant status (oxidant-antioxidant balance) of these 2 biota was assessed. Results indicated that BDE-209 significantly affected the activities of antioxidant enzymes (superoxide dismutase and catalase) and enhanced the levels of malondialdehyde in both species. The present study may facilitate a better understanding of the toxicity of BDE-209 toward the soil environment. Environ Toxicol Chem 2016;35:1349-1357. © 2015 SETAC. © 2015 SETAC.

In vitro assembly of catalase.

PubMed

Baureder, Michael; Barane, Elisabeth; Hederstedt, Lars

2014-10-10

Most aerobic organisms contain catalase, which functions to decompose hydrogen peroxide. Typical catalases are structurally complex homo-tetrameric enzymes with one heme prosthetic group buried in each subunit. It is not known how catalase in the cell is assembled from its constituents. The bacterium Enterococcus faecalis cannot synthesize heme but can acquire it from the environment to form a cytoplasmic catalase. We have in E. faecalis monitored production of the enzyme polypeptide (KatA) depending on the availability of heme and used our findings to devise a procedure for the purification of preparative amounts of in vivo-synthesized apocatalase. We show that fully active catalase can be obtained in vitro by incubating isolated apoprotein with hemin. We have characterized features of the assembly process and describe a temperature-trapped hemylated intermediate of the enzyme maturation process. Hemylation of apocatalase does not require auxiliary cell components, but rapid assembly of active enzyme seemingly is assisted in the cell. Our findings provide insight about catalase assembly and offer new experimental possibilities for detailed studies of this process. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Biochemical characterization of the purple form of Marinobacter hydrocarbonoclasticus nitrous oxide reductase

PubMed Central

Dell'Acqua, Simone; Pauleta, Sofia R.; Moura, José J. G.; Moura, Isabel

2012-01-01

Nitrous oxide reductase (N2OR) catalyses the final step of the denitrification pathway—the reduction of nitrous oxide to nitrogen. The catalytic centre (CuZ) is a unique tetranuclear copper centre bridged by inorganic sulphur in a tetrahedron arrangement that can have different oxidation states. Previously, Marinobacter hydrocarbonoclasticus N2OR was isolated with the CuZ centre as CuZ*, in the [1Cu2+ : 3Cu+] redox state, which is redox inert and requires prolonged incubation under reductive conditions to be activated. In this work, we report, for the first time, the isolation of N2OR from M. hydrocarbonoclasticus in the ‘purple’ form, in which the CuZ centre is in the oxidized [2Cu2+ : 2Cu+] redox state and is redox active. This form of the enzyme was isolated in the presence of oxygen from a microaerobic culture in the presence of nitrate and also from a strictly anaerobic culture. The purple form of the enzyme was biochemically characterized and was shown to be a redox active species, although it is still catalytically non-competent, as its specific activity is lower than that of the activated fully reduced enzyme and comparable with that of the enzyme with the CuZ centre in either the [1Cu2+ : 3Cu+] redox state or in the redox inactive CuZ* state. PMID:22451106

Biochemical Characterization of the Lactobacillus reuteri Glycoside Hydrolase Family 70 GTFB Type of 4,6-α-Glucanotransferase Enzymes That Synthesize Soluble Dietary Starch Fibers.

PubMed

Bai, Yuxiang; van der Kaaij, Rachel Maria; Leemhuis, Hans; Pijning, Tjaard; van Leeuwen, Sander Sebastiaan; Jin, Zhengyu; Dijkhuizen, Lubbert

2015-10-01

4,6-α-Glucanotransferase (4,6-α-GTase) enzymes, such as GTFB and GTFW of Lactobacillus reuteri strains, constitute a new reaction specificity in glycoside hydrolase family 70 (GH70) and are novel enzymes that convert starch or starch hydrolysates into isomalto/maltopolysaccharides (IMMPs). These IMMPs still have linear chains with some α1→4 linkages but mostly (relatively long) linear chains with α1→6 linkages and are soluble dietary starch fibers. 4,6-α-GTase enzymes and their products have significant potential for industrial applications. Here we report that an N-terminal truncation (amino acids 1 to 733) strongly enhances the soluble expression level of fully active GTFB-ΔN (approximately 75-fold compared to full-length wild type GTFB) in Escherichia coli. In addition, quantitative assays based on amylose V as the substrate are described; these assays allow accurate determination of both hydrolysis (minor) activity (glucose release, reducing power) and total activity (iodine staining) and calculation of the transferase (major) activity of these 4,6-α-GTase enzymes. The data show that GTFB-ΔN is clearly less hydrolytic than GTFW, which is also supported by nuclear magnetic resonance (NMR) analysis of their final products. From these assays, the biochemical properties of GTFB-ΔN were characterized in detail, including determination of kinetic parameters and acceptor substrate specificity. The GTFB enzyme displayed high conversion yields at relatively high substrate concentrations, a promising feature for industrial application. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Activating Intrinsic Carbohydrate-Active Enzymes of the Smut Fungus Ustilago maydis for the Degradation of Plant Cell Wall Components

PubMed Central

Geiser, Elena; Reindl, Michèle; Blank, Lars M.; Feldbrügge, Michael

2016-01-01

ABSTRACT The microbial conversion of plant biomass to valuable products in a consolidated bioprocess could greatly increase the ecologic and economic impact of a biorefinery. Current strategies for hydrolyzing plant material mostly rely on the external application of carbohydrate-active enzymes (CAZymes). Alternatively, production organisms can be engineered to secrete CAZymes to reduce the reliance on externally added enzymes. Plant-pathogenic fungi have a vast repertoire of hydrolytic enzymes to sustain their lifestyle, but expression of the corresponding genes is usually highly regulated and restricted to the pathogenic phase. Here, we present a new strategy in using the biotrophic smut fungus Ustilago maydis for the degradation of plant cell wall components by activating its intrinsic enzyme potential during axenic growth. This fungal model organism is fully equipped with hydrolytic enzymes, and moreover, it naturally produces value-added substances, such as organic acids and biosurfactants. To achieve the deregulated expression of hydrolytic enzymes during the industrially relevant yeast-like growth in axenic culture, the native promoters of the respective genes were replaced by constitutively active synthetic promoters. This led to an enhanced conversion of xylan, cellobiose, and carboxymethyl cellulose to fermentable sugars. Moreover, a combination of strains with activated endoglucanase and β-glucanase increased the release of glucose from carboxymethyl cellulose and regenerated amorphous cellulose, suggesting that mixed cultivations could be a means for degrading more complex substrates in the future. In summary, this proof of principle demonstrates the potential applicability of activating the expression of native CAZymes from phytopathogens in a biocatalytic process. IMPORTANCE This study describes basic experiments that aim at the degradation of plant cell wall components by the smut fungus Ustilago maydis. As a plant pathogen, this fungus contains a set of lignocellulose-degrading enzymes that may be suited for biomass degradation. However, its hydrolytic enzymes are specifically expressed only during plant infection. Here, we provide the proof of principle that these intrinsic enzymes can be synthetically activated during the industrially relevant yeast-like growth. The fungus is known to naturally synthesize valuable compounds, such as itaconate or glycolipids. Therefore, it could be suited for use in a consolidated bioprocess in which more complex and natural substrates are simultaneously converted to fermentable sugars and to value-added compounds in the future. PMID:27316952

Astaxanthin Induces the Nrf2/HO-1 Antioxidant Pathway in Human Umbilical Vein Endothelial Cells by Generating Trace Amounts of ROS.

PubMed

Niu, Tingting; Xuan, Rongrong; Jiang, Ligang; Wu, Wei; Zhen, Zhanghe; Song, Yuling; Hong, Lili; Zheng, Kaiqin; Zhang, Jiaxing; Xu, Qingshan; Tan, Yinghong; Yan, Xiaojun; Chen, Haimin

2018-02-14

Astaxanthin is a powerful antioxidant that possesses potent protective effects against various human diseases and physiological disorders. However, the mechanisms underlying its antioxidant functions in cells are not fully understood. In the present study, the effects of astaxanthin on reactive oxygen species (ROS) production and antioxidant enzyme activity, as well as mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K)/Akt, and the nuclear factor erythroid 2-related factor 2 (Nrf-2)/heme oxygenase-1 (HO-1) pathways in human umbilical vein endothelial cells (HUVECs), were examined. It was shown that astaxanthin (0.1, 1, and 10 μM) induced ROS production by 9.35%, 14.8%, and 18.06% compared to control, respectively, in HUVECs. In addition, astaxanthin increased the mRNA levels of phase II enzymes HO-1 and also promoted GSH-Px enzyme activity. Furthermore, we observed ERK phosphorylation, nuclear translocation of Nrf-2, and activation of antioxidant response element-driven luciferase activity upon astaxanthin treatment. Knockdown of Nrf-2 by small interfering RNA inhibited HO-1 mRNA expression by 60%, indicating that the Nrf-2/ARE signaling pathway is activated by astaxanthin. Our results suggest that astaxanthin activates the Nrf-2/HO-1 antioxidant pathway by generating small amounts of ROS.

Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular Lego.

PubMed

Dodhia, Vikash Rajnikant; Fantuzzi, Andrea; Gilardi, Gianfranco

2006-10-01

The membrane-bound human cytochrome P450s have essential roles in the metabolism of endogenous compounds and drugs. Presented here are the results on the construction and characterization of three fusion proteins containing the N-terminally modified human cytochrome P450s CYP2C9, CY2C19 and CYP3A4 fused to the soluble NADPH-dependent oxidoreductase domain of CYP102A1 from Bacillus megaterium. The constructs, CYP2C9/BMR, CYP2C19/BMR and CYP3A4/BMR are well expressed in Escherichia coli as holo proteins. The chimeras can be purified in the absence of detergent and the purified enzymes are both active and correctly folded in the absence of detergent, as demonstrated by circular dichroism and functional studies. Additionally, in comparison with the parent P450 enzyme, these chimeras have greatly improved solubility properties. The chimeras are catalytically self-sufficient and present turnover rates similar to those reported for the native enzymes in reconstituted systems, unlike previously reported mammalian cytochrome P450 fusion proteins. Furthermore the specific activities of these chimeras are not dependent on the enzyme concentration present in the reaction buffer and they do not require the addition of accessory proteins, detergents or phospholipids to be fully active. The solubility, catalytic self-sufficiency and wild-type like activities of these chimeras would greatly simplify the studies of cytochrome P450 mediated drug metabolism in solution.

Cold and Hot Extremozymes: Industrial Relevance and Current Trends

PubMed Central

Sarmiento, Felipe; Peralta, Rocío; Blamey, Jenny M.

2015-01-01

The development of enzymes for industrial applications relies heavily on the use of microorganisms. The intrinsic properties of microbial enzymes, e.g., consistency, reproducibility, and high yields along with many others, have pushed their introduction into a wide range of products and industrial processes. Extremophilic microorganisms represent an underutilized and innovative source of novel enzymes. These microorganisms have developed unique mechanisms and molecular means to cope with extreme temperatures, acidic and basic pH, high salinity, high radiation, low water activity, and high metal concentrations among other environmental conditions. Extremophile-derived enzymes, or extremozymes, are able to catalyze chemical reactions under harsh conditions, like those found in industrial processes, which were previously not thought to be conducive for enzymatic activity. Due to their optimal activity and stability under extreme conditions, extremozymes offer new catalytic alternatives for current industrial applications. These extremozymes also represent the cornerstone for the development of environmentally friendly, efficient, and sustainable industrial technologies. Many advances in industrial biocatalysis have been achieved in recent years; however, the potential of biocatalysis through the use of extremozymes is far from being fully realized. In this article, the adaptations and significance of psychrophilic, thermophilic, and hyperthermophilic enzymes, and their applications in selected industrial markets will be reviewed. Also, the current challenges in the development and mass production of extremozymes as well as future prospects and trends for their biotechnological application will be discussed. PMID:26539430

Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate.

PubMed

Vandavasi, Venu Gopal; Langan, Patricia S; Weiss, Kevin L; Parks, Jerry M; Cooper, Jonathan B; Ginell, Stephan L; Coates, Leighton

2017-01-01

The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M β-lactamase but not for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A β-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, as previously proposed. Copyright © 2016 Vandavasi et al.

Cellobiohydrolase (CBH) Activity Assays.

PubMed

Sharma, Hem Kanta; Qin, Wensheng; Xu, Chunbao Charles

2018-01-01

Cellulosic biomass is the most abundant biopolymer on the earth. It has great potential to quench the thirst of liquid energy by producing biofuels and thus help to mitigate human reliance on fossil fuels. Although several cellulase activity assay methods have been used to disintegrate the glycosidic bonds, the appropriate selection of substrates and synergistic involvement of multiple enzymes in hydrolytic activity is not yet fully understood. The proper quantification of hydrolytic enzymes and hydrolysates is challenging because of the heterogeneity of cellulose, changes in enzyme-substrate ratio and the presence of some inhibitory compounds like cellobiose and cellodextran. In the glycosyl hydrolase (GH) family, cellobiohydrolase (CBH) is expected to disrupt the crystalline cellulose and release the sugar molecules. Several methods have been proposed for CBH assay with slight modification in substrate and quantification of hydrolysates. However, the Avicel method is still considered as the most promising and efficient hydrolytic technique so far. The most commonly used CBH assays including Avicel and other recent methods for proper quantification are outlined in this chapter. Also a qualitative screening of CBH producing bacteria using carboxymethyl cellulose (CMC) agar plates is described.

Transcriptome, antioxidant enzyme activity and histopathology analysis of hepatopancreas from the white shrimp Litopenaeus vannamei fed with aflatoxin B1(AFB1).

PubMed

Zhao, Wei; Wang, Lei; Liu, Mei; Jiang, Keyong; Wang, Mengqiang; Yang, Guang; Qi, Cancan; Wang, Baojie

2017-09-01

Aflatoxin produced by Aspergillus flavus or Aspergillus parasiticus fungi during grain and feed processing and storage. Aflatoxins cause severe health problems reducing the yield and profitability of shrimp cultures. We sought to understand the interaction between shrimp immunity and aflatoxin B1 (AFB1), analyzing transcriptome expression, antioxidant enzyme activity, and histological features of the hepatopancreas of shrimp fed with AFB1. From over 4 million high-quality reads, de novo unigene assembly produced 103,644 fully annotated genes. A total of 1024 genes were differentially expressed in shrimp fed with AFB1, being involved in functions, such as peroxidase metabolism, signal transduction, transcriptional control, apoptosis, proteolysis, endocytosis, and cell adhesion and cell junction. Upon AFB1 challenge, there were severe histological alterations in shrimp hepatopancreas. AFB1 challenge increased the activity of several antioxidant enzymes. Our data contribute to improve the current understanding of host-AFB1 interaction, providing an abundant source for identification of novel genes. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Clinically Relevant Variant of the Human Hydrogen Sulfide-Synthesizing Enzyme Cystathionine β-Synthase: Increased CO Reactivity as a Novel Molecular Mechanism of Pathogenicity?

PubMed Central

Malagrinò, Francesca; Santo, Paulo E.; Gutierres, André; Bandeiras, Tiago M.; Leandro, Paula

2017-01-01

The human disease classical homocystinuria results from mutations in the gene encoding the pyridoxal 5′-phosphate- (PLP-) dependent cystathionine β-synthase (CBS), a key enzyme in the transsulfuration pathway that controls homocysteine levels, and is a major source of the signaling molecule hydrogen sulfide (H2S). CBS activity, contributing to cellular redox homeostasis, is positively regulated by S-adenosyl-L-methionine (AdoMet) but fully inhibited upon CO or NO• binding to a noncatalytic heme moiety. Despite extensive studies, the molecular basis of several pathogenic CBS mutations is not yet fully understood. Here we found that the ferrous heme of the reportedly mild p.P49L CBS variant has altered spectral properties and markedly increased affinity for CO, making the protein much more prone than wild type (WT) CBS to inactivation at physiological CO levels. The higher CO affinity could result from the slightly higher flexibility in the heme surroundings revealed by solving at 2.80-Å resolution the crystallographic structure of a truncated p.P49L. Additionally, we report that p.P49L displays impaired H2S-generating activity, fully rescued by PLP supplementation along the purification, despite a minor responsiveness to AdoMet. Altogether, the results highlight how increased propensity to CO inactivation of an otherwise WT-like variant may represent a novel pathogenic mechanism in classical homocystinuria. PMID:28421128

Enzyme cascades activated on topologically programmed DNA scaffolds

NASA Astrophysics Data System (ADS)

Wilner, Ofer I.; Weizmann, Yossi; Gill, Ron; Lioubashevski, Oleg; Freeman, Ronit; Willner, Itamar

2009-04-01

The ability of DNA to self-assemble into one-, two- and three-dimensional nanostructures, combined with the precision that is now possible when positioning nanoparticles or proteins on DNA scaffolds, provide a promising approach for the self-organization of composite nanostructures. Predicting and controlling the functions that emerge in self-organized biomolecular nanostructures is a major challenge in systems biology, and although a number of innovative examples have been reported, the emergent properties of systems in which enzymes are coupled together have not been fully explored. Here, we report the self-assembly of a DNA scaffold made of DNA strips that include `hinges' to which biomolecules can be tethered. We attach either two enzymes or a cofactor-enzyme pair to the scaffold, and show that enzyme cascades or cofactor-mediated biocatalysis can proceed effectively; similar processes are not observed in diffusion-controlled homogeneous mixtures of the same components. Furthermore, because the relative position of the two enzymes or the cofactor-enzyme pair is determined by the topology of the DNA scaffold, it is possible to control the reactivity of the system through the design of the individual DNA strips. This method could lead to the self-organization of complex multi-enzyme cascades.

An enzyme-mediated protein-fragment complementation assay for substrate screening of sortase A.

PubMed

Li, Ning; Yu, Zheng; Ji, Qun; Sun, Jingying; Liu, Xiao; Du, Mingjuan; Zhang, Wei

2017-04-29

Enzyme-mediated protein conjugation has gained great attention recently due to the remarkable site-selectivity and mild reaction condition affected by the nature of enzyme. Among all sorts of enzymes reported, sortase A from Staphylococcus aureus (SaSrtA) is the most popular enzyme due to its selectivity and well-demonstrated applications. Position scanning has been widely applied to understand enzyme substrate specificity, but the low throughput of chemical synthesis of peptide substrates and analytical methods (HPLC, LC-ESI-MS) have been the major hurdle to fully decode enzyme substrate profile. We have developed a simple high-throughput substrate profiling method to reveal novel substrates of SaSrtA 7M, a widely used hyperactive peptide ligase, by modified protein-fragment complementation assay (PCA). A small library targeting the LPATG motif recognized by SaSrtA 7M was generated and screened against proteins carrying N-terminal glycine. Using this method, we have confirmed all currently known substrates of the enzyme, and moreover identified some previously unknown substrates with varying activities. The method provides an easy, fast and highly-sensitive way to determine substrate profile of a peptide ligase in a high-throughput manner. Copyright © 2017 Elsevier Inc. All rights reserved.

Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site

PubMed Central

Sunden, Fanny; Peck, Ariana; Salzman, Julia; Ressl, Susanne; Herschlag, Daniel

2015-01-01

Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called ‘catalytic residues’ are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes. DOI: http://dx.doi.org/10.7554/eLife.06181.001 PMID:25902402

Synergistic Enhancement of Enzyme Performance and Resilience via Orthogonal Peptide-Protein Chemistry Enabled Multilayer Construction.

PubMed

Zhang, Xue-Jian; Wang, Xiao-Wei; Sun, Jiaxing; Su, Chao; Yang, Shuguang; Zhang, Wen-Bin

2018-05-16

Protein immobilization is critical to utilize their unique functions in diverse applications. Herein, we report that orthogonal peptide-protein chemistry enabled multilayer construction can facilitate the incorporation of various folded structural domains, including calmodulin in different states, affibody and dihydrofolate reductase (DHFR). An extended conformation is found to be the most advantageous for steady film growth. The resulting protein thin films exhibit sensitive and selective responsive behaviors to bio-signals (Ca2+, TFP, NADPH, etc.) and fully maintain the catalytic activity of DHFR. The approach is applicable to different substrates such as hydrophobic gold and hydrophilic silica microparticles. The DHFR enzyme can be immobilized onto silica microparticles with tunable amounts. The multi-layer set-up exhibits a synergistic enhancement of DHFR activity with increasing number of bilayers and also makes the embedded DHFR more resilient to lyophilization. Therefore, this is a convenient and versatile method for protein immobilization with potential benefits of synergistic enhancement in enzyme performance and resilience.

Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site

DOE PAGES

Sunden, Fanny; Peck, Ariana; Salzman, Julia; ...

2015-04-22

Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called ‘catalytic residues’ are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modesmore » of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes.« less

An extraovarian aspartic protease accumulated in tick oocytes with vitellin-degradation activity.

PubMed

Pohl, Paula C; Sorgine, Marcos Henrique F; Leal, Alexandre T; Logullo, Carlos; Oliveira, Pedro L; Vaz, Itabajara da Silva; Masuda, Aoi

2008-12-01

An aspartic endopeptidase named THAP, from the eggs of the tick Riphicephalus (Boophilus) microplus, has been suggested to be involved in vitellin-degradation. Here we characterized this enzyme further, showing that THAP mRNA is present in the fat body, midgut and ovary of ticks, in two developmental stages (partially and fully engorged females). However, higher transcription levels were found in fully engorged vitellogenic females. The THAP protein was detected in the haemolymph, midgut and fat body and, in higher quantity, in the ovary of fully engorged females, and it was present throughout embryo development. The protein is synthesized as a higher molecular mass form and after the onset of embryogenesis THAP is converted into an active form by autocatalysis. We also produced a recombinant protein (rTHAP) in E. coli that was active in the fluorogenic peptide substrate and able to hydrolyze vitellin from 7-day-old eggs in a reaction that is heme-sensitive and inhibited by pepstatin A. However, rTHAP does not hydrolyze vitellin from 1 and 12-day-old eggs. As a result, we suggest a model for THAP synthesis, transport, storage and activation and for the role it plays in embryonic development by participating in vitellin processing.

Light-driven enzymatic catalysis of DNA repair: a review of recent biophysical studies on photolyase.

PubMed

Weber, Stefan

2005-02-25

More than 50 years ago, initial experiments on enzymatic photorepair of ultraviolet (UV)-damaged DNA were reported [Proc. Natl. Acad. Sci. U. S. A. 35 (1949) 73]. Soon after this discovery, it was recognized that one enzyme, photolyase, is able to repair UV-induced DNA lesions by effectively reversing their formation using blue light. The enzymatic process named DNA photoreactivation depends on a non-covalently bound cofactor, flavin adenine dinucleotide (FAD). Flavins are ubiquitous redox-active catalysts in one- and two-electron transfer reactions of numerous biological processes. However, in the case of photolyase, not only the ground-state redox properties of the FAD cofactor are exploited but also, and perhaps more importantly, its excited-state properties. In the catalytically active, fully reduced redox form, the FAD absorbs in the blue and near-UV ranges of visible light. Although there is no direct experimental evidence, it appears generally accepted that starting from the excited singlet state, the chromophore initiates a reductive cleavage of the two major DNA photodamages, cyclobutane pyrimidine dimers and (6-4) photoproducts, by short-distance electron transfer to the DNA lesion. Back electron transfer from the repaired DNA segment is believed to eventually restore the initial redox states of the cofactor and the DNA nucleobases, resulting in an overall reaction with net-zero exchanged electrons. Thus, the entire process represents a true catalytic cycle. Many biochemical and biophysical studies have been carried out to unravel the fundamentals of this unique mode of action. The work has culminated in the elucidation of the three-dimensional structure of the enzyme in 1995 that revealed remarkable details, such as the FAD-cofactor arrangement in an unusual U-shaped configuration. With the crystal structure of the enzyme at hand, research on photolyases did not come to an end but, for good reason, intensified: the geometrical structure of the enzyme alone is not sufficient to fully understand the enzyme's action on UV-damaged DNA. Much effort has therefore been invested to learn more about, for example, the geometry of the enzyme-substrate complex, and the mechanism and pathways of intra-enzyme and enzyme <-->DNA electron transfer. Many of the key results from biochemical and molecular biology characterizations of the enzyme or the enzyme-substrate complex have been summarized in a number of reviews. Complementary to these articles, this review focuses on recent biophysical studies of photoreactivation comprising work performed from the early 1990s until the present.

Identification of a mitochondrial alcohol dehydrogenase in Schizosaccharomyces pombe: new insights into energy metabolism

PubMed Central

Crichton, Paul G.; Affourtit, Charles; Moore, Anthony L.

2006-01-01

In the present study we have shown that mitochondria isolated from Schizosaccharomyces pombe exhibit antimycin A-sensitive oxygen uptake activity that is exclusively dependent on ethanol and is inhibited by trifluoroethanol, a potent inhibitor of ADH (alcohol dehydrogenase). Ethanol-dependent respiratory activity has, to our knowledge, not been reported in S. pombe mitochondria to date, which is surprising as it has been concluded previously that only one ADH gene, encoding a cytosolic enzyme, occurs in this yeast. Spectrophotometric enzyme assays reveal that ADH activity in isolated mitochondria is increased ∼16-fold by Triton X-100, which demonstrates that the enzyme is located in the matrix. Using genetic knockouts, we show conclusively that the novel mitochondrial ADH is encoded by adh4 and, as such, is unrelated to ADH isoenzymes found in mitochondria of other yeasts. By performing a modular-kinetic analysis of mitochondrial electron transfer, we furthermore show how ethanol-dependent respiratory activity (which involves oxidation of matrix-located NADH) compares with that observed when succinate or externally added NADH are used as substrates. This analysis reveals distinct kinetic differences between substrates which fully explain the lack of respiratory control generally observed during ethanol oxidation in yeast mitochondria. PMID:16999687

A novel cold-adapted and glucose-tolerant GH1 β-glucosidase from Exiguobacterium antarcticum B7.

PubMed

Crespim, Elaine; Zanphorlin, Letícia M; de Souza, Flavio H M; Diogo, José A; Gazolla, Alex C; Machado, Carla B; Figueiredo, Fernanda; Sousa, Amanda S; Nóbrega, Felipe; Pellizari, Vivian H; Murakami, Mário T; Ruller, Roberto

2016-01-01

A novel GH1 β-glucosidase (EaBgl1A) from a bacterium isolated from Antarctica soil samples was recombinantly overexpressed in Escherichia coli cells and characterized. The enzyme showed unusual pH dependence with maximum activity at neutral pH and retention of high catalytic activity in the pH range 6 to 9, indicating a catalytic machinery compatible with alkaline conditions. EaBgl1A is also a cold-adapted enzyme, exhibiting activity in the temperature range from 10 to 40°C with optimal activity at 30°C, which allows its application in industrial processes using low temperatures. Kinetic characterization revealed an enzymatic turnover (Kcat) of 6.92s(-1) (cellobiose) and 32.98s(-1) (pNPG) and a high tolerance for product inhibition, which is an extremely desirable feature for biotechnological purposes. Interestingly, the enzyme was stimulated by up to 200 mM glucose, whereas the commercial cocktails tested were found fully inhibited at this concentration. These properties indicate EaBgl1A as a promising biocatalyst for biotechnological applications where low temperatures are required. Copyright © 2015 Elsevier B.V. All rights reserved.

Temperature-dependent endogenous oxygen concentration regulates microsomal oleate desaturase in developing sunflower seeds.

PubMed

Rolletschek, Hardy; Borisjuk, Ljudmilla; Sánchez-García, Alicia; Gotor, Cecilia; Romero, Luis C; Martínez-Rivas, José M; Mancha, Manuel

2007-01-01

Oleoyl-phosphatidylcholine desaturase (FAD2) is a key enzyme involved in fatty acid desaturation in oilseeds, which is affected by environmental temperature. The results of this study show that FAD2 is regulated in vivo via temperature-dependent endogenous oxygen concentrations in developing sunflower (Helianthus annuus L.) seeds. By combining in vivo oxygen profiling, in situ hybridization of FAD2 genes, an assay of energy status, fatty acid analysis, and an in vitro FAD2 enzyme activity assay, it is shown that: (i) the oil-storing embryo is characterized by a very low oxygen level that is developmentally regulated. Oxygen supply is mainly limited by the thin seed coat. (ii) Elevations of external oxygen supply raised the energy status of seed and produced a dramatic increase of the FAD2 enzyme activity as well as the linoleic acid content. (iii) A clear negative correlation exists between temperature and internal oxygen concentration. The changes occurred almost instantly and the effect was fully reversible. The results indicate that the internal oxygen level acts as a key regulator for the activity of the FAD2 enzyme. It is concluded that a major mechanism by which temperature modifies the unsaturation degree of the sunflower oil is through its effect on dissolved oxygen levels in the developing seed.

Entrapment of Carbon Dioxide in the Active Site of Carbonic Anhydrase II*♦

PubMed Central

Domsic, John F.; Avvaru, Balendu Sankara; Kim, Chae Un; Gruner, Sol M.; Agbandje-McKenna, Mavis; Silverman, David N.; McKenna, Robert

2008-01-01

The visualization at near atomic resolution of transient substrates in the active site of enzymes is fundamental to fully understanding their mechanism of action. Here we show the application of using CO2-pressurized, cryo-cooled crystals to capture the first step of CO2 hydration catalyzed by the zinc-metalloenzyme human carbonic anhydrase II, the binding of substrate CO2, for both the holo and the apo (without zinc) enzyme to 1.1Å resolution. Until now, the feasibility of such a study was thought to be technically too challenging because of the low solubility of CO2 and the fast turnover to bicarbonate by the enzyme (Liang, J. Y., and Lipscomb, W. N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3675–3679). These structures provide insight into the long hypothesized binding of CO2 in a hydrophobic pocket at the active site and demonstrate that the zinc does not play a critical role in the binding or orientation of CO2. This method may also have a much broader implication for the study of other enzymes for which CO2 is a substrate or product and for the capturing of transient substrates and revealing hydrophobic pockets in proteins. PMID:18768466

Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cooper, Jonathan B.; Weiss, Kevin L.; Coates, Leighton

The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. Several active site residues in class A β-lactamases have been proposed to play key roles in monobactam hydrolysis. The protonation states of these residues have been determined previously for the apo form of a CTX-M β-lactamase. However, they have not yet been determined for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a classmore » A β-lactamase in acyl enzyme complex with aztreonam we directly observed most of the hydrogen atoms (as deuterium) within the active site in the captured acyl-enzyme state between Toho-1 β-lactamase and aztreonam. Although Lys 234 is fully protonated in the acyl-intermediate, we find that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, in agreement with previous mechanistic proposals.« less

Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate

DOE PAGES

Cooper, Jonathan B.; Weiss, Kevin L.; Coates, Leighton; ...

2016-10-24

The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. Several active site residues in class A β-lactamases have been proposed to play key roles in monobactam hydrolysis. The protonation states of these residues have been determined previously for the apo form of a CTX-M β-lactamase. However, they have not yet been determined for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a classmore » A β-lactamase in acyl enzyme complex with aztreonam we directly observed most of the hydrogen atoms (as deuterium) within the active site in the captured acyl-enzyme state between Toho-1 β-lactamase and aztreonam. Although Lys 234 is fully protonated in the acyl-intermediate, we find that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, in agreement with previous mechanistic proposals.« less

Relevance of Local Flexibility Near the Active Site for Enzymatic Catalysis: Biochemical Characterization and Engineering of Cellulase Cel5A From Bacillus agaradherans.

PubMed

Saavedra, Juan M; Azócar, Mauricio A; Rodríguez, Vida; Ramírez-Sarmiento, César A; Andrews, Barbara A; Asenjo, Juan A; Parra, Loreto P

2018-03-25

Detailed molecular mechanisms underpinning enzymatic reactions are still a central problem in biochemistry. The need for active site flexibility to sustain catalytic activity constitutes a notion of wide acceptance, although its direct influence remains to be fully understood. With the aim of studying the relationship between structural dynamics and enzyme catalysis, the cellulase Cel5A from Bacillus agaradherans is used as a model for in silico comparative analysis with mesophilic and psychrophilic counterparts. Structural features that determine flexibility are related to kinetic and thermodynamic parameters of catalysis. As a result, three specific positions in the vicinity of the active site of Cel5A are selected for protein engineering via site-directed mutagenesis. Three Cel5A variants are generated, N141L, A137Y and I102A/A137Y, showing a concomitant increase in the catalytic activity at low temperatures and a decrease in activation energy and activation enthalpy, similar to cold-active enzymes. These results are interpreted in structural terms by molecular dynamics simulations, showing that disrupting a hydrogen bond network in the vicinity of the active site increases local flexibility. These results provide a structural framework for explaining the changes in thermodynamic parameters observed between homologous enzymes with varying temperature adaptations. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Changes in Biochemical Characteristics and Activities of Ripening Associated Enzymes in Mango Fruit during the Storage at Different Temperatures

PubMed Central

Kimura, Yoshinobu

2014-01-01

As a part of the study to explore the possible strategy for enhancing the shelf life of mango fruits, we investigated the changes in biochemical parameters and activities of ripening associated enzymes of Ashwina hybrid mangoes at 4-day regular intervals during storage at −10°C, 4°C, and 30 ± 1°C. Titratable acidity, vitamin C, starch content, and reducing sugar were higher at unripe state and gradually decreased with the increasing of storage time at all storage temperatures while phenol content, total soluble solid, total sugar, and nonreducing sugar contents gradually increased. The activities of amylase, α-mannosidase, α-glucosidase, and invertase increased sharply within first few days and decreased significantly in the later stage of ripening at 30 ± 1°C. Meanwhile polyphenol oxidase, β-galactosidase, and β-hexosaminidase predominantly increased significantly with the increasing days of storage till later stage of ripening. At −10°C and 4°C, the enzymes as well as carbohydrate contents of storage mango changed slightly up to 4 days and thereafter the enzyme became fully dormant. The results indicated that increase in storage temperature and time correlated with changes in biochemical parameters and activities of glycosidases suggested the suppression of β-galactosidase and β-hexosaminidase might enhance the shelf life of mango fruits. PMID:25136564

Regulation of accumulation of ammonium-inducible glutamate dehydrogenase catalytic activity and antigen during the cell cycle of fully induced, synchronous Chlorella sorokiniana cells.

PubMed

Yeung, A T; Bascomb, N F; Turner, K J; Schmidt, R R

1981-05-01

By use of a rocket immunoelectrophoresis-activity stain procedure, it was shown that catalytic activity of an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) was accompanied by a coincident increase in enzyme antigen during the cell cycle of preinduced synchronous Chlorella sorokiniana cells growing in the continuous presence of ammonia. Between the fourth and fifth hours of the G-1 phase of the cell cycle, a three- to fourfold increase in linear accumulation of enzyme antigen was observed. Pulse-chase studies with [35S]sulfate, coupled with a specific indirect immunoadsorption procedure for enzyme antigen, showed that NADP-GDH antigen undergoes continuous degradation (i.e., a half-life of 88 to 110 min) during its linear pattern of accumulation during the cell cycle. The apparent half-life of the enzyme increased by approximately 23% of the 4.5-h positive rate change in antigen accumulation during the cell cycle. This increase in half-life is insufficient in itself to account for the large change in rate of NADP-GDH antigen accumulation. The data from immunoelectrophoresis, pulse-chase, and initial 35S incorporation rate experiments taken together support the inference that changes in the rate of NADP-GDH synthesis are primarily responsible for the accumulation patterns of NADP-GDH activity during the C. sorokiniana cell cycle.

Genetic organization and regulation of a meta cleavage pathway for catechols produced from catabolism of toluene, benzene, phenol, and cresols by Pseudomonas pickettii PKO1.

PubMed Central

Kukor, J J; Olsen, R H

1991-01-01

Plasmid pRO1957 contains a 26.5-kb BamHI restriction endonuclease-cleaved DNA fragment cloned from the chromosome of Pseudomonas pickettii PKO1 that allows P. aeruginosa PAO1c to grow on toluene, benzene, phenol, or m-cresol as the sole carbon source. The genes encoding enzymes for meta cleavage of catechol or 3-methylcatechol, derived from catabolism of these substrates, were subcloned from pRO1957 and were shown to be organized into a single operon with the promoter proximal to tbuE. Deletion and analysis of subclones demonstrated that the order of genes in the meta cleavage operon was tbuEFGKIHJ, which encoded catechol 2,3-dioxygenase, 2-hydroxymuconate semialdehyde hydrolase, 2-hydroxymuconate semialdehyde dehydrogenase, 4-hydroxy-2-oxovalerate aldolase, 4-oxalocrotonate decarboxylase, 4-oxalocrotonate isomerase, and 2-hydroxypent-2,4-dienoate hydratase, respectively. The regulatory gene for the tbuEFGKIHJ operon, designated tbuS, was subcloned into vector plasmid pRO2317 from pRO1957 as a 1.3-kb PstI fragment, designated pRO2345. When tbuS was not present, meta pathway enzyme expression was partially derepressed, but these activity levels could not be fully induced. However, when tbuS was present in trans with tbuEFGKIHJ, meta pathway enzymes were repressed in the absence of an effector and were fully induced when an effector was present. This behavior suggests that the gene product of tbuS acts as both a repressor and an activator. Phenol and m-cresol were inducers of meta pathway enzymatic activity. Catechol, 3-methylcatechol, 4-methylcatechol, o-cresol, and p-cresol were not inducers but could be metabolized by cells previously induced by phenol or m-cresol. PMID:1856161

Dual allosteric activation mechanisms in monomeric human glucokinase.

PubMed

Whittington, A Carl; Larion, Mioara; Bowler, Joseph M; Ramsey, Kristen M; Brüschweiler, Rafael; Miller, Brian G

2015-09-15

Cooperativity in human glucokinase (GCK), the body's primary glucose sensor and a major determinant of glucose homeostatic diseases, is fundamentally different from textbook models of allostery because GCK is monomeric and contains only one glucose-binding site. Prior work has demonstrated that millisecond timescale order-disorder transitions within the enzyme's small domain govern cooperativity. Here, using limited proteolysis, we map the site of disorder in unliganded GCK to a 30-residue active-site loop that closes upon glucose binding. Positional randomization of the loop, coupled with genetic selection in a glucokinase-deficient bacterium, uncovers a hyperactive GCK variant with substantially reduced cooperativity. Biochemical and structural analysis of this loop variant and GCK variants associated with hyperinsulinemic hypoglycemia reveal two distinct mechanisms of enzyme activation. In α-type activation, glucose affinity is increased, the proteolytic susceptibility of the active site loop is suppressed and the (1)H-(13)C heteronuclear multiple quantum coherence (HMQC) spectrum of (13)C-Ile-labeled enzyme resembles the glucose-bound state. In β-type activation, glucose affinity is largely unchanged, proteolytic susceptibility of the loop is enhanced, and the (1)H-(13)C HMQC spectrum reveals no perturbation in ensemble structure. Leveraging both activation mechanisms, we engineer a fully noncooperative GCK variant, whose functional properties are indistinguishable from other hexokinase isozymes, and which displays a 100-fold increase in catalytic efficiency over wild-type GCK. This work elucidates specific structural features responsible for generating allostery in a monomeric enzyme and suggests a general strategy for engineering cooperativity into proteins that lack the structural framework typical of traditional allosteric systems.

Bactericidal activity of electrolyzed acid water from solution containing sodium chloride at low concentration, in comparison with that at high concentration.

PubMed

Kiura, Hiromasa; Sano, Kouichi; Morimatsu, Shinichi; Nakano, Takashi; Morita, Chizuko; Yamaguchi, Masaki; Maeda, Toyoyuki; Katsuoka, Yoji

2002-05-01

Electrolyzed strong acid water (ESW) containing free chlorine at various concentrations is becoming to be available in clinical settings as a disinfectant. ESW is prepared by electrolysis of a NaCl solution, and has a corrosive activity against medical instruments. Although lower concentrations of NaCl and free chlorine are desired to eliminate corrosion, the germicidal effect of ESW with low NaCl and free-chlorine concentrations (ESW-L) has not been fully clarified. In this study, we demonstrated that ESW-L possesses bactericidal activity against Mycobacteria and spores of Bacillus subtilis. The effect was slightly weaker than that of ESW containing higher NaCl and free-chlorine concentrations (ESW-H), but acceptable as a disinfectant. To clarify the mechanism of the bactericidal activity, we investigated ESW-L-treated Pseudomonas aeruginosa by transmission electron microscopy, a bacterial enzyme assay and restriction fragment length polymorphism pattern (RFLP) assay. Since the bacterium, whose growth was completely inhibited by ESW-L, revealed the inactivation of cytoplasmic enzyme, blebs and breaks in its outer membrane and remained complete RFLP of DNA, damage of the outer membrane and inactivation of cytoplasmic enzyme are the important determinants of the bactericidal activity.

The plant cell-wall enzyme AtXTH3 catalyses covalent cross-linking between cellulose and cello-oligosaccharide

NASA Astrophysics Data System (ADS)

Shinohara, Naoki; Sunagawa, Naoki; Tamura, Satoru; Yokoyama, Ryusuke; Ueda, Minoru; Igarashi, Kiyohiko; Nishitani, Kazuhiko

2017-04-01

Cellulose is an economically important material, but routes of its industrial processing have not been fully explored. The plant cell wall - the major source of cellulose - harbours enzymes of the xyloglucan endotransglucosylase/hydrolase (XTH) family. This class of enzymes is unique in that it is capable of elongating polysaccharide chains without the requirement for activated nucleotide sugars (e.g., UDP-glucose) and in seamlessly splitting and reconnecting chains of xyloglucan, a naturally occurring soluble analogue of cellulose. Here, we show that a recombinant version of AtXTH3, a thus far uncharacterized member of the Arabidopsis XTH family, catalysed the transglycosylation between cellulose and cello-oligosaccharide, between cellulose and xyloglucan-oligosaccharide, and between xyloglucan and xyloglucan-oligosaccharide, with the highest reaction rate observed for the latter reaction. In addition, this enzyme formed cellulose-like insoluble material from a soluble cello-oligosaccharide in the absence of additional substrates. This newly found activity (designated “cellulose endotransglucosylase,” or CET) can potentially be involved in the formation of covalent linkages between cellulose microfibrils in the plant cell wall. It can also comprise a new route of industrial cellulose functionalization.

Efficient heterologous expression and one-step purification of fully active c-terminal histidine-tagged uridine monophosphate kinase from Mycobacterium tuberculosis.

PubMed

Penpassakarn, Praweenuch; Chaiyen, Pimchai; Palittapongarnpim, Prasit

2011-11-01

Tuberculosis has long been recognized as one of the most significant public health problems. Finding novel antituberculous drugs is always a necessary approach for controlling the disease. Mycobacterium tuberculosis pyrH gene (Rv2883c) encodes for uridine monophosphate kinase (UMK), which is a key enzyme in the uridine nucleotide interconversion pathway. The enzyme is essential for M. tuberculosis to sustain growth and hence is a potential drug target. In this study, we have developed a rapid protocol for production and purification of M. tuberculosis UMK by cloning pyrH (Rv2883c) of M. tuberculosis H37Rv with the addition of 6-histidine residues to the C-terminus of the protein, and expressing in E. coli BL21-CodonPlus (DE3)-RIPL using an auto-induction medium. The enzyme was efficiently purified by a single-step TALON cobalt affinity chromatography with about 8 fold increase in specific activity, which was determined by a coupled assay with the pyruvate kinase and lactate dehydrogenase. The molecular mass of monomeric UMK was 28.2 kDa and that of the native enzyme was 217 kDa. The enzyme uses UMP as a substrate but not CMP and TMP and activity was enhanced by GTP. Measurements of enzyme kinetics revealed the kcat value of 7.6 +/- 0.4 U mg(-1) or 0.127 +/- 0.006 sec(-1).The protocol reported here can be used for expression of M. tuberculosis UMK in large quantity for formulating a high throughput target-based assay for screening anti-tuberculosis UMK compounds.

11-β hydroxysteroid type 1 knockout mice display an antidepressant-like phenotype in the forced swim test.

PubMed

Slattery, David A; Uzunov, Doncho P; Cryan, John F

2016-02-01

11β-dehydroxysteroid dehydrogenase (HSD) types 1 and 2, enzymes are involved in the activation and inactivation of glucocorticoids in vivo, respectively. Indirect evidence implicates two enzymes in the aetiology of depression but no study has directly assessed the potential role of 11 β-HSD1 in animal tests. We assessed 11 β-HSD1 knockout mice in the forced swim test (FST), tail suspension test (TST) and for locomotor activity. Genetic ablation of the 11β-HSD1 gene results in an antidepressant-like phenotype in the FST; the most widely utilised animal test of antidepressant activity, but not in the related TST. This may be related to the different biological substrates underlying these tests. The decreased FST immobility was not due to alterations in general activity. Taken together these results suggest that 11β-HSD1 may play an important role in depression-related behaviours and further studies are necessary to fully characterise its role in such behaviour.

Contrasting respirable quartz and kaolin retention of lecithin surfactant and expression of membranolytic activity following phospholipase A2 digestion.

PubMed

Wallace, W E; Keane, M J; Mike, P S; Hill, C A; Vallyathan, V; Regad, E D

1992-11-01

Respirable-sized quartz, a well-established fibrogenic mineral dust, is compared with kaolin in erythrocyte hemolysis assays after treatment with saline dispersion of dipalmitoyl phosphatidylcholine, a primary phospholipid component of pulmonary surfactant. Both dusts are rendered inactive after treatment, but the membranolytic activity is partly to fully restored after treatment with phospholipase A2, an enzyme normally associated with cellular plasma membranes and lysosomes. Phospholipid-coated dusts were incubated for periods of 2-72 h at a series of applied enzyme concentrations, and the adsorbed lipid species and hemolytic activity were quantitated at each time for both dusts. Surfactant was lost more readily from quartz than from kaolin, with consequent more rapid restoration of mineral surface hemolytic activity for quartz. Interactions of surfactant and mineral surface functional groups responsible for the mineral-specific rate differences, and implications for determining the mineral surface bioavailability of silica and silicate dusts, are discussed.

Salicylic acid induces vanillin synthesis through the phospholipid signaling pathway in Capsicum chinense cell cultures

PubMed Central

Rodas-Junco, Beatriz A; Cab-Guillen, Yahaira; Muñoz-Sanchez, J Armando; Vázquez-Flota, Felipe; Monforte-Gonzalez, Miriam; Hérnandez-Sotomayor, S M Teresa

2013-01-01

Signal transduction via phospholipids is mediated by phospholipases such as phospholipase C (PLC) and D (PLD), which catalyze hydrolysis of plasma membrane structural phospholipids. Phospholipid signaling is also involved in plant responses to phytohormones such as salicylic acid (SA). The relationships between phospholipid signaling, SA, and secondary metabolism are not fully understood. Using a Capsicum chinense cell suspension as a model, we evaluated whether phospholipid signaling modulates SA-induced vanillin production through the activation of phenylalanine ammonia lyase (PAL), a key enzyme in the biosynthetic pathway. Salicylic acid was found to elicit PAL activity and consequently vanillin production, which was diminished or reversed upon exposure to the phosphoinositide-phospholipase C (PI-PLC) signaling inhibitors neomycin and U73122. Exposure to the phosphatidic acid inhibitor 1-butanol altered PLD activity and prevented SA-induced vanillin production. Our results suggest that PLC and PLD-generated secondary messengers may be modulating SA-induced vanillin production through the activation of key biosynthetic pathway enzymes.

Ineffective Degradation of Immunogenic Gluten Epitopes by Currently Available Digestive Enzyme Supplements

PubMed Central

Janssen, George; Christis, Chantal; Kooy-Winkelaar, Yvonne; Edens, Luppo; Smith, Drew

2015-01-01

Background Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP). Methods Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1) enzyme assays and 2) mass spectrometric identification. Gluten epitope degradation was monitored by 1) R5 ELISA, 2) mass spectrometric analysis of the degradation products and 3) T cell proliferation assays. Findings The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data. Conclusion Currently available digestive enzyme supplements are ineffective in degrading immunogenic gluten epitopes. PMID:26030273

Structure of a Thermobifida fusca lytic polysaccharide monooxygenase and mutagenesis of key residues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kruer-Zerhusen, Nathan; Alahuhta, Markus; Lunin, Vladimir V.

Auxiliary activity (AA) enzymes are produced by numerous bacterial and fungal species to assist in the degradation of biomass. These enzymes are abundant but have yet to be fully characterized. Here, we report the X-ray structure of Thermobifida fusca AA10A (TfAA10A), investigate mutational characterization of key surface residues near its active site, and explore the importance of the various domains of Thermobifida fusca AA10B (TfAA10B). The structure of TfAA10A is similar to other bacterial LPMOs (lytic polysaccharide monooxygenases), including signs of photo-reduction and a distorted active site, with mixed features showing both type I and II copper coordination. The pointmore » mutation experiments of TfAA10A show that Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for the binding of substrate, but that the X1 module does not affect binding or activity. In TfAA10A, Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for substrate binding, but that the X1 module does not affect binding or activity. The structure of TfAA10A is similar to other bacterial lytic polysaccharide monooxygenases with mixed features showing both type I and II copper coordination. The role of LPMOs and the variability of abundance in genomes are not fully explored. LPMOs likely perform initial attacks into crystalline cellulose to allow larger processive cellulases to bind and attack, but the precise nature of their synergistic behavior remains to be definitively characterized.« less

Structure of a Thermobifida fusca lytic polysaccharide monooxygenase and mutagenesis of key residues

DOE PAGES

Kruer-Zerhusen, Nathan; Alahuhta, Markus; Lunin, Vladimir V.; ...

2017-11-30

Auxiliary activity (AA) enzymes are produced by numerous bacterial and fungal species to assist in the degradation of biomass. These enzymes are abundant but have yet to be fully characterized. Here, we report the X-ray structure of Thermobifida fusca AA10A (TfAA10A), investigate mutational characterization of key surface residues near its active site, and explore the importance of the various domains of Thermobifida fusca AA10B (TfAA10B). The structure of TfAA10A is similar to other bacterial LPMOs (lytic polysaccharide monooxygenases), including signs of photo-reduction and a distorted active site, with mixed features showing both type I and II copper coordination. The pointmore » mutation experiments of TfAA10A show that Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for the binding of substrate, but that the X1 module does not affect binding or activity. In TfAA10A, Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for substrate binding, but that the X1 module does not affect binding or activity. The structure of TfAA10A is similar to other bacterial lytic polysaccharide monooxygenases with mixed features showing both type I and II copper coordination. The role of LPMOs and the variability of abundance in genomes are not fully explored. LPMOs likely perform initial attacks into crystalline cellulose to allow larger processive cellulases to bind and attack, but the precise nature of their synergistic behavior remains to be definitively characterized.« less

Structure and function of a CE4 deacetylase isolated from a marine environment

PubMed Central

Tuveng, Tina Rise; Rothweiler, Ulli; Udatha, Gupta; Vaaje-Kolstad, Gustav; Smalås, Arne

2017-01-01

Chitin, a polymer of β(1–4)-linked N-acetylglucosamine found in e.g. arthropods, is a valuable resource that may be used to produce chitosan and chitooligosaccharides, two compounds with considerable industrial and biomedical potential. Deacetylating enzymes may be used to tailor the properties of chitin and its derived products. Here, we describe a novel CE4 enzyme originating from a marine Arthrobacter species (ArCE4A). Crystal structures of this novel deacetylase were determined, with and without bound chitobiose [(GlcNAc)2], and refined to 2.1 Å and 1.6 Å, respectively. In-depth biochemical characterization showed that ArCE4A has broad substrate specificity, with higher activity against longer oligosaccharides. Mass spectrometry-based sequencing of reaction products generated from a fully acetylated pentamer showed that internal sugars are more prone to deacetylation than the ends. These enzyme properties are discussed in the light of the structure of the enzyme-ligand complex, which adds valuable information to our still rather limited knowledge on enzyme-substrate interactions in the CE4 family. PMID:29107991

Synthetic cascades are enabled by combining biocatalysts with artificial metalloenzymes

NASA Astrophysics Data System (ADS)

Köhler, V.; Wilson, Y. M.; Dürrenberger, M.; Ghislieri, D.; Churakova, E.; Quinto, T.; Knörr, L.; Häussinger, D.; Hollmann, F.; Turner, N. J.; Ward, T. R.

2013-02-01

Enzymatic catalysis and homogeneous catalysis offer complementary means to address synthetic challenges, both in chemistry and in biology. Despite its attractiveness, the implementation of concurrent cascade reactions that combine an organometallic catalyst with an enzyme has proven challenging because of the mutual inactivation of both catalysts. To address this, we show that incorporation of a d6-piano stool complex within a host protein affords an artificial transfer hydrogenase (ATHase) that is fully compatible with and complementary to natural enzymes, thus enabling efficient concurrent tandem catalysis. To illustrate the generality of the approach, the ATHase was combined with various NADH-, FAD- and haem-dependent enzymes, resulting in orthogonal redox cascades. Up to three enzymes were integrated in the cascade and combined with the ATHase with a view to achieving (i) a double stereoselective amine deracemization, (ii) a horseradish peroxidase-coupled readout of the transfer hydrogenase activity towards its genetic optimization, (iii) the formation of L-pipecolic acid from L-lysine and (iv) regeneration of NADH to promote a monooxygenase-catalysed oxyfunctionalization reaction.

Renaturation and one step purification of the chicken GIIA secreted phospholipase A2 from inclusion bodies.

PubMed

Karray, Aida; Amara, Sawsan; Carrière, Frédéric; Gargouri, Youssef; Bezzine, Sofiane

2014-06-01

The cDNA coding for a mature protein of 123 amino acids, containing all of the structural features of catalytically active group IIA sPLA2, has been amplified from chicken intestine. The gene has been cloned into the bacterial expression vector pET-21a(+), which allows protein over-expression as inclusion bodies and enables about 3mg/l of pure refolded fully active enzyme to be obtained. Recombinant expression of chicken intestinal sPLA2-IIA (ChPLA2-IIA) in Escherichia coli shows that the enzyme is Ca(2+) dependent, maximally active at pH 8-9, and hydrolyses phosphatidylglycerol versus phosphatidylcholine with a 10-fold preference. Indeed, we report in this work, a comparative kinetic study between the wild type and the recombinant ChPLA2-IIA, on zwitterionic head group phospholipids (DDPC) and negatively charged phospholipids (POPG) using the monomolecular film technique. The ability to express reasonably large amounts of the sPLA2 Group IIA, compared to that obtained with the classical purification will provide a basis for future site directed mutagenesis studies of this important enzyme. Copyright © 2014 Elsevier B.V. All rights reserved.

Expression and purification of spinach nitrite reductase in E. coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bellissimo, D.; Privalle, L.

1991-03-11

The study of structure-function relationships in nitrite reductase (NiR) by site-directed mutagenesis requires an expression system from which suitable quantities of active enzyme can be purified. Spinach NiR cDNA was cloned into pUC18 and expressed in E.coli JM109 as a beta-galactosidase fusion protein. The IPTG-induced fusion protein contains five additional amino acids at the N-terminus. The expressed NiR in aerobic cultures was mostly insoluble and inactive indicating the presence of inclusion bodies. By altering growth conditions, active NiR could represent 0.5-1.0% of the total E.coli protein, Effects of the addition of delta-aminolevulinic acid, a heme precursor, and anaerobic growth weremore » also examined. Spinach NiR was purified approximately 200 fold to homogeneity. When subjected to electrophoresis on SDS polyacrylamide gels, the NiR migrated as a single band with similar mobility to pure spinach enzyme. The expressed enzyme also reacted with rabbit anti-spinach NiR antibody as visualized by Western blot analysis. The absorption spectrum of the E.coli-expressed enzyme was identical to spinach enzyme with a Soret and alpha band a 386 and 573 nm, respectively, and an A{sub 278}/A{sub 386} = 1.9. The addition of nitrite produced the characteristic shifts in the spectrum. The E. coli-expressed NiR catalyzed the methylviologen-dependent reduction of nitrite. The specific activity was 100 U/mg. The K{sub m} determined for nitrite was 0.3 mM which is in agreement with values reported for the enzyme. These results indicate that the E.coli-expressed NiR is fully comparable to spinach NiR in purity, catalytic activity and physical state. Site-directed mutants have been made using PCR to examine structure-function relationships in this enzyme.« less

A highly efficient sorbitol dehydrogenase from Gluconobacter oxydans G624 and improvement of its stability through immobilization

PubMed Central

Kim, Tae-Su; Patel, Sanjay K. S.; Selvaraj, Chandrabose; Jung, Woo-Suk; Pan, Cheol-Ho; Kang, Yun Chan; Lee, Jung-Kul

2016-01-01

A sorbitol dehydrogenase (GoSLDH) from Gluconobacter oxydans G624 (G. oxydans G624) was expressed in Escherichia coli BL21(DE3)-CodonPlus RIL. The complete 1455-bp codon-optimized gene was amplified, expressed, and thoroughly characterized for the first time. GoSLDH exhibited Km and kcat values of 38.9 mM and 3820 s−1 toward L-sorbitol, respectively. The enzyme exhibited high preference for NADP+ (vs. only 2.5% relative activity with NAD+). GoSLDH sequencing, structure analyses, and biochemical studies, suggested that it belongs to the NADP+-dependent polyol-specific long-chain sorbitol dehydrogenase family. GoSLDH is the first fully characterized SLDH to date, and it is distinguished from other L-sorbose-producing enzymes by its high activity and substrate specificity. Isothermal titration calorimetry showed that the protein binds more strongly to D-sorbitol than other L-sorbose-producing enzymes, and substrate docking analysis confirmed a higher turnover rate. The high oxidation potential of GoSLDH for D-sorbitol was confirmed by cyclovoltametric analysis. Further, stability of GoSLDH significantly improved (up to 13.6-fold) after cross-linking of immobilized enzyme on silica nanoparticles and retained 62.8% residual activity after 10 cycles of reuse. Therefore, immobilized GoSLDH may be useful for L-sorbose production from D-sorbitol. PMID:27633501

A highly efficient sorbitol dehydrogenase from Gluconobacter oxydans G624 and improvement of its stability through immobilization.

PubMed

Kim, Tae-Su; Patel, Sanjay K S; Selvaraj, Chandrabose; Jung, Woo-Suk; Pan, Cheol-Ho; Kang, Yun Chan; Lee, Jung-Kul

2016-09-16

A sorbitol dehydrogenase (GoSLDH) from Gluconobacter oxydans G624 (G. oxydans G624) was expressed in Escherichia coli BL21(DE3)-CodonPlus RIL. The complete 1455-bp codon-optimized gene was amplified, expressed, and thoroughly characterized for the first time. GoSLDH exhibited Km and kcat values of 38.9 mM and 3820 s(-1) toward L-sorbitol, respectively. The enzyme exhibited high preference for NADP(+) (vs. only 2.5% relative activity with NAD(+)). GoSLDH sequencing, structure analyses, and biochemical studies, suggested that it belongs to the NADP(+)-dependent polyol-specific long-chain sorbitol dehydrogenase family. GoSLDH is the first fully characterized SLDH to date, and it is distinguished from other L-sorbose-producing enzymes by its high activity and substrate specificity. Isothermal titration calorimetry showed that the protein binds more strongly to D-sorbitol than other L-sorbose-producing enzymes, and substrate docking analysis confirmed a higher turnover rate. The high oxidation potential of GoSLDH for D-sorbitol was confirmed by cyclovoltametric analysis. Further, stability of GoSLDH significantly improved (up to 13.6-fold) after cross-linking of immobilized enzyme on silica nanoparticles and retained 62.8% residual activity after 10 cycles of reuse. Therefore, immobilized GoSLDH may be useful for L-sorbose production from D-sorbitol.

Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Guitot, Karine; Scarabelli, Silvia; Drujon, Thierry; Bolbach, Gérard; Amoura, Mehdi; Burlina, Fabienne; Jeltsch, Albert; Sagan, Sandrine; Guianvarc'h, Dominique

2014-07-01

Histone lysine methyltransferases (HKMTs) are enzymes that play an essential role in epigenetic regulation. Thus, identification of inhibitors specifically targeting these enzymes represents a challenge for the development of new antitumor therapeutics. Several methods for measuring HKMT activity are already available. Most of them use indirect measurement of the enzymatic reaction through radioactive labeling or antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach because it allows direct detection and quantification of enzymatic reactions and can be used to determine kinetics and to screen small molecules as potential inhibitors. Application of mass spectrometry to the study of HKMTs has not been fully explored yet. We describe here the development of a simple reliable label-free MALDI-TOF MS-based assay for the detection and quantification of peptide methylation, using SET7/9 as a model enzyme. Importantly, the use of expensive internal standard often required in mass spectrometry quantitative analysis is not necessary in this assay. This MS assay allowed us to determine enzyme kinetic parameters as well as IC50 for a known inhibitor of this enzyme. Furthermore, a comparative study with an antibody-based immunosorbent assay showed that the MS assay is more reliable and suitable for the screening of inhibitors. Copyright © 2014 Elsevier Inc. All rights reserved.

Catalytic site interactions in yeast OMP synthase.

PubMed

Hansen, Michael Riis; Barr, Eric W; Jensen, Kaj Frank; Willemoës, Martin; Grubmeyer, Charles; Winther, Jakob R

2014-01-15

The enigmatic kinetics, half-of-the-sites binding, and structural asymmetry of the homodimeric microbial OMP synthases (orotate phosphoribosyltransferase, EC 2.4.2.10) have been proposed to result from an alternating site mechanism in these domain-swapped enzymes [R.W. McClard et al., Biochemistry 45 (2006) 5330-5342]. This behavior was investigated in the yeast enzyme by mutations in the conserved catalytic loop and 5-phosphoribosyl-1-diphosphate (PRPP) binding motif. Although the reaction is mechanistically sequential, the wild-type (WT) enzyme shows parallel lines in double reciprocal initial velocity plots. Replacement of Lys106, the postulated intersubunit communication device, produced intersecting lines in kinetic plots with a 2-fold reduction of kcat. Loop (R105G K109S H111G) and PRPP-binding motif (D131N D132N) mutant proteins, each without detectable enzymatic activity and ablated ability to bind PRPP, complemented to produce a heterodimer with a single fully functional active site showing intersecting initial velocity plots. Equilibrium binding of PRPP and orotidine 5'-monophosphate showed a single class of two binding sites per dimer in WT and K106S enzymes. Evidence here shows that the enzyme does not follow half-of-the-sites cooperativity; that interplay between catalytic sites is not an essential feature of the catalytic mechanism; and that parallel lines in steady-state kinetics probably arise from tight substrate binding. Copyright © 2013. Published by Elsevier Inc.

Mutual synergy between catalase and peroxidase activities of the bifunctional enzyme KatG is facilitated by electron hole-hopping within the enzyme.

PubMed

Njuma, Olive J; Davis, Ian; Ndontsa, Elizabeth N; Krewall, Jessica R; Liu, Aimin; Goodwin, Douglas C

2017-11-10

KatG is a bifunctional, heme-dependent enzyme in the front-line defense of numerous bacterial and fungal pathogens against H 2 O 2 -induced oxidative damage from host immune responses. Contrary to the expectation that catalase and peroxidase activities should be mutually antagonistic, peroxidatic electron donors (PxEDs) enhance KatG catalase activity. Here, we establish the mechanism of synergistic cooperation between these activities. We show that at low pH values KatG can fully convert H 2 O 2 to O 2 and H 2 O only if a PxED is present in the reaction mixture. Stopped-flow spectroscopy results indicated rapid initial rates of H 2 O 2 disproportionation slowing concomitantly with the accumulation of ferryl-like heme states. These states very slowly returned to resting ( i.e. ferric) enzyme, indicating that they represented catalase-inactive intermediates. We also show that an active-site tryptophan, Trp-321, participates in off-pathway electron transfer. A W321F variant in which the proximal tryptophan was replaced with a non-oxidizable phenylalanine exhibited higher catalase activity and less accumulation of off-pathway heme intermediates. Finally, rapid freeze-quench EPR experiments indicated that both WT and W321F KatG produce the same methionine-tyrosine-tryptophan (MYW) cofactor radical intermediate at the earliest reaction time points and that Trp-321 is the preferred site of off-catalase protein oxidation in the native enzyme. Of note, PxEDs did not affect the formation of the MYW cofactor radical but could reduce non-productive protein-based radical species that accumulate during reaction with H 2 O 2 Our results suggest that catalase-inactive intermediates accumulate because of off-mechanism oxidation, primarily of Trp-321, and PxEDs stimulate KatG catalase activity by preventing the accumulation of inactive intermediates. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sunden, Fanny; Peck, Ariana; Salzman, Julia

Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called ‘catalytic residues’ are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modesmore » of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes.« less

Hypermutation by intersegmental transfer of APOBEC3G cytidine deaminase.

PubMed

Nowarski, Roni; Britan-Rosich, Elena; Shiloach, Tamar; Kotler, Moshe

2008-10-01

Deamination of cytidine residues in single-stranded DNA (ssDNA) is an important mechanism by which apolipoprotein B mRNA-editing, catalytic polypeptide-like (APOBEC) enzymes restrict endogenous and exogenous viruses. The dynamic process underlying APOBEC-induced hypermutation is not fully understood. Here we show that enzymatically active APOBEC3G can be detected in wild-type Vif(+) HIV-1 virions, albeit at low levels. In vitro studies showed that single enzyme-DNA encounters result in distributive deamination of adjacent cytidines. Nonlinear translocation of APOBEC3G, however, directed scattered deamination of numerous targets along the DNA. Increased ssDNA concentrations abolished enzyme processivity in the case of short, but not long, DNA substrates, emphasizing the key role of rapid intersegmental transfer in targeting the deaminase. Our data support a model by which APOBEC3G intersegmental transfer via monomeric binding to two ssDNA segments results in dispersed hypermutation of viral genomes.

Xenobiotic metabolism capacities of human skin in comparison with a 3D-epidermis model and keratinocyte-based cell culture as in vitro alternatives for chemical testing: phase II enzymes.

PubMed

Götz, Christine; Pfeiffer, Roland; Tigges, Julia; Ruwiedel, Karsten; Hübenthal, Ulrike; Merk, Hans F; Krutmann, Jean; Edwards, Robert J; Abel, Josef; Pease, Camilla; Goebel, Carsten; Hewitt, Nicola; Fritsche, Ellen

2012-05-01

The 7th Amendment to the EU Cosmetics Directive prohibits the use of animals in cosmetic testing for certain endpoints, such as genotoxicity. Therefore, skin in vitro models have to replace chemical testing in vivo. However, the metabolic competence neither of human skin nor of alternative in vitro models has so far been fully characterized, although skin is the first-pass organ for accidentally or purposely (cosmetics and pharmaceuticals) applied chemicals. Thus, there is an urgent need to understand the xenobiotic-metabolizing capacities of human skin and to compare these activities to models developed to replace animal testing. We have measured the activity of the phase II enzymes glutathione S-transferase, UDP-glucuronosyltransferase and N-acetyltransferase in ex vivo human skin, the 3D epidermal model EpiDerm 200 (EPI-200), immortalized keratinocyte-based cell lines (HaCaT and NCTC 2544) and primary normal human epidermal keratinocytes. We show that all three phase II enzymes are present and highly active in skin as compared to phase I. Human skin, therefore, represents a more detoxifying than activating organ. This work systematically compares the activities of three important phase II enzymes in four different in vitro models directly to human skin. We conclude from our studies that 3D epidermal models, like the EPI-200 employed here, are superior over monolayer cultures in mimicking human skin xenobiotic metabolism and thus better suited for dermatotoxicity testing. © 2012 John Wiley & Sons A/S.

The toxicology and immunology of detergent enzymes.

PubMed

Basketter, David; Berg, Ninna; Kruszewski, Francis H; Sarlo, Katherine; Concoby, Beth

2012-01-01

Detergent enzymes have a very good safety profile, with almost no capacity to generate adverse acute or chronic responses in humans. The exceptions are the limited ability of some proteases to produce irritating effects at high concentrations, and the intrinsic potential of these bacterial and fungal proteins to act as respiratory sensitizers, demonstrated in humans during the early phase of the industrial use of enzymes during the 1960s and 1970s. How enzymes generate these responses are beginning to become a little clearer, with a developing appreciation of the cell surface mechanism(s) by which the enzymatic activity promotes the T-helper (T(H))-2 cell responses, leading to the generation of IgE. It is a reasonable assumption that the majority of enzyme proteins possess this intrinsic hazard. However, toxicological methods for characterizing further the respiratory sensitization hazard of individual enzymes remains a problematic area, with the consequence that the information feeding into risk assessment/management, although sufficient, is limited. Most of this information was in the past generated in animal models and in vitro immunoassays that assess immunological cross-reactivity. Ultimately, by understanding more fully the mechanisms which drive the IgE response to enzymes, it will be possible to develop better methods for hazard characterization and consequently for risk assessment and management.

Functional characterization of GH7 endo-1,4-β-glucanase from Aspergillus fumigatus and its potential industrial application.

PubMed

Bernardi, Aline Vianna; de Gouvêa, Paula Fagundes; Gerolamo, Luis Eduardo; Yonamine, Deborah Kimie; de Lourdes de Lima Balico, Laís; Uyemura, Sergio Akira; Dinamarco, Taisa Magnani

2018-04-30

A gene encoding an endo-1,4-β-glucanase (Afu6g01800) from A. fumigatus was cloned into the vector pET-28a(+) and expressed in the E. coli strain RosettaTM (DE3) pLysS. Sequence analysis indicated that the enzyme Af-EGL7 belonged to the GH7 family. The gene Af-egl7 encoded a protein comprising 460 amino acids, with a CBM1 domain at residues 424-460 and molecular mass of 52 kDa, as estimated by SDS-PAGE. This enzyme was optimally active at pH and temperatures ranging from 4.5 to 5.5 and from 40 to 60 °C, respectively. Mn 2+ addition significantly enhanced the Af-EGL7 cellulase activity by 233%, whereas SDS addition fully inhibited this activity. Higher activity was observed toward β-glucan than toward xyloglucan and CM-Cellulose, suggesting that the enzyme corresponds to a β-1,3-1,4-glucanase. qRT-PCR in different culture media helped to establish the time-course expression profile. Different polysaccharides induced the gene Af-egl7 in a time-dependent manner; in the particular case of the substrate sugarcane exploded bagasse (SEB), Af-egl7 was induced 2500-fold. Upon addition to a commercial cellulase cocktail, Af-EGL7 significantly improved SEB saccharification, which suggested that the enzyme Af-EGL7 had great potential to hydrolyze complex biomass. From a biotechnological point of view, A. fumigatus Af-EGL7 is a promising candidate to enhance enzyme cocktails used in biorefineries such as consolidated bioprocessing. Copyright © 2018 Elsevier Inc. All rights reserved.

Distinct roles of N- and O-glycans in cellulase activity and stability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Amore, Antonella; Knott, Brandon C.; Supekar, Nitin T.

In nature, many microbes secrete mixtures of glycoside hydrolases, oxidoreductases, and accessory enzymes to deconstruct polysaccharides and lignin in plants. These enzymes are often decorated with N- and O-glycosylation, the roles of which have been broadly attributed to protection from proteolysis, as the extracellular milieu is an aggressive environment. Glycosylation has been shown to sometimes affect activity, but these effects are not fully understood. In this paper, we examine N- and O-glycosylation on a model, multimodular glycoside hydrolase family 7 cellobiohydrolase (Cel7A), which exhibits an O-glycosylated carbohydrate-binding module (CBM) and an O-glycosylated linker connected to an N- and O-glycosylated catalyticmore » domain (CD) - a domain architecture common to many biomass-degrading enzymes. We report consensus maps for Cel7A glycosylation that include glycan sites and motifs. Additionally, we examine the roles of glycans on activity, substrate binding, and thermal and proteolytic stability. N-glycan knockouts on the CD demonstrate that N-glycosylation has little impact on cellulose conversion or binding, but does have major stability impacts. O-glycans on the CBM have little impact on binding, proteolysis, or activity in the whole-enzyme context. However, linker O-glycans greatly impact cellulose conversion via their contribution to proteolysis resistance. Molecular simulations predict an additional role for linker O-glycans, namely that they are responsible for maintaining separation between ordered domains when Cel7A is engaged on cellulose, as models predict a-helix formation and decreased cellulose interaction for the nonglycosylated linker. In conclusion, this study reveals key roles for N- and O-glycosylation that are likely broadly applicable to other plant cell-wall-degrading enzymes.« less

Distinct roles of N- and O-glycans in cellulase activity and stability

DOE PAGES

Amore, Antonella; Knott, Brandon C.; Supekar, Nitin T.; ...

2017-12-11

In nature, many microbes secrete mixtures of glycoside hydrolases, oxidoreductases, and accessory enzymes to deconstruct polysaccharides and lignin in plants. These enzymes are often decorated with N- and O-glycosylation, the roles of which have been broadly attributed to protection from proteolysis, as the extracellular milieu is an aggressive environment. Glycosylation has been shown to sometimes affect activity, but these effects are not fully understood. In this paper, we examine N- and O-glycosylation on a model, multimodular glycoside hydrolase family 7 cellobiohydrolase (Cel7A), which exhibits an O-glycosylated carbohydrate-binding module (CBM) and an O-glycosylated linker connected to an N- and O-glycosylated catalyticmore » domain (CD) - a domain architecture common to many biomass-degrading enzymes. We report consensus maps for Cel7A glycosylation that include glycan sites and motifs. Additionally, we examine the roles of glycans on activity, substrate binding, and thermal and proteolytic stability. N-glycan knockouts on the CD demonstrate that N-glycosylation has little impact on cellulose conversion or binding, but does have major stability impacts. O-glycans on the CBM have little impact on binding, proteolysis, or activity in the whole-enzyme context. However, linker O-glycans greatly impact cellulose conversion via their contribution to proteolysis resistance. Molecular simulations predict an additional role for linker O-glycans, namely that they are responsible for maintaining separation between ordered domains when Cel7A is engaged on cellulose, as models predict a-helix formation and decreased cellulose interaction for the nonglycosylated linker. In conclusion, this study reveals key roles for N- and O-glycosylation that are likely broadly applicable to other plant cell-wall-degrading enzymes.« less

Genetics Home Reference: adenosine deaminase 2 deficiency

MedlinePlus

... not fully understand how a loss of this enzyme's function leads to the features of ADA2 deficiency. They speculate that a lack of this enzyme may disrupt the balance between pro-inflammatory and ...

A highly Conserved Aspartic Acid Residue of the Chitosanase from Bacillus Sp. TS Is Involved in the Substrate Binding.

PubMed

Zhou, Zhanping; Zhao, Shuangzhi; Liu, Yang; Chang, Zhengying; Ma, Yanhe; Li, Jian; Song, Jiangning

2016-11-01

The chitosanase from Bacillus sp. TS (CsnTS) is an enzyme belonging to the glycoside hydrolase family 8. The sequence of CsnTS shares 98 % identity with the chitosanase from Bacillus sp. K17. Crystallography analysis and site-direct mutagenesis of the chitosanase from Bacillus sp. K17 identified the important residues involved in the catalytic interaction and substrate binding. However, despite progress in understanding the catalytic mechanism of the chitosanase from the family GH8, the functional roles of some residues that are highly conserved throughout this family have not been fully elucidated. This study focused on one of these residues, i.e., the aspartic acid residue at position 318. We found that apart from asparagine, mutation of Asp318 resulted in significant loss of enzyme activity. In-depth investigations showed that mutation of this residue not only impaired enzymatic activity but also affected substrate binding. Taken together, our results showed that Asp318 plays an important role in CsnTS activity.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rejda, J.M.; Johal, S.; Chollet, R.

Homogeneous preparations of ribulose 1,5-bisphosphate carboxylase/oxygenase were isolated from several diploid and tetraploid cultivars of perennial ryegrass by three different purification protocols. The apparent K/sub m/ values for substrate CO/sub 2/ were essentially identical for the fully CO/sub 2//Mg/sup 2 +/-activated diploid and tetraploid enzymes, as were the kinetics for deactivation and activation of the CO/sub 2//Mg/sup 2 +/-activated and -depleted carboxylases, respectively. Similarly, virtually indistinguishable electrophoretic properties were observed for both the native and dissociated diploid and tetraploid ryegrass proteins, including native and subunit molecular weights and the isoelectric points of the native proteins and the large and smallmore » subunit component polypeptides. The quantity of carboxylase protein or total soluble leaf protein did not differ significantly between the diploid and tetraploid cultivars. Contrary to a previous report, these results indicate that increased ploidy level has had essentially no effect on the quantity or enzymic and physicochemical properties of ribulosebisphosphate carboxylase/oxygenase in perennial ryegrass.« less

Biochemical composition and antioxidant activity affected by spraying potassium sulfate in black grape (Vitis vinifera L. cv. Rasha).

PubMed

Zareei, Elnaz; Javadi, Taimoor; Aryal, Rishi

2018-04-27

The physiological and metabolic processes involved with grapevine growth and production are influenced by key macro and micro-nutrients. Potassium is an essential plant nutrient that affects growth and fruit quality. In this study, the impact of foliar spraying of potassium sulfate (K 2 SO 4 ) on qualitative characteristics of grape berries was evaluated in the cultivar 'Rasha', a commonly cultivated cultivar in Kurdistan province of Iran. Leaves of the fully-grown vines were sprayed with each of the 1.5 g L -1 and 3 g L -1 potassium sulfate solution once (one month after petal senescence) and twice (15 days after first spraying). The control plants were sprayed with distilled water. Various biochemical content and enzyme activities on the ripe berries were analyzed. Significant increase in anthocyanin, total protein content and antioxidant enzyme activities were observed in the berries treated twice with 3 g L -1 K 2 SO 4 . Concentrations of total carbohydrate, phenol and antioxidant activity in berries sprayed with K 2 SO 4 were higher compared to the controls. We observed a strong correlation between antioxidant activity and different phenolic compounds. These findings suggest that K 2 SO 4 treatment influences biosynthesis of phenolic compounds and antioxidant enzymes. Thus treatment by K 2 SO 4 could improve nutritional and qualitative attributes of grape. This article is protected by copyright. All rights reserved.

Inhibition of Human and Rat Sucrase and Maltase Activities To Assess Antiglycemic Potential: Optimization of the Assay Using Acarbose and Polyphenols.

PubMed

Pyner, Alison; Nyambe-Silavwe, Hilda; Williamson, Gary

2017-10-04

We optimized the assays used to measure inhibition of rat and human α-glucosidases (sucrase and maltase activities), intestinal enzymes which catalyze the final steps of carbohydrate digestion. Cell-free extracts from fully differentiated intestinal Caco-2/TC7 monolayers were shown to be a suitable source of sucrase-isomaltase, with the same sequence as human small intestine, and were compared to a rat intestinal extract. The kinetic conditions of the assay were optimized, including comparison of enzymatic and chromatographic methods to detect the monosaccharide products. Human sucrase activity was more susceptible than the rat enzyme to inhibition by acarbose (IC 50 (concentration required for 50% inhibition) = 2.5 ± 0.5 and 12.3 ± 0.6 μM, respectively), by a polyphenol-rich green tea extract, and by pure (-)-epigallocatechin gallate (EGCG) (IC 50 = 657 ± 150 and 950 ± 86 μM respectively). In contrast, the reverse was observed when assessing maltase activity (e.g. IC 50 = 677 ± 241 and 14.0 ± 2.0 μM for human and rat maltase, respectively). 5-Caffeoylquinic acid did not significantly inhibit maltase and was only a very weak inhibitor of sucrase. The data show that for sucrase and maltase activities, inhibition patterns of rat and human enzymes are generally qualitatively similar but can be quantitatively different.

Trichomonas vaginalis: metronidazole and other nitroimidazole drugs are reduced by the flavin enzyme thioredoxin reductase and disrupt the cellular redox system. Implications for nitroimidazole toxicity and resistance.

PubMed

Leitsch, David; Kolarich, Daniel; Binder, Marina; Stadlmann, Johannes; Altmann, Friedrich; Duchêne, Michael

2009-04-01

Infections with the microaerophilic parasite Trichomonas vaginalis are treated with the 5-nitroimidazole drug metronidazole, which is also in use against Entamoeba histolytica, Giardia intestinalis and microaerophilic/anaerobic bacteria. Here we report that in T. vaginalis the flavin enzyme thioredoxin reductase displays nitroreductase activity with nitroimidazoles, including metronidazole, and with the nitrofuran drug furazolidone. Reactive metabolites of metronidazole and other nitroimidazoles form covalent adducts with several proteins that are known or assumed to be associated with thioredoxin-mediated redox regulation, including thioredoxin reductase itself, ribonucleotide reductase, thioredoxin peroxidase and cytosolic malate dehydrogenase. Disulphide reducing activity of thioredoxin reductase was greatly diminished in extracts of metronidazole-treated cells and intracellular non-protein thiol levels were sharply decreased. We generated a highly metronidazole-resistant cell line that displayed only minimal thioredoxin reductase activity, not due to diminished expression of the enzyme but due to the lack of its FAD cofactor. Reduction of free flavins, readily observed in metronidazole-susceptible cells, was also absent in the resistant cells. On the other hand, iron-depleted T. vaginalis cells, expressing only minimal amounts of PFOR and hydrogenosomal malate dehydrogenase, remained fully susceptible to metronidazole. Thus, taken together, our data suggest a flavin-based mechanism of metronidazole activation and thereby challenge the current model of hydrogenosomal activation of nitroimidazole drugs.

Purification and characterization of alpha-amylase from Bacillus licheniformis CUMC305

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krishnan, T.; Chandra, A.K.

Alpha-amylase produced by Bacillus licheniformis CUMC305 was purified 212-fold with a 42% yield through a series of four steps. The purified enzyme was homogeneous as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and discontinuous gel electrophoresis. The purified enzyme showed maximal activity at 90 degrees C and pH 9.0, and 91% of this activity remained at 100 degrees C. In the presence of substrate (soluble starch), the alpha-amylase enzyme was fully stable after a 4-hour incubation at 100 degrees C. The enzyme showed 100% stability in the pH range 7 to 9; 95% stability at pH 10; and 84, 74,more » 68, and 50% stability at pH values of 6, 5, 4, and 3, respectively, after 18 hours of treatment. The activation energy for this enzyme was calculated as 5.1 x 10 to the power of 5 J/mol. The molecular weight was estimated to be 28,000 by sodium dodecyl sulfate-gel electrophoresis. The relative rates of hydrolysis of soluble starch, amylose, amylopectin, and glycogen were 1.27, 1.8, 1.94, and 2.28 mg/ml, respectively. Vmax values for hydrolysis of these substrates were calculated as 0.738, 1.08, 0.8, and 0.5 mg of maltose/ml per min, respectively. Of the cations, Na+, Ca(2+), and Mg(2+), showed stimulatory effect, wheras Hg(2+), Cu(2+), Ni(2+), Zn(2+), Ag+, Fe(2+), Co(2+), Cd(2+), Al(3+), and Mn(2+) were inhibitory. Of the anions, azide, F-, SO/sub 3/(2-), SO/sub 4/(3-), S/sub 2/O/sub 3/(2-), MoO/sub 4/(2-), and Wo/sub 4/(2-) showed an excitant effect. p-Chloromercuribenzoic acid and sodium iodoacetate were inhibitory, whereas cysteine, reduced glutathione, thiourea, beta-mercaptoethanol, and sodium glycerophosphate afforded protection to enzyme activity. Alpha-amylase was fairly resistant to EDTA treatment at 30 degrees C, but heating at 90 degrees C in presence of EDTA resulted in the complete loss of enzyme activity. (Refs. 32).« less

Lentiviral Gene Therapy Using Cellular Promoters Cures Type 1 Gaucher Disease in Mice

PubMed Central

Dahl, Maria; Doyle, Alexander; Olsson, Karin; Månsson, Jan-Eric; Marques, André R A; Mirzaian, Mina; Aerts, Johannes M; Ehinger, Mats; Rothe, Michael; Modlich, Ute; Schambach, Axel; Karlsson, Stefan

2015-01-01

Gaucher disease is caused by an inherited deficiency of the enzyme glucosylceramidase. Due to the lack of a fully functional enzyme, there is progressive build-up of the lipid component glucosylceramide. Insufficient glucosylceramidase activity results in hepatosplenomegaly, cytopenias, and bone disease in patients. Gene therapy represents a future therapeutic option for patients unresponsive to enzyme replacement therapy and lacking a suitable bone marrow donor. By proof-of-principle experiments, we have previously demonstrated a reversal of symptoms in a murine disease model of type 1 Gaucher disease, using gammaretroviral vectors harboring strong viral promoters to drive glucosidase β-acid (GBA) gene expression. To investigate whether safer vectors can correct the enzyme deficiency, we utilized self-inactivating lentiviral vectors (SIN LVs) with the GBA gene under the control of human phosphoglycerate kinase (PGK) and CD68 promoter, respectively. Here, we report prevention of, as well as reversal of, manifest disease symptoms after lentiviral gene transfer. Glucosylceramidase activity above levels required for clearance of glucosylceramide from tissues resulted in reversal of splenomegaly, reduced Gaucher cell infiltration and a restoration of hematological parameters. These findings support the use of SIN-LVs with cellular promoters in future clinical gene therapy protocols for type 1 Gaucher disease. PMID:25655314

Isolation and characterization of a novel metagenomic enzyme capable of degrading bacterial phytotoxin toxoflavin

PubMed Central

Lee, Boyoung; Park, Ji Hyun; Oh, Joon Young; Choi, Jung Sup; Kim, Jin-Cheol

2018-01-01

Toxoflavin, a 7-azapteridine phytotoxin produced by the bacterial pathogens such as Burkholderia glumae and Burkholderia gladioli, has been known as one of the key virulence factors in crop diseases. Because the toxoflavin had an antibacterial activity, a metagenomic E. coli clone capable of growing well in the presence of toxoflavin (30 μg/ml) was isolated and the first metagenome-derived toxoflavin-degrading enzyme, TxeA of 140 amino acid residues, was identified from the positive E. coli clone. The conserved amino acids for metal-binding and extradiol dioxygenase activity, Glu-12, His-8 and Glu-130, were revealed by the sequence analysis of TxeA. The optimum conditions for toxoflavin degradation were evaluated with the TxeA purified in E. coli. Toxoflavin was totally degraded at an initial toxoflavin concentration of 100 μg/ml and at pH 5.0 in the presence of Mn2+, dithiothreitol and oxygen. The final degradation products of toxoflavin and methyltoxoflavin were fully identified by MS and NMR as triazines. Therefore, we suggested that the new metagenomic enzyme, TxeA, provided the clue to applying the new metagenomic enzyme to resistance development of crop plants to toxoflavin-mediated disease as well as to biocatalysis for Baeyer-Villiger type oxidation. PMID:29293506

Detailed kinetics and regulation of mammalian 2-oxoglutarate dehydrogenase

PubMed Central

2011-01-01

Background Mitochondrial 2-oxoglutarate (α-ketoglutarate) dehydrogenase complex (OGDHC), a key regulatory point of tricarboxylic acid (TCA) cycle, plays vital roles in multiple pathways of energy metabolism and biosynthesis. The catalytic mechanism and allosteric regulation of this large enzyme complex are not fully understood. Here computer simulation is used to test possible catalytic mechanisms and mechanisms of allosteric regulation of the enzyme by nucleotides (ATP, ADP), pH, and metal ion cofactors (Ca2+ and Mg2+). Results A model was developed based on an ordered ter-ter enzyme kinetic mechanism combined with con-formational changes that involve rotation of one lipoic acid between three catalytic sites inside the enzyme complex. The model was parameterized using a large number of kinetic data sets on the activity of OGDHC, and validated by comparison of model predictions to independent data. Conclusions The developed model suggests a hybrid rapid-equilibrium ping-pong random mechanism for the kinetics of OGDHC, consistent with previously reported mechanisms, and accurately describes the experimentally observed regulatory effects of cofactors on the OGDHC activity. This analysis provides a single consistent theoretical explanation for a number of apparently contradictory results on the roles of phosphorylation potential, NAD (H) oxidation-reduction state ratio, as well as the regulatory effects of metal ions on ODGHC function. PMID:21943256

Continuous activation of Nrf2 and its target antioxidant enzymes leads to arsenite-induced malignant transformation of human bronchial epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Xu; Wang, Dapeng; Department of Toxicology, School of Public Health, Guizhou Medical University, Guiyang, Guizhou

Long-term exposure to arsenite leads to human lung cancer, but the underlying mechanisms of carcinogenesis remain obscure. The transcription factor of nuclear factor-erythroid-2 p45-related factor (Nrf2)-mediated antioxidant response represents a critical cellular defense mechanism and protection against various diseases. Paradoxically, emerging data suggest that the constitutive activation of Nrf2 is associated with cancer development, progression and chemotherapy resistance. However, the role of Nrf2 in the occurrence of cancer induced by long-term arsenite exposure remains to be fully understood. By establishing transformed human bronchial epithelial (HBE) cells via chronic low-dose arsenite treatment, we showed that, in acquiring this malignant phenotype, continuousmore » low level of ROS and sustained enhancement of Nrf2 and its target antioxidant enzyme levels were observed in the later-stage of arsenite-induced cell transformation. The downregulation of Keap1 level may be responsible for the over-activation of Nrf2 and its target enzymes. To validate these observations, Nrf2 was knocked down in arsenite-transformed HBE cells by SiRNA transfection, and the levels of Nrf2 and its target antioxidant enzymes, ROS, cell proliferation, migration, and colony formation were determined following these treatments. Results showed that blocked Nrf2 expression significantly reduced Nrf2 and its target antioxidant enzyme levels, restored ROS levels, and eventually suppressed cell proliferation, migration, and colony formation of the transformed cells. In summary, the results of the study strongly suggested that the continuous activation of Nrf2 and its target antioxidant enzymes led to the over-depletion of intracellular ROS levels, which contributed to arsenite-induced HBE cell transformation. - Highlights: • Low level, long term arsenite exposure induces malignant transformation in vitro. • Long term arsenite exposure reduces ROS and MDA levels. • Long term arsenite exposure enhances Nrf2-mediated antioxidant levels. • Knockdown of Nrf2 reduces malignant degree of arsenite-transformed cells.« less

Mechanism of salt-induced activity enhancement of a marine-derived laccase, Lac15.

PubMed

Li, Jie; Xie, Yanan; Wang, Rui; Fang, Zemin; Fang, Wei; Zhang, Xuecheng; Xiao, Yazhong

2018-04-01

Laccase (benzenediol: oxygen oxidoreductases, EC1.10.3.2) is a multi-copper oxidase capable of oxidizing a variety of phenolic and other aromatic organic compounds. The catalytic power of laccase makes it an attractive candidate for potential applications in many areas of industry including biodegradation of organic pollutants and synthesis of novel drugs. Most laccases are vulnerable to high salt and have limited applications. However, some laccases are not only tolerant to but also activated by certain concentrations of salt and thus have great application potential. The mechanisms of salt-induced activity enhancement of laccases are unclear as yet. In this study, we used dynamic light scattering, size exclusion chromatography, analytical ultracentrifugation, intrinsic fluorescence emission, circular dichroism, ultraviolet-visible light absorption, and an enzymatic assay to investigate the potential correlation between the structure and activity of the marine-derived laccase, Lac15, whose activity is promoted by low concentrations of NaCl. The results showed that low concentrations of NaCl exert little influence on the protein structure, which was partially folded in the absence of the salt; moreover, the partially folded rather than the fully folded state seemed to be favorable for enzyme activity, and this partially folded state was distinctive from the so-called 'molten globule' occasionally observed in active enzymes. More data indicated that salt might promote laccase activity through mechanisms involving perturbation of specific local sites rather than a change in global structure. Potential binding sites for chloride ions and their roles in enzyme activity promotion are proposed.

Aryl acylamidase activity of human serum albumin with o-nitrotrifluoroacetanilide as the substrate.

PubMed

Masson, Patrick; Froment, Marie-Thérèse; Darvesh, Sultan; Schopfer, Lawrence M; Lockridge, Oksana

2007-08-01

Albumin is generally regarded as an inert protein with no enzyme activity. However, albumin has esterase activity as well as aryl acylamidase activity. A new acetanilide substrate, o-nitrotrifluoroacetanilide (o-NTFNAC), which is more reactive than the classical o-nitroacetanilide, made it possible to determine the catalytic parameters for hydrolysis by fatty-acid free human serum albumin. Owing to the low enzymatic activity of albumin, kinetic studies were performed at high albumin concentration (0.075 mM). The albumin behavior with this substrate was Michaelis-Menten like. Kinetic analysis was performed according to the formalism used for catalysis at high enzyme concentration. This approach provided values for the turnover and dissociation constant of the albumin-substrate complex: k(cat) = 0.13 +/- 0.02 min(-1) and Ks = 0.67 +/- 0.04 mM. MALDI-TOF experiments showed that unlike the ester substrate p-nitrophenyl acetate, o-NTFNAC does not form a stable adduct (acetylated enzyme). Kinetic analysis and MALDI-TOF experiments demonstrated that hydrolysis of o-NTFNAC by albumin is fully rate-limited by the acylation step (k(cat) = k2). Though the aryl acylamidase activity of albumin is low (k(cat)/Ks = 195 M(-1)min(-1)), because of its high concentration in human plasma (0.6-1 mM), albumin may participate in hydrolysis of aryl acylamides through second-order kinetics. This suggests that albumin may have a role in the metabolism of endogenous and exogenous aromatic amides, including drugs and xenobiotics.

Evidence for the cytotoxic effects of Mycobacterium tuberculosis phospholipase C towards macrophages.

PubMed

Bakala N'goma, J C; Schué, M; Carrière, F; Geerlof, A; Canaan, S

2010-12-01

Phospholipase Cs (PLCs) contribute importantly to the virulence and pathogenicity of several bacteria. It has been reported in previous studies that mutations in the four predicted plc genes of Mycobacterium tuberculosis inhibit the growth of these bacteria during the late phase of infection in mice. These enzymes have not yet been fully characterised, mainly because they are not easy to produce in large quantities. With a view to elucidating the role of all Mycobacterium tuberculosis phospholipase Cs (PLC-A, PLC-B, PLC-C and PLC-D), a large amount of active, soluble recombinant PLCs, were expressed and purified using Mycobacterium smegmatis as expression system. These enzymes showed different pH activity profiles. PLC-C was found to be the most active of the four recombinant PLCs under acidic conditions. All the enzymes tested induced cytotoxic effects on mouse macrophage RAW 264.7 cell lines, via direct or indirect enzymatic hydrolysis of cell membrane phospholipids. These results open new prospects for characterising biochemical and structural features of Mycobacterium tuberculosis PLCs, which might lead to the identification of novel anti-tuberculosis drug targets. All mycobacterial phospholipase Cs can now be studied in order to determine their role in the virulence and pathogenicity of bacteria of this kind. 2010 Elsevier B.V. All rights reserved.

The expression and activity of antioxidant enzymes in the liver of rats exposed to high-fructose diet in the period from weaning to adulthood.

PubMed

Glban, Alhadi M; Vasiljević, Ana; Veličković, Nataša; Nikolić-Kokić, Aleksandra; Blagojević, Duško; Matić, Gordana; Nestorov, Jelena

2015-08-30

Increased fructose consumption correlates with rising prevalence of various metabolic disorders, some of which were linked to oxidative stress. The relationship between fructose consumption and oxidative stress is complex and effects of a fructose-rich diet on the young population have not been fully elucidated. The aim of this study was to investigate whether high-fructose diet applied in the period from weaning to adulthood induces oxidative stress in the liver, thus contributing to induction or aggravation of metabolic disturbances in later adulthood. To that end we examined the effects of high-fructose diet on expression and activity of antioxidant enzymes, markers of lipid peroxidation and protein damage in the liver as the main fructose metabolizing tissue. High-fructose diet increased only SOD2 (mitochondrial manganese superoxide dismutase) activity, with no effect on other antioxidant enzymes, lipid peroxidation or accumulation of damaged proteins in the liver. The results show that fructose-induced metabolic disturbances could not be attributed to oxidative stress, at least not at young age. The absence of oxidative stress in the liver observed herein implies that young organisms are capable of maintaining redox homeostasis when challenged by fructose-derived energy overload. © 2014 Society of Chemical Industry.

Structure of the endonuclease IV homologue from Thermotoga maritima in the presence of active-site divalent metal ions

PubMed Central

Tomanicek, Stephen J.; Hughes, Ronny C.; Ng, Joseph D.; Coates, Leighton

2010-01-01

The most frequent lesion in DNA is at apurinic/apyrimidinic (AP) sites resulting from DNA-base losses. These AP-site lesions can stall DNA replication and lead to genome instability if left unrepaired. The AP endonucleases are an important class of enzymes that are involved in the repair of AP-site intermediates during damage-general DNA base-excision repair pathways. These enzymes hydrolytically cleave the 5′-phosphodiester bond at an AP site to generate a free 3′-­hydroxyl group and a 5′-terminal sugar phosphate using their AP nuclease activity. Specifically, Thermotoga maritima endonuclease IV is a member of the second conserved AP endonuclease family that includes Escherichia coli endonuclease IV, which is the archetype of the AP endonuclease superfamily. In order to more fully characterize the AP endonuclease family of enzymes, two X-­ray crystal structures of the T. maritima endonuclease IV homologue were determined in the presence of divalent metal ions bound in the active-site region. These structures of the T. maritima endonuclease IV homologue further revealed the use of the TIM-barrel fold and the trinuclear metal binding site as important highly conserved structural elements that are involved in DNA-binding and AP-site repair processes in the AP endonuclease superfamily. PMID:20823514

Study of orotidine 5'-monophosphate decarboxylase in complex with the top three OMP, BMP, and PMP ligands by molecular dynamics simulation.

PubMed

Jamshidi, Shirin; Jalili, Seifollah; Rafii-Tabar, Hashem

2015-01-01

Catalytic mechanism of orotidine 5'-monophosphate decarboxylase (OMPDC), one of the nature most proficient enzymes which provides large rate enhancement, has not been fully understood yet. A series of 30 ns molecular dynamics (MD) simulations were run on X-ray structure of the OMPDC from Saccharomyces cerevisiae in its free form as well as in complex with different ligands, namely 1-(5'-phospho-D-ribofuranosyl) barbituric acid (BMP), orotidine 5'-monophosphate (OMP), and 6-phosphonouridine 5'-monophosphate (PMP). The importance of this biological system is justified both by its high rate enhancement and its potential use as a target in chemotherapy. This work focuses on comparing two physicochemical states of the enzyme (protonated and deprotonated Asp91) and three ligands (substrate OMP, inhibitor, and transition state analog BMP and substrate analog PMP). Detailed analysis of the active site geometry and its interactions is properly put in context by extensive comparison with relevant experimental works. Our overall results show that in terms of hydrogen bond occupancy, electrostatic interactions, dihedral angles, active site configuration, and movement of loops, notable differences among different complexes are observed. Comparison of the results obtained from these simulations provides some detailed structural data for the complexes, the enzyme, and the ligands, as well as useful insights into the inhibition mechanism of the OMPDC enzyme. Furthermore, these simulations are applied to clarify the ambiguous mechanism of the OMPDC enzyme, and imply that the substrate destabilization and transition state stabilization contribute to the mechanism of action of the most proficient enzyme, OMPDC.

Effects of hexamethonium, phenothiazines, propranolol and ephedrine on acetylcholinesterase carbamylation by physostigmine, aldicarb and carbaryl: interaction between the active site and the functionally distinct peripheral sites in acetylcholinesterase.

PubMed

Singh, A K; Spassova, D

1998-01-01

Physostigmine, aldicarb and carbaryl were potent inhibitors of acetylcholinesterase (AChE). The physostigmine-inhibited AChE fluoresced at 300 nm excitation and 500 nm emission wavelengths, but the aldicarb and carbaryl inhibited enzyme did not. This suggests that the carbamylated active center is not the fluorescing site in AChE. The fluorescence intensity of physostigmine-inhibited AChE decreased with increasing the substrate (acetylthiocholine) concentration, thus indicating that physostigmine binding to the active site is essential for the development of fluorescence. Thus, the physostigmine-inhibited AChE fluoresces due to the binding of trimethylpyrrolo[2,3-b]indol (TMPI) moiety, formed by the hydrolysis of physostigmine, to a peripheral site in AChE. The fluorescence intensity of the physostigmine-inhibited enzyme decreased when the inhibited-enzyme was dialyzed for either 30 min that poorly reactivated the enzyme or 180 min that fully reactivated the enzyme. This suggests that dialysis dissociates the AChE-TMPI complex much faster than it reactivates the carbamylated AChE. Ephedrine, propranolol and phenothiazines including trifluoparazine (TPZ) caused non-competitive inhibition, while hexamethonium caused an uncompetitive inhibition of AChE activity. TPZ, upon binding with AChE, formed a fluorescent TPZ-enzyme complex. The fluorescence intensity of TPZ-AChE complex was effectively decreased by ephedrine, but not by propranolol or hexamethonium. This indicates that TPZ and ephedrine bind to the same site in AChE which is different from the site/or sites to which propranolol or hexamethonium bind. Hexamethonium protected AChE from inhibition by carbamates and decreased the fluorescence intensity of the physostigmine-inhibited AChE. Phenothiazines and ephedrine did not modulate the enzyme inhibition or the fluorescence intensity of the physostigmine-inhibited AChE. Propranolol and TPZ potentiated the enzyme inhibition and increased the fluorescence intensity in the presence of physostigmine. These compounds, however, did not affect the inhibition of AChE by carbaryl or aldicarb. Ephedrine blocked the effects of TPZ, but did not alter the effects of propranolol on physostigmine-inhibited AChE. AChE, therefore, contains multiple peripheral binding sites which, upon binding to specific ligands, transduce differential signals to the active center.

[Purine and pyrimidine nucleoside phosphorylases - remarkable enzymes still not fully understood].

PubMed

Bzowska, Agnieszka

2015-01-01

Purine and pyrimidine nucleoside phosphorylases catalyze the reversible phosphorolytic cleavage of the glycosidic bond of purine and pyrimidine nucleosides, and are key enzymes of the nucleoside salvage pathway. This metabolic route is the less costly alternative to the de novo synthesis of nucleosides and nucleotides, supplying cells with these important building blocks. Interest in nucleoside phosphorylases is not only due to their important role in metabolism of nucleosides and nucleotides, but also due to the potential medical use of the enzymes (all phosphorylases in activating prodrugs - nucleoside and nucleic base analogs, high-molecular mass purine nucleoside phosphorylases in gene therapy of some solid tumors) and their inhibitors (as selective immunosuppressive, anticancer and antiparasitic agents, and preventing inactivation of other nucleoside drugs). Phosphorylases are also convenient tools for efficient enzymatic synthesis of otherwise inaccessible nucleoside analogues. In this paper the contribution of Professor David Shugar and some of his colleagues and coworkers in studies of these remarkable enzymes carried out over nearly 40 years is discussed on the background of global research in this field.

Functional and Structural Characterization of a (+)-Limonene Synthase from Citrus sinensis.

PubMed

Morehouse, Benjamin R; Kumar, Ramasamy P; Matos, Jason O; Olsen, Sarah Naomi; Entova, Sonya; Oprian, Daniel D

2017-03-28

Terpenes make up the largest and most diverse class of natural compounds and have important commercial and medical applications. Limonene is a cyclic monoterpene (C 10 ) present in nature as two enantiomers, (+) and (-), which are produced by different enzymes. The mechanism of production of the (-)-enantiomer has been studied in great detail, but to understand how enantiomeric selectivity is achieved in this class of enzymes, it is important to develop a thorough biochemical description of enzymes that generate (+)-limonene, as well. Here we report the first cloning and biochemical characterization of a (+)-limonene synthase from navel orange (Citrus sinensis). The enzyme obeys classical Michaelis-Menten kinetics and produces exclusively the (+)-enantiomer. We have determined the crystal structure of the apoprotein in an "open" conformation at 2.3 Å resolution. Comparison with the structure of (-)-limonene synthase (Mentha spicata), which is representative of a fully closed conformation (Protein Data Bank entry 2ONG ), reveals that the short H-α1 helix moves nearly 5 Å inward upon substrate binding, and a conserved Tyr flips to point its hydroxyl group into the active site.

Antagonistic Enzymes in a Biocatalytic pH Feedback System Program Autonomous DNA Hydrogel Life Cycles.

PubMed

Heinen, Laura; Heuser, Thomas; Steinschulte, Alexander; Walther, Andreas

2017-08-09

Enzymes regulate complex functions and active behavior in natural systems and have shown increasing prospect for developing self-regulating soft matter systems. Striving for advanced autonomous hydrogel materials with fully programmable, self-regulated life cycles, we combine two enzymes with an antagonistic pH-modulating effect in a feedback-controlled biocatalytic reaction network (BRN) and couple it to pH-responsive DNA hydrogels to realize hydrogel systems with distinct preprogrammable lag times and lifetimes in closed systems. The BRN enables precise and orthogonal internal temporal control of the "ON" and "OFF" switching times of the temporary gel state by modulation of programmable, nonlinear pH changes. The time scales are tunable by variation of the enzyme concentrations and additional buffer substances. The resulting material system operates in full autonomy after injection of the chemical fuels driving the BRN. The concept may open new applications inherent to DNA hydrogels, for instance, autonomous shape memory behavior for soft robotics. We further foresee general applicability to achieve autonomous life cycles in other pH switchable systems.

Leach-proof magnetic thrombolytic nanoparticles and coatings of enhanced activity

NASA Astrophysics Data System (ADS)

Drozdov, Andrey S.; Vinogradov, Vasiliy V.; Dudanov, Ivan P.; Vinogradov, Vladimir V.

2016-06-01

Despite the fact that magnetic thrombolytic composites is an emerging area, all known so far systems are based on the similar mechanism of action: thrombolytic enzyme releases from the magnetic carrier leaving non-active matrix, thus making the whole system active only for a limited period of time. Such systems often have very complex structure organization and composition, consisting of materials not approved for parenteral injection, making them poor candidates for real clinical trials and implementation. Here we report, for the first time, the production of thrombolytic magnetic composite material with non-releasing behavior and prolonged action. Obtained composite shows good thrombolytic activity, consists of fully biocompatible materials and could be applied as infinitely active thrombolytic coatings or magnetically-targetable thrombolytic agents.

A Novel N-Acetylglutamate Synthase Architecture Revealed by the Crystal Structure of the Bifunctional Enzyme from Maricaulis maris

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Dashuang; Li, Yongdong; Cabrera-Luque, Juan

2012-05-24

Novel bifunctional N-acetylglutamate synthase/kinases (NAGS/K) that catalyze the first two steps of arginine biosynthesis and are homologous to vertebrate N-acetylglutamate synthase (NAGS), an essential cofactor-producing enzyme in the urea cycle, were identified in Maricaulis maris and several other bacteria. Arginine is an allosteric inhibitor of NAGS but not NAGK activity. The crystal structure of M. maris NAGS/K (mmNAGS/K) at 2.7 {angstrom} resolution indicates that it is a tetramer, in contrast to the hexameric structure of Neisseria gonorrhoeae NAGS. The quaternary structure of crystalline NAGS/K from Xanthomonas campestris (xcNAGS/K) is similar, and cross-linking experiments indicate that both mmNAGS/K and xcNAGS aremore » tetramers in solution. Each subunit has an amino acid kinase (AAK) domain, which is likely responsible for N-acetylglutamate kinase (NAGK) activity and has a putative arginine binding site, and an N-acetyltransferase (NAT) domain that contains the putative NAGS active site. These structures and sequence comparisons suggest that the linker residue 291 may determine whether arginine acts as an allosteric inhibitor or activator in homologous enzymes in microorganisms and vertebrates. In addition, the angle of rotation between AAK and NAT domains varies among crystal forms and subunits within the tetramer. A rotation of 26{sup o} is sufficient to close the predicted AcCoA binding site, thus reducing enzymatic activity. Since mmNAGS/K has the highest degree of sequence homology to vertebrate NAGS of NAGS and NAGK enzymes whose structures have been determined, the mmNAGS/K structure was used to develop a structural model of human NAGS that is fully consistent with the functional effects of the 14 missense mutations that were identified in NAGS-deficient patients.« less

Oxidative Stress and Digestive Enzyme Activity of Flatfish Larvae in a Changing Ocean

PubMed Central

Pimentel, Marta S.; Faleiro, Filipa; Diniz, Mário; Machado, Jorge; Pousão-Ferreira, Pedro; Peck, Myron A.; Pörtner, Hans O.; Rosa, Rui

2015-01-01

Until now, it is not known how the antioxidant and digestive enzymatic machinery of fish early life stages will change with the combined effects of future ocean acidification and warming. Here we show that high pCO2 (~1600 μatm) significantly decreased metabolic rates (up to 27.4 %) of flatfish larvae, Solea senegalensis, at both present (18 °C) and warmer temperatures (+4 °C). Moreover, both warming and hypercapnia increased the heat shock response and the activity of antioxidant enzymes, namely catalase (CAT) and glutathione S-transferase (GST), mainly in post-metamorphic larvae (30 dph). The lack of changes in the activity of CAT and GST of pre-metamorphic larvae (10 dph) seems to indicate that earlier stages lack a fully-developed antioxidant defense system. Nevertheless, the heat shock and antioxidant responses of post-metamorphic larvae were not enough to avoid the peroxidative damage, which was greatly increased under future environmental conditions. Digestive enzymatic activity of S. senegalensis larvae was also affected by future predictions. Hypercapnic conditions led to a decrease in the activity of digestive enzymes, both pancreatic (up to 26.1 % for trypsin and 74.5 % for amylase) and intestinal enzymes (up to 36.1 % for alkaline phosphatase) in post-metamorphic larvae. Moreover, the impact of ocean acidification and warming on some of these physiological and biochemical variables (namely, lower OCR and higher HSP and MDA levels) were translated into larvae performance, being significantly correlated with decreased larval growth and survival or increased incidence of skeletal deformities. The increased vulnerability of flatfish early life stages under future ocean conditions is expected to potentially determine recruitment and population dynamics in marine ecosystems. PMID:26221723

Oxidative Stress and Digestive Enzyme Activity of Flatfish Larvae in a Changing Ocean.

PubMed

Pimentel, Marta S; Faleiro, Filipa; Diniz, Mário; Machado, Jorge; Pousão-Ferreira, Pedro; Peck, Myron A; Pörtner, Hans O; Rosa, Rui

2015-01-01

Until now, it is not known how the antioxidant and digestive enzymatic machinery of fish early life stages will change with the combined effects of future ocean acidification and warming. Here we show that high pCO2 (~1600 μatm) significantly decreased metabolic rates (up to 27.4 %) of flatfish larvae, Solea senegalensis, at both present (18 °C) and warmer temperatures (+4 °C). Moreover, both warming and hypercapnia increased the heat shock response and the activity of antioxidant enzymes, namely catalase (CAT) and glutathione S-transferase (GST), mainly in post-metamorphic larvae (30 dph). The lack of changes in the activity of CAT and GST of pre-metamorphic larvae (10 dph) seems to indicate that earlier stages lack a fully-developed antioxidant defense system. Nevertheless, the heat shock and antioxidant responses of post-metamorphic larvae were not enough to avoid the peroxidative damage, which was greatly increased under future environmental conditions. Digestive enzymatic activity of S. senegalensis larvae was also affected by future predictions. Hypercapnic conditions led to a decrease in the activity of digestive enzymes, both pancreatic (up to 26.1 % for trypsin and 74.5 % for amylase) and intestinal enzymes (up to 36.1 % for alkaline phosphatase) in post-metamorphic larvae. Moreover, the impact of ocean acidification and warming on some of these physiological and biochemical variables (namely, lower OCR and higher HSP and MDA levels) were translated into larvae performance, being significantly correlated with decreased larval growth and survival or increased incidence of skeletal deformities. The increased vulnerability of flatfish early life stages under future ocean conditions is expected to potentially determine recruitment and population dynamics in marine ecosystems.

Tubular and Spherical SiO₂ Obtained by Sol Gel Method for Lipase Immobilization and Enzymatic Activity.

PubMed

Anastasescu, Crina; Preda, Silviu; Rusu, Adriana; Culita, Dana; Plavan, Gabriel; Strungaru, Stefan; Calderon-Moreno, Jose Maria; Munteanu, Cornel; Gifu, Catalina; Enache, Mirela; Socoteanu, Radu; Angelescu, Daniel; Anastasescu, Mihai; Gartner, Mariuca; Balint, Ioan; Zaharescu, Maria

2018-06-05

A wide range of hybrid biomaterials has been designed in order to sustain bioremediation processes by associating sol-gel SiO₂ matrices with various biologically active compounds (enzymes, antibodies). SiO₂ is a widespread, chemically stable and non-toxic material; thus, the immobilization of enzymes on silica may lead to improving the efficiency of biocatalysts in terms of endurance and economic costs. Our present work explores the potential of different hybrid morphologies, based on hollow tubes and solid spheres of amorphous SiO₂, for enzyme immobilization and the development of competitive biocatalysts. The synthesis protocol and structural characterization of spherical and tubular SiO₂ obtained by the sol gel method were fully investigated in connection with the subsequent immobilization of lipase from Rhizopus orizae . The immobilization is conducted at pH 6, lower than the isoelectric point of lipase and higher than the isoelectric point of silica, which is meant to sustain the physical interactions of the enzyme with the SiO₂ matrix. The morphological, textural and surface properties of spherical and tubular SiO₂ were investigated by SEM, nitrogen sorption, and electrokinetic potential measurements, while the formation and characterization of hybrid organic-inorganic complexes were studied by UV-VIS, FTIR-ATR and fluorescence spectroscopy. The highest degree of enzyme immobilization (as depicted from total organic carbon) was achieved for tubular morphology and the hydrolysis of p-nitrophenyl acetate was used as an enzymatic model reaction conducted in the presence of hybrid lipase⁻SiO₂ complex.

The effect of β-N-methylamino-L-alanine (BMAA) on oxidative stress response enzymes of the macrophyte Ceratophyllum demersum.

PubMed

Esterhuizen-Londt, M; Pflugmacher, S; Downing, T G

2011-04-01

Cyanobacteria are known to produce bioactive secondary metabolites such as hepatotoxins, cytotoxins and neurotoxins. The newly recognized neurotoxin β-N-methylamino-L-alanine (BMAA) is a naturally occurring non-protein amino acid found in the majority of cyanobacterial genera tested. Evidence that exists for implication of BMAA in neurodegenerative disorders relies on bioaccumulation and biomagnification from symbiotic cyanobacteria. Uptake and accumulation of free BMAA by various non-symbiotic organisms, including aquatic macrophytes, has been documented but to date limited evidence of ecotoxicology exists. We therefore investigated the effect of BMAA on the oxidative stress responses of the macrophyte, Ceratophyllum demersum. Markers for oxidative stress in this study are the antioxidative enzymes superoxide dismutase, catalase, guaiacol peroxidase, glutathione peroxidase and glutathione reductase. We found that BMAA had an inhibitory effect on all the oxidative stress response enzymes tested in plants exposed to BMAA. However enzymes not related to oxidative stress response were not affected by BMAA in in vitro experiments. Binding studies in the presence of BMAA showed reduced enzyme specific activity over time compared to the control. This study shows that BMAA causes oxidative stress indirectly as it inhibits antioxidant enzymes required to combat reactive oxygen species that cause damage to cells. Further investigations are required to fully understand the inhibitory effect of BMAA on these enzymes. Copyright © 2011 Elsevier Ltd. All rights reserved.

Integrated microdroplet-based system for enzyme synthesis and sampling

NASA Astrophysics Data System (ADS)

Lapierre, Florian; Best, Michel; Stewart, Robert; Oakeshott, John; Peat, Thomas; Zhu, Yonggang

2013-12-01

Microdroplet-based microfluidic devices are emerging as powerful tools for a wide range of biochemical screenings and analyses. Monodispersed aqueous microdroplets from picoliters to nanoliters in volume are generated inside microfluidic channels within an immiscible oil phase. This results in the formation of emulsions which can contain various reagents for chemical reactions and can be considered as discrete bioreactors. In this paper an integrated microfluidic platform for the synthesis, screening and sorting of libraries of an organophosphate degrading enzyme is presented. The variants of the selected enzyme are synthesized from a DNA source using in-vitro transcription and translation method. The synthesis occurs inside water-in-oil emulsion droplets, acting as bioreactors. Through a fluorescence based detection system, only the most efficient enzymes are selected. All the necessary steps from the enzyme synthesis to selection of the best genes (producing the highest enzyme activity) are thus integrated inside a single and unique device. In the second part of the paper, an innovative design of the microfluidic platform is presented, integrating an electronic prototyping board for ensuring the communication between the various components of the platform (camera, syringe pumps and high voltage power supply), resulting in a future handheld, user-friendly, fully automated device for enzyme synthesis, screening and selection. An overview on the capabilities as well as future perspectives of this new microfluidic platform is provided.

Active Site Mapping of Xylan-Deconstructing Enzymes with Arabinoxylan Oligosaccharides Produced by Automated Glycan Assembly.

PubMed

Senf, Deborah; Ruprecht, Colin; de Kruijff, Goswinus H M; Simonetti, Sebastian O; Schuhmacher, Frank; Seeberger, Peter H; Pfrengle, Fabian

2017-03-02

Xylan-degrading enzymes are crucial for the deconstruction of hemicellulosic biomass, making the hydrolysis products available for various industrial applications such as the production of biofuel. To determine the substrate specificities of these enzymes, we prepared a collection of complex xylan oligosaccharides by automated glycan assembly. Seven differentially protected building blocks provided the basis for the modular assembly of 2-substituted, 3-substituted, and 2-/3-substituted arabino- and glucuronoxylan oligosaccharides. Elongation of the xylan backbone relied on iterative additions of C4-fluorenylmethoxylcarbonyl (Fmoc) protected xylose building blocks to a linker-functionalized resin. Arabinofuranose and glucuronic acid residues have been selectively attached to the backbone using fully orthogonal 2-(methyl)naphthyl (Nap) and 2-(azidomethyl)benzoyl (Azmb) protecting groups at the C2 and C3 hydroxyls of the xylose building blocks. The arabinoxylan oligosaccharides are excellent tools to map the active site of glycosyl hydrolases involved in xylan deconstruction. The substrate specificities of several xylanases and arabinofuranosidases were determined by analyzing the digestion products after incubation of the oligosaccharides with glycosyl hydrolases. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hydrolysis of phosphatidylcholine during LDL oxidation is mediated by platelet-activating factor acetylhydrolase.

PubMed

Steinbrecher, U P; Pritchard, P H

1989-03-01

Degradation of phosphatidylcholine to lysophosphatidylcholine occurs during oxidative modification of low density lipoproteins (LDL). In this study, we have shown that this phospholipid hydrolysis is brought about by an LDL-associated phospholipase A2 that can hydrolyze oxidized but not intact LDL phosphatidylcholine. The chemical nature of the oxidized phospholipids that can act as substrates for this enzyme was not fully characterized, but we hypothesized that the specificity of the enzyme for oxidized LDL phosphatidylcholine might be explained by fragmentation of polyunsaturated sn-2 fatty acyl groups in LDL phosphatidylcholine during oxidation. To facilitate characterization of this enzyme, we therefore selected a fluorescent phosphatidylcholine substrate that had a short-chain, polar residue in the sn-2 position: 1-palmitoyl 2-(6-[7-nitrobenzoxadiazolyl]amino) caproyl phosphatidylcholine, (C6NBD PC). This substrate was efficiently hydrolyzed by LDL, but the dodecanoyl analogue of C6NBD PC, which differed only in that a 12-carbon rather than a 6-carbon acyl derivative was present in the sn-2 position, was not hydrolyzed. The phospholipase activity was heat-stable, calcium-independent, and was inhibited by the serine esterase inhibitors phenylmethylsulfonyl-fluoride and diisopropylfluorophosphate, but was resistant to p-bromophenacylbromide and dithiobisnitrobenzoic acid. The phospholipid hydrolysis could not be attributed to the action of lecithin:cholesterol acyltransferase or lipoprotein lipase. Nearly all of the activity in EDTA-anticoagulated normal plasma was physically associated with apoB-containing lipoproteins, but this apoprotein was not essential as enzyme activity was present in plasma from abetalipoproteinemic patients. These properties are very similar to those recently reported for human plasma platelet-activating factor (PAF) acetylhydrolase. In the present study, we found that acylhydrolase activity against C6NBD PC, PAF, and oxidized phosphatidylcholine copurfied through gel filtration and ion-exchange chromatography. Substrate competition was demonstrated between C6NBD PC, PAF, and oxidized 2-arachidonyl phosphatidylcholine, suggesting that a single enzyme was active against all three substrates. The enzyme had an apparent molecular weight of 40,000-45,000 by high pressure gel exclusion chromatography. Inhibition of this activity with disopropyfluorophosphate prior to oxidative modification of LDL prevented phospholipid hydrolysis but did not affect the production of thiobarbituric acid reactive compounds or the change in electrophoretic mobility. In addition, this inhibition of phospholipase did not prevent the rapid degradati

Over-expressing the C3 photosynthesis cycle enzyme Sedoheptulose-1-7 Bisphosphatase improves photosynthetic carbon gain and yield under fully open air CO2 fumigation (FACE)

PubMed Central

2011-01-01

Background Biochemical models predict that photosynthesis in C3 plants is most frequently limited by the slower of two processes, the maximum capacity of the enzyme Rubisco to carboxylate RuBP (Vc,max), or the regeneration of RuBP via electron transport (J). At current atmospheric [CO2] levels Rubisco is not saturated; consequently, elevating [CO2] increases the velocity of carboxylation and inhibits the competing oxygenation reaction which is also catalyzed by Rubisco. In the future, leaf photosynthesis (A) should be increasingly limited by RuBP regeneration, as [CO2] is predicted to exceed 550 ppm by 2050. The C3 cycle enzyme sedoheptulose-1,7 bisphosphatase (SBPase, EC 3.1.3.17) has been shown to exert strong metabolic control over RuBP regeneration at light saturation. Results We tested the hypothesis that tobacco transformed to overexpressing SBPase will exhibit greater stimulation of A than wild type (WT) tobacco when grown under field conditions at elevated [CO2] (585 ppm) under fully open air fumigation. Growth under elevated [CO2] stimulated instantaneous A and the diurnal photosynthetic integral (A') more in transformants than WT. There was evidence of photosynthetic acclimation to elevated [CO2] via downregulation of Vc,max in both WT and transformants. Nevertheless, greater carbon assimilation and electron transport rates (J and Jmax) for transformants led to greater yield increases than WT at elevated [CO2] compared to ambient grown plants. Conclusion These results provide proof of concept that increasing content and activity of a single photosynthesis enzyme can enhance carbon assimilation and yield of C3 crops grown at [CO2] expected by the middle of the 21st century. PMID:21884586

Transcriptional regulation of respiration in yeast metabolizing differently repressive carbon substrates.

PubMed

Fendt, Sarah-Maria; Sauer, Uwe

2010-02-18

Depending on the carbon source, Saccharomyces cerevisiae displays various degrees of respiration. These range from complete respiration as in the case of ethanol, to almost complete fermentation, and thus very low degrees of respiration on glucose. While many key regulators are known for these extreme cases, we focus here on regulators that are relevant at intermediate levels of respiration. We address this question by linking the functional degree of respiration to transcriptional regulation via enzyme abundances. Specifically, we investigated aerobic batch cultures with the differently repressive carbon sources glucose, mannose, galactose and pyruvate. Based on 13C flux analysis, we found that the respiratory contribution to cellular energy production was largely absent on glucose and mannose, intermediate on galactose and highest on pyruvate. In vivo abundances of 40 respiratory enzymes were quantified by GFP-fusions under each condition. During growth on the partly and fully respired substrates galactose and pyruvate, several TCA cycle and respiratory chain enzymes were significantly up-regulated. From these enzyme levels and the known regulatory network structure, we determined the probability for a given transcription factor to cause the coordinated expression changes. The most probable transcription factors to regulate the different degrees of respiration were Gcr1p, Cat8p, the Rtg-proteins and the Hap-complex. For the latter three ones we confirmed their importance for respiration by quantifying the degree of respiration and biomass yields in the corresponding deletion strains. Cat8p is required for wild-type like respiration, independent of its known activation of gluconeogenic genes. The Rtg-proteins and the Hap-complex are essential for wild-type like respiration under partially respiratory conditions. Under fully respiratory conditions, the Hap-complex, but not the Rtg-proteins are essential for respiration.

The oligomerization state determines regulatory properties and inhibitor sensitivity of type 4 cAMP-specific phosphodiesterases.

PubMed

Richter, Wito; Conti, Marco

2004-07-16

PDE4 splice variants are classified into long and short forms depending on the presence or absence of two unique N-terminal domains termed upstream conserved regions 1 and 2 (UCR1 and -2). We have shown previously that the UCR module mediates dimerization of PDE4 long forms, whereas short forms, which lack UCR1, behave as monomers. In the present study, we demonstrate that dimerization is an essential structural element that determines the regulatory properties and inhibitor sensitivities of PDE4 enzymes. Comparing the properties of the dimeric wild type PDE4D3 with several monomeric mutant PDE4D3 constructs revealed that disruption of dimerization ablates the activation of PDE4 long forms by either protein kinase A phosphorylation or phosphatidic acid binding. Moreover, the analysis of heterodimers consisting of a catalytically active and a catalytically inactive PDE4D3 subunit indicates that protein kinase A phosphorylation of both subunits is essential to fully activate PDE4 enzymes. In addition to affecting enzyme regulation, disruption of dimerization reduces the sensitivity of the enzymes toward the prototypical PDE4 inhibitor rolipram. Parallel binding assays indicated that this shift in rolipram sensitivity is likely mediated by a decrease in the number of inhibitor binding sites in the high affinity rolipram binding state. Thus, although dimerization is not a requirement for high affinity rolipram binding, it functions to stabilize PDE4 long forms in their high affinity rolipram binding conformation. Taken together, our data indicate that dimerization defines the properties of PDE4 enzymes and suggest a common structural and functional organization for all PDEs.

Dual allosteric activation mechanisms in monomeric human glucokinase

PubMed Central

Whittington, A. Carl; Larion, Mioara; Bowler, Joseph M.; Ramsey, Kristen M.; Brüschweiler, Rafael; Miller, Brian G.

2015-01-01

Cooperativity in human glucokinase (GCK), the body’s primary glucose sensor and a major determinant of glucose homeostatic diseases, is fundamentally different from textbook models of allostery because GCK is monomeric and contains only one glucose-binding site. Prior work has demonstrated that millisecond timescale order-disorder transitions within the enzyme’s small domain govern cooperativity. Here, using limited proteolysis, we map the site of disorder in unliganded GCK to a 30-residue active-site loop that closes upon glucose binding. Positional randomization of the loop, coupled with genetic selection in a glucokinase-deficient bacterium, uncovers a hyperactive GCK variant with substantially reduced cooperativity. Biochemical and structural analysis of this loop variant and GCK variants associated with hyperinsulinemic hypoglycemia reveal two distinct mechanisms of enzyme activation. In α-type activation, glucose affinity is increased, the proteolytic susceptibility of the active site loop is suppressed and the 1H-13C heteronuclear multiple quantum coherence (HMQC) spectrum of 13C-Ile–labeled enzyme resembles the glucose-bound state. In β-type activation, glucose affinity is largely unchanged, proteolytic susceptibility of the loop is enhanced, and the 1H-13C HMQC spectrum reveals no perturbation in ensemble structure. Leveraging both activation mechanisms, we engineer a fully noncooperative GCK variant, whose functional properties are indistinguishable from other hexokinase isozymes, and which displays a 100-fold increase in catalytic efficiency over wild-type GCK. This work elucidates specific structural features responsible for generating allostery in a monomeric enzyme and suggests a general strategy for engineering cooperativity into proteins that lack the structural framework typical of traditional allosteric systems. PMID:26283387

Substituting Tyr138 in the active site loop of human phenylalanine hydroxylase affects catalysis and substrate activation.

PubMed

Leandro, João; Stokka, Anne J; Teigen, Knut; Andersen, Ole A; Flatmark, Torgeir

2017-07-01

Mammalian phenylalanine hydroxylase (PAH) is a key enzyme in l-phenylalanine (l-Phe) metabolism and is active as a homotetramer. Biochemical and biophysical work has demonstrated that it cycles between two states with a variably low and a high activity, and that the substrate l-Phe is the key player in this transition. X-ray structures of the catalytic domain have shown mobility of a partially intrinsically disordered Tyr 138 -loop to the active site in the presence of l-Phe. The mechanism by which the loop dynamics are coupled to substrate binding at the active site in tetrameric PAH is not fully understood. We have here conducted functional studies of four Tyr 138 point mutants. A high linear correlation ( r 2 = 0.99) was observed between their effects on the catalytic efficiency of the catalytic domain dimers and the corresponding effect on the catalytic efficiency of substrate-activated full-length tetramers. In the tetramers, a correlation ( r 2 = 0.96) was also observed between the increase in catalytic efficiency (activation) and the global conformational change (surface plasmon resonance signal response) at the same l-Phe concentration. The new data support a similar functional importance of the Tyr 138 -loop in the catalytic domain and the full-length enzyme homotetramer.

Adaptation of Phenylalanine and Tyrosine Catabolic Pathway to Hibernation in Bats

PubMed Central

Cui, Jie; Liu, Yang; McAllan, Bronwyn M.; Liao, Chen-Chung; Zhang, Shuyi

2013-01-01

Some mammals hibernate in response to harsh environments. Although hibernating mammals may metabolize proteins, the nitrogen metabolic pathways commonly activated during hibernation are not fully characterized. In contrast to the hypothesis of amino acid preservation, we found evidence of amino acid metabolism as three of five key enzymes, including phenylalanine hydroxylase (PAH), homogentisate 1,2-dioxygenase (HGD), fumarylacetoacetase (FAH), involved in phenylalanine and tyrosine catabolism were co-upregulated during hibernation in two distantly related species of bats, Myotis ricketti and Rhinolophus ferrumequinum. In addition, the levels of phenylalanine in the livers of these bats were significantly decreased during hibernation. Because phenylalanine and tyrosine are both glucogenic and ketogenic, these results indicate the role of this catabolic pathway in energy supply. Since any deficiency in the catabolism of these two amino acids can cause accumulations of toxic metabolites, these results also suggest the detoxification role of these enzymes during hibernation. A higher selective constraint on PAH, HPD, and HGD in hibernators than in non-hibernators was observed, and hibernators had more conserved amino acid residues in each of these enzymes than non-hibernators. These conserved amino acid residues are mostly located in positions critical for the structure and activity of the enzymes. Taken together, results of this work provide novel insights in nitrogen metabolism and removal of harmful metabolites during bat hibernation. PMID:23620802

Differential repression of arylsulphatase synthesis in Aspergillus oryzae.

PubMed

Burns, G R; Wynn, C H

1977-09-15

1. The activities of the three arylsulphatases (arylsulphate sulphohydrolase, EC 3.1.6.1) of Aspergillus oryzae produced under a variety of repressing and non-repressing conditions were determined. 2. These enzymes exhibit different sensitivities to repression by inorganic sulphate. 3. Arylsulphatase I, but not arylsulphatases II and III, exhibits a transient de-repression in the early growth phase in sulphate media. 4. When the fungus is cultured in repressing media and subsequently transferred to non-repressing media, the synthesis of the three enzymes is non-co-ordinate. 5. Growth of the fungus in media containing choline O-sulphate or tyrosine O-sulphate as the sole source of sulphur results in complete de-repression of arylsulphatase I, But the synthesis of arylsulphatases II and III is essentially fully repressed. 6. The marked similarities between the repression characteristics of arylsulphatases II and III, contrasted with those of arylsulphatase I, indicate that the genetic locus of arylsulphatase I is distinct from that of arylsulphatases II and III, suggesting that there are distinct physiological roles for the enzyme.

Induction of Protease Release of the Resistant Diatom Chaetoceros didymus in Response to Lytic Enzymes from an Algicidal Bacterium

PubMed Central

Paul, Carsten; Pohnert, Georg

2013-01-01

Marine lytic bacteria can have a substantial effect on phytoplankton and are even capable to terminate blooms of microalgae. The bacterium Kordia algicida was reported to lyse cells of the diatom Skeletonema costatum and several other diatoms by a quorum sensing controlled excretion of proteases. However the diatom Chaetoceros didymus is fully resistant against the bacterial enzymes. We show that the growth curve of this diatom is essentially unaffected by addition of bacterial filtrates that are active against other diatoms. By monitoring proteases from the medium using zymography and fluorescence based activity assays we demonstrate that C. didymus responds to the presence of the lytic bacteria with the induced production of algal proteases. These proteases exhibit a substantially increased activity compared to the bacterial counterparts. The induction is also triggered by signals in the supernatant of a K. algicida culture. Size fractionation shows that only the >30 kD fraction of the bacterial exudates acts as an inducing cue. Implications for a potential induced defense of the diatom C. didymus are discussed. PMID:23469204

Sequence Search and Comparative Genomic Analysis of SUMO-Activating Enzymes Using CoGe.

PubMed

Carretero-Paulet, Lorenzo; Albert, Victor A

2016-01-01

The growing number of genome sequences completed during the last few years has made necessary the development of bioinformatics tools for the easy access and retrieval of sequence data, as well as for downstream comparative genomic analyses. Some of these are implemented as online platforms that integrate genomic data produced by different genome sequencing initiatives with data mining tools as well as various comparative genomic and evolutionary analysis possibilities.Here, we use the online comparative genomics platform CoGe ( http://www.genomevolution.org/coge/ ) (Lyons and Freeling. Plant J 53:661-673, 2008; Tang and Lyons. Front Plant Sci 3:172, 2012) (1) to retrieve the entire complement of orthologous and paralogous genes belonging to the SUMO-Activating Enzymes 1 (SAE1) gene family from a set of species representative of the Brassicaceae plant eudicot family with genomes fully sequenced, and (2) to investigate the history, timing, and molecular mechanisms of the gene duplications driving the evolutionary expansion and functional diversification of the SAE1 family in Brassicaceae.

Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase.

PubMed

Coelho, Catarina; Foti, Alessandro; Hartmann, Tobias; Santos-Silva, Teresa; Leimkühler, Silke; Romão, Maria João

2015-10-01

Aldehyde oxidase (AOX) is a xanthine oxidase (XO)-related enzyme with emerging importance due to its role in the metabolism of drugs and xenobiotics. We report the first crystal structures of human AOX1, substrate free (2.6-Å resolution) and in complex with the substrate phthalazine and the inhibitor thioridazine (2.7-Å resolution). Analysis of the protein active site combined with steady-state kinetic studies highlight the unique features, including binding and substrate orientation at the active site, that characterize human AOX1 as an important drug-metabolizing enzyme. Structural analysis of the complex with the noncompetitive inhibitor thioridazine revealed a new, unexpected and fully occupied inhibitor-binding site that is structurally conserved among mammalian AOXs and XO. The new structural insights into the catalytic and inhibition mechanisms of human AOX that we now report will be of great value for the rational analysis of clinical drug interactions involving inhibition of AOX1 and for the prediction and design of AOX-stable putative drugs.

Optimized Target Residence Time: Type I1/2 Inhibitors for p38α MAP Kinase with Improved Binding Kinetics through Direct Interaction with the R-Spine.

PubMed

Wentsch, Heike K; Walter, Niklas M; Bührmann, Mike; Mayer-Wrangowski, Svenja; Rauh, Daniel; Zaman, Guido J R; Willemsen-Seegers, Nicole; Buijsman, Rogier C; Henning, Melanie; Dauch, Daniel; Zender, Lars; Laufer, Stefan

2017-05-02

Skepinone-L was recently reported to be a p38α MAP kinase inhibitor with high potency and excellent selectivity in vitro and in vivo. However, this class of compounds still act as fully ATP-competitive Type I binders which, furthermore, suffer from short residence times at the enzyme. We herein describe a further development with the first Type I1/2 binders for p38α MAP kinase. Type I1/2 inhibitors interfere with the R-spine, inducing a glycine flip and occupying both hydrophobic regions I and II. This design approach leads to prolonged target residence time, binding to both the active and inactive states of the kinase, excellent selectivity, excellent potency on the enzyme level, and low nanomolar activity in a human whole blood assay. This promising binding mode is proven by X-ray crystallography. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization and mutational analysis of the UDP-Glc(NAc) 4-epimerase from Marinithermus hydrothermalis.

PubMed

Beerens, Koen; Soetaert, Wim; Desmet, Tom

2013-09-01

UDP-hexose 4-epimerases are important enzymes that play key roles in various biological pathways, including lipopolysaccharide biosynthesis, galactose metabolism through the Leloir pathway, and biofilm formation. Unfortunately, the determinants of their substrate specificity are not yet fully understood. They can be classified into three groups, with groups 1 and 3 preferring non-acetylated and acetylated UDP-hexoses, respectively, whereas members of group 2 are equally active on both types of substrates. In this study, the UDP-Glc(NAc) 4-epimerase from Marinithermus hydrothermalis (mGalE) was functionally expressed in Escherichia coli and thoroughly characterized. The enzyme was found to be thermostable, displaying its highest activity at 70 °C and having a half-life of 23 min at 60 °C. Activity could be detected on both acetylated and non-acetylated UDP-hexoses, meaning that this epimerase belongs to group 2. This observation correlates well with the identity of the so-called "gatekeeper" residue (Ser279), which has previously been suggested to influence substrate specificity (Schulz et al., J Biol Chem 279:32796-32803, 2004). Furthermore, substituting this serine to a tyrosine brings about a significant preference for non-acetylated sugars, thereby demonstrating that a single residue can determine substrate specificity among type 1 and type 2 epimerases. In addition, two consecutive glycine residues (Gly118 and Gly119) were identified as a unique feature of GalE enzymes from Thermus species, and their importance for activity as well as affinity was confirmed by mutagenesis. Finally, homology modeling and mutational analysis has revealed that the enzyme's catalytic triad contains a threonine residue (Thr117) instead of the usual serine.

Conversion of nicotinic acid to trigonelline is catalyzed by N-methyltransferase belonged to motif B′ methyltransferase family in Coffea arabica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mizuno, Kouichi, E-mail: koumno@akita-pu.ac.jp; Matsuzaki, Masahiro; Kanazawa, Shiho

Graphical abstract: Trigonelline synthase catalyzes the conversion of nicotinic acid to trigonelline. We isolated and characterized trigonelline synthase gene(s) from Coffea arabica. - Highlights: • Trigonelline is a major compound in coffee been same as caffeine is. • We isolated and characterized trigonelline synthase gene. • Coffee trigonelline synthases are highly homologous with coffee caffeine synthases. • This study contributes the fully understanding of pyridine alkaloid metabolism. - Abstract: Trigonelline (N-methylnicotinate), a member of the pyridine alkaloids, accumulates in coffee beans along with caffeine. The biosynthetic pathway of trigonelline is not fully elucidated. While it is quite likely that themore » production of trigonelline from nicotinate is catalyzed by N-methyltransferase, as is caffeine synthase (CS), the enzyme(s) and gene(s) involved in N-methylation have not yet been characterized. It should be noted that, similar to caffeine, trigonelline accumulation is initiated during the development of coffee fruits. Interestingly, the expression profiles for two genes homologous to caffeine synthases were similar to the accumulation profile of trigonelline. We presumed that these two CS-homologous genes encoded trigonelline synthases. These genes were then expressed in Escherichiacoli, and the resulting recombinant enzymes that were obtained were characterized. Consequently, using the N-methyltransferase assay with S-adenosyl[methyl-{sup 14}C]methionine, it was confirmed that these recombinant enzymes catalyzed the conversion of nicotinate to trigonelline, coffee trigonelline synthases (termed CTgS1 and CTgS2) were highly identical (over 95% identity) to each other. The sequence homology between the CTgSs and coffee CCS1 was 82%. The pH-dependent activity curve of CTgS1 and CTgS2 revealed optimum activity at pH 7.5. Nicotinate was the specific methyl acceptor for CTgSs, and no activity was detected with any other nicotinate derivatives, or with any of the typical substrates of B′-MTs. It was concluded that CTgSs have strict substrate specificity. The K{sub m} values of CTgS1 and CTgS2 were 121 and 184 μM with nicotinic acid as a substrate, and 68 and 120 μM with S-adenosyl-L-methionine as a substrate, respectively.« less

Autodigestion: Proteolytic Degradation and Multiple Organ Failure in Shock

PubMed Central

Altshuler, Angelina E.; Kistler, Erik B.; Schmid-Schönbein, Geert W.

2015-01-01

There is currently no effective treatment for multiorgan failure following shock other than alleviation supportive care. A better understanding of the pathogenesis of these sequelae to shock is required. The intestine plays a central role in multiorgan failure. It was previously suggested that bacteria and their toxins are responsible for the organ failure seen in circulatory shock, but clinical trials in septic patients have not confirmed this hypothesis. Instead, we review here evidence that the digestive enzymes, synthesized in the pancreas and discharged into the small intestine as requirement for normal digestion, may play a role in multi-organ failure. These powerful enzymes are non-specific, highly concentrated and fully activated in the lumen of the intestine. During normal digestion they are compartmentalized in the lumen of the intestine by the mucosal epithelial barrier. However, if this barrier becomes permeable, e.g. in an ischemic state, the digestive enzymes escape into the wall of the intestine. They digest tissues in the mucosa and generate small molecular weight cytotoxic fragments such as unbound free fatty acids. Digestive enzymes may also escape into the systemic circulation and activate other degrading proteases. These proteases have the ability to clip the ectodomain of surface receptors and compromise their function; for example cleaving the insulin receptor causing insulin resistance. The combination of digestive enzymes and cytotoxic fragments leaking into the central circulation causes cell and organ dysfunction, and ultimately may lead to complete organ failure and death. We summarize current evidence suggesting that enteral blockade of digestive enzymes inside the lumen of the intestine may serve to reduce acute cell and organ damage and improve survival in experimental shock. PMID:26717111

Demonstration that CobG, the monooxygenase associated with the ring contraction process of the aerobic cobalamin (vitamin B12) biosynthetic pathway, contains an Fe-S center and a mononuclear non-heme iron center.

PubMed

Schroeder, Susanne; Lawrence, Andrew D; Biedendieck, Rebekka; Rose, Ruth-Sarah; Deery, Evelyne; Graham, Ross M; McLean, Kirsty J; Munro, Andrew W; Rigby, Stephen E J; Warren, Martin J

2009-02-20

The ring contraction process that occurs during cobalamin (vitamin B(12)) biosynthesis is mediated via the action of two enzymes, CobG and CobJ. The first of these generates a tertiary alcohol at the C-20 position of precorrin-3A by functioning as a monooxygenase, a reaction that also forms a gamma lactone with the acetic acid side chain on ring A. The product, precorrin-3B, is then acted upon by CobJ, which methylates at the C-17 position and promotes ring contraction of the macrocycle by catalyzing a masked pinacol rearrangement. Here, we report the characterization of CobG enzymes from Pseudomonas denitrificans and Brucella melitensis. We show that both contain a [4Fe-4S] center as well as a mononuclear non-heme iron. Although both enzymes are active in vivo, the P. denitrificans enzyme was found to be inactive in vitro. Further analysis of this enzyme revealed that the mononuclear non-heme iron was not reducible, and it was concluded that it is rapidly inactivated once it is released from the bacterial cell. In contrast, the B. melitensis enzyme was found to be fully active in vitro and the mononuclear non-heme iron was reducible by dithionite. The reduced mononuclear non-heme was able to react with the oxygen analogue NO, but only in the presence of the substrate precorrin-3A. The cysteine residues responsible for binding the Fe-S center were identified by site-directed mutagenesis. A mechanism for CobG is presented.

Mild Hypothermia Attenuates Mitochondrial Oxidative Stress by Protecting Respiratory Enzymes and Upregulating MnSOD in a Pig Model of Cardiac Arrest

PubMed Central

Gong, Ping; Li, Chun-Sheng; Hua, Rong; Zhao, Hong; Tang, Zi-Ren; Mei, Xue; Zhang, Ming-Yue; Cui, Juan

2012-01-01

Mild hypothermia is the only effective treatment confirmed clinically to improve neurological outcomes for comatose patients with cardiac arrest. However, the underlying mechanism is not fully elucidated. In this study, our aim was to determine the effect of mild hypothermia on mitochondrial oxidative stress in the cerebral cortex. We intravascularly induced mild hypothermia (33°C), maintained this temperature for 12 h, and actively rewarmed in the inbred Chinese Wuzhishan minipigs successfully resuscitated after 8 min of untreated ventricular fibrillation. Cerebral samples were collected at 24 and 72 h following return of spontaneous circulation (ROSC). We found that mitochondrial malondialdehyde and protein carbonyl levels were significantly increased in the cerebral cortex in normothermic pigs even at 24 h after ROSC, whereas mild hypothermia attenuated this increase. Moreover, mild hypothermia attenuated the decrease in Complex I and Complex III (i.e., major sites of reactive oxygen species production) activities of the mitochondrial respiratory chain and increased antioxidant enzyme manganese superoxide dismutase (MnSOD) activity. This increase in MnSOD activity was consistent with the upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA and protein expressions, and with the increase of Nrf2 nuclear translocation in normothermic pigs at 24 and 72 h following ROSC, whereas mild hypothermia enhanced these tendencies. Thus, our findings indicate that mild hypothermia attenuates mitochondrial oxidative stress in the cerebral cortex, which may be associated with reduced impairment of mitochondrial respiratory chain enzymes, and enhancement of MnSOD activity and expression via Nrf2 activation. PMID:22532848

Understanding how the complex molecular architecture of mannan-degrading hydrolases contributes to plant cell wall degradation.

PubMed

Zhang, Xiaoyang; Rogowski, Artur; Zhao, Lei; Hahn, Michael G; Avci, Utku; Knox, J Paul; Gilbert, Harry J

2014-01-24

Microbial degradation of plant cell walls is a central component of the carbon cycle and is of increasing importance in environmentally significant industries. Plant cell wall-degrading enzymes have a complex molecular architecture consisting of catalytic modules and, frequently, multiple non-catalytic carbohydrate binding modules (CBMs). It is currently unclear whether the specificities of the CBMs or the topology of the catalytic modules are the primary drivers for the specificity of these enzymes against plant cell walls. Here, we have evaluated the relationship between CBM specificity and their capacity to enhance the activity of GH5 and GH26 mannanases and CE2 esterases against intact plant cell walls. The data show that cellulose and mannan binding CBMs have the greatest impact on the removal of mannan from tobacco and Physcomitrella cell walls, respectively. Although the action of the GH5 mannanase was independent of the context of mannan in tobacco cell walls, a significant proportion of the polysaccharide was inaccessible to the GH26 enzyme. The recalcitrant mannan, however, was fully accessible to the GH26 mannanase appended to a cellulose binding CBM. Although CE2 esterases display similar specificities against acetylated substrates in vitro, only CjCE2C was active against acetylated mannan in Physcomitrella. Appending a mannan binding CBM27 to CjCE2C potentiated its activity against Physcomitrella walls, whereas a xylan binding CBM reduced the capacity of esterases to deacetylate xylan in tobacco walls. This work provides insight into the biological significance for the complex array of hydrolytic enzymes expressed by plant cell wall-degrading microorganisms.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Yu; Wang, Wenhui; Wang, Qi

Highlights: Black-Right-Pointing-Pointer 5-LOX is able to upregulate expression of NF-{kappa}B p65. Black-Right-Pointing-Pointer 5-LOX enhances nuclear translocation of NF-{kappa}B p65 via increasing p-I{kappa}B-{alpha} level. Black-Right-Pointing-Pointer 5-LOX stimulates transcriptional activity of NF-{kappa}B in hepatoma cells. Black-Right-Pointing-Pointer LTB4 activates transcriptional activity of NF-{kappa}B in hepatoma cells. -- Abstract: The issue that lipid metabolism enzyme and its metabolites regulate transcription factors in cancer cell is not fully understood. In this study, we first report that the lipid metabolism enzyme 5-Lipoxygenase (5-LOX) and its metabolite leukotriene B4 (LTB4) are capable of activating nuclear factor-{kappa}B (NF-{kappa}B) in hepatoma cells. We found that the treatment of MK886more » (an inhibitor of 5-LOX) or knockdown of 5-LOX was able to downregulate the expression of NF-{kappa}B p65 at the mRNA level and decreased the phosphorylation level of inhibitor {kappa}B{alpha} (I{kappa}B{alpha}) in the cytoplasm of hepatoma HepG2 or H7402 cells, which resulted in the decrease of the level of nuclear NF-{kappa}B p65. These were confirmed by immunofluorescence staining in HepG2 cell. Moreover, the above treatments were able to decrease the transcriptional activity of NF-{kappa}B in the cells. The LTB4, one of metabolites of 5-LOX, is responsible for 5-LOX-activated NF-{kappa}B in a dose-dependent manner. Thus, we conclude that the lipid metabolism enzyme 5-LOX and its metabolite LTB4 are capable of activating transcription factor NF-{kappa}B in hepatoma cells. Our finding provides new insight into the significance of lipid metabolism in activation of transcription factors in cancer.« less

Oligomeric state regulated trafficking of human platelet-activating factor acetylhydrolase type-II.

PubMed

Monillas, Elizabeth S; Caplan, Jeffrey L; Thévenin, Anastasia F; Bahnson, Brian J

2015-05-01

The intracellular enzyme platelet-activating factor acetylhydrolase type-II (PAFAH-II) hydrolyzes platelet-activating factor and oxidatively fragmented phospholipids. PAFAH-II in its resting state is mainly cytoplasmic, and it responds to oxidative stress by becoming increasingly bound to endoplasmic reticulum and Golgi membranes. Numerous studies have indicated that this enzyme is essential for protecting cells from oxidative stress induced apoptosis. However, the regulatory mechanism of the oxidative stress response by PAFAH-II has not been fully resolved. Here, changes to the oligomeric state of human PAFAH-II were investigated as a potential regulatory mechanism toward enzyme trafficking. Native PAGE analysis in vitro and photon counting histogram within live cells showed that PAFAH-II is both monomeric and dimeric. A Gly-2-Ala site-directed mutation of PAFAH-II demonstrated that the N-terminal myristoyl group is required for homodimerization. Additionally, the distribution of oligomeric PAFAH-II is distinct within the cell; homodimers of PAFAH-II were localized to the cytoplasm while monomers were associated to the membranes of the endoplasmic reticulum and Golgi. We propose that the oligomeric state of PAFAH-II drives functional protein trafficking. PAFAH-II localization to the membrane is critical for substrate acquisition and effective oxidative stress protection. It is hypothesized that the balance between monomer and dimer serves as a regulatory mechanism of a PAFAH-II oxidative stress response. Copyright © 2015 Elsevier B.V. All rights reserved.

Possible involvement of membrane lipids peroxidation and oxidation of catalytically essential thiols of the cerebral transmembrane sodium pump as component mechanisms of iron-mediated oxidative stress-linked dysfunction of the pump's activity

PubMed Central

Omotayo, T.I.; Akinyemi, G.S.; Omololu, P.A.; Ajayi, B.O.; Akindahunsi, A.A.; Rocha, J.B.T.; Kade, I.J.

2014-01-01

The precise molecular events defining the complex role of oxidative stress in the inactivation of the cerebral sodium pump in radical-induced neurodegenerative diseases is yet to be fully clarified and thus still open. Herein we investigated the modulation of the activity of the cerebral transmembrane electrogenic enzyme in Fe2+-mediated in vitro oxidative stress model. The results show that Fe2+ inhibited the transmembrane enzyme in a concentration dependent manner and this effect was accompanied by a biphasic generation of aldehydic product of lipid peroxidation. While dithiothreitol prevented both Fe2+ inhibitory effect on the pump and lipid peroxidation, vitamin E prevented only lipid peroxidation but not inhibition of the pump. Besides, malondialdehyde (MDA) inhibited the pump by a mechanism not related to oxidation of its critical thiols. Apparently, the low activity of the pump in degenerative diseases mediated by Fe2+ may involve complex multi-component mechanisms which may partly involve an initial oxidation of the critical thiols of the enzyme directly mediated by Fe2+ and during severe progression of such diseases; aldehydic products of lipid peroxidation such as MDA may further exacerbate this inhibitory effect by a mechanism that is likely not related to the oxidation of the catalytically essential thiols of the ouabain-sensitive cerebral electrogenic pump. PMID:25618580

Synthesis and tyrosinase inhibitory properties of some novel derivatives of kojic acid

PubMed Central

Saghaie, L; Pourfarzam, M.; Fassihi, A.; Sartippour, B.

2013-01-01

Tyrosinase is a multifunctional oxidase that is widely distributed in nature. It is a key enzyme in melanin biosynthesis and is involved in determining the color of mammalian skin and hair. In addition it is responsible for the undesirable enzymatic browning that occurs in plant-derived foods, limiting the shelf-life of fresh-cut products with the resultant economic loss. In recent years there has been considerable interest to study the inhibitory activity of tyrosinase and a number of inhibitory compounds derived from natural sources or partly/fully synthetic have been described. However, the current conventional methods to control tyrosinase action are inadequate. Considering the significant industrial and economic impact of the inhibitors of tyrosinase, this study was set to seek new potent inhibitors of this enzyme. A series of 3-hydroxypyridine-4-one derivatives were prepared in high yield and evaluated for their inhibitory activity on tyrosinase enzyme using dopachrome method. Our results show that all synthesized compounds have inhibitory effect on tyrosinase activity for the oxidation of L-DOPA. Among compounds studied those containing two free hydroxyl group (ie Va and V’a) were more potent than their analogues with one hydroxyl group (ie Vb and V’b). Also substitution of a methyl group on position N1 of the hydroxypyridinone ring seems to confer more inhibitory potency. PMID:24082892

Synthesis and tyrosinase inhibitory properties of some novel derivatives of kojic acid.

PubMed

Saghaie, L; Pourfarzam, M; Fassihi, A; Sartippour, B

2013-10-01

Tyrosinase is a multifunctional oxidase that is widely distributed in nature. It is a key enzyme in melanin biosynthesis and is involved in determining the color of mammalian skin and hair. In addition it is responsible for the undesirable enzymatic browning that occurs in plant-derived foods, limiting the shelf-life of fresh-cut products with the resultant economic loss. In recent years there has been considerable interest to study the inhibitory activity of tyrosinase and a number of inhibitory compounds derived from natural sources or partly/fully synthetic have been described. However, the current conventional methods to control tyrosinase action are inadequate. Considering the significant industrial and economic impact of the inhibitors of tyrosinase, this study was set to seek new potent inhibitors of this enzyme. A series of 3-hydroxypyridine-4-one derivatives were prepared in high yield and evaluated for their inhibitory activity on tyrosinase enzyme using dopachrome method. Our results show that all synthesized compounds have inhibitory effect on tyrosinase activity for the oxidation of L-DOPA. Among compounds studied those containing two free hydroxyl group (ie Va and V'a) were more potent than their analogues with one hydroxyl group (ie Vb and V'b). Also substitution of a methyl group on position N(1) of the hydroxypyridinone ring seems to confer more inhibitory potency.

Mitochondrial Reactive Oxygen Species Trigger Hypoxia-Induced Transcription

NASA Astrophysics Data System (ADS)

Chandel, N. S.; Maltepe, E.; Goldwasser, E.; Mathieu, C. E.; Simon, M. C.; Schumacker, P. T.

1998-09-01

Transcriptional activation of erythropoietin, glycolytic enzymes, and vascular endothelial growth factor occurs during hypoxia or in response to cobalt chloride (CoCl2) in Hep3B cells. However, neither the mechanism of cellular O2 sensing nor that of cobalt is fully understood. We tested whether mitochondria act as O2 sensors during hypoxia and whether hypoxia and cobalt activate transcription by increasing generation of reactive oxygen species (ROS). Results show (i) wild-type Hep3B cells increase ROS generation during hypoxia (1.5% O2) or CoCl2 incubation, (ii) Hep3B cells depleted of mitochondrial DNA (ρ 0 cells) fail to respire, fail to activate mRNA for erythropoietin, glycolytic enzymes, or vascular endothelial growth factor during hypoxia, and fail to increase ROS generation during hypoxia; (iii) ρ 0 cells increase ROS generation in response to CoCl2 and retain the ability to induce expression of these genes; and (iv) the antioxidants pyrrolidine dithiocarbamate and ebselen abolish transcriptional activation of these genes during hypoxia or CoCl2 in wild-type cells, and abolish the response to CoCl2 in ρ 0 cells. Thus, hypoxia activates transcription via a mitochondria-dependent signaling process involving increased ROS, whereas CoCl2 activates transcription by stimulating ROS generation via a mitochondria-independent mechanism.

Cascade catalysis in membranes with enzyme immobilization for multi-enzymatic conversion of CO2 to methanol.

PubMed

Luo, Jianquan; Meyer, Anne S; Mateiu, R V; Pinelo, Manuel

2015-05-25

Facile co-immobilization of enzymes is highly desirable for bioconversion methods involving multi-enzymatic cascade reactions. Here we show for the first time that three enzymes can be immobilized in flat-sheet polymeric membranes simultaneously or separately by simple pressure-driven filtration (i.e. by directing membrane fouling formation), without any addition of organic solvent. Such co-immobilization and sequential immobilization systems were examined for the production of methanol from CO2 with formate dehydrogenase (FDH), formaldehyde dehydrogenase (FaldDH) and alcohol dehydrogenase (ADH). Enzyme activity was fully retained by this non-covalent immobilization strategy. The two immobilization systems had similar catalytic efficiencies because the second reaction (formic acid→formaldehyde) catalyzed by FaldDH was found to be the cascade bottleneck (a threshold substrate concentration was required). Moreover, the trade-off between the mitigation of product inhibition and low substrate concentration for the adjacent enzymes probably made the co-immobilization meaningless. Thus, sequential immobilization could be used for multi-enzymatic cascade reactions, as it allowed the operational conditions for each single step to be optimized, not only during the enzyme immobilization but also during the reaction process, and the pressure-driven mass transfer (flow-through mode) could overcome the diffusion resistance between enzymes. This study not only offers a green and facile immobilization method for multi-enzymatic cascade systems, but also reveals the reaction bottleneck and provides possible solutions for the bioconversion of CO2 to methanol. Copyright © 2015 Elsevier B.V. All rights reserved.

Kinetic and spectroscopic characterization of tungsten-substituted DMSO reductase from Rhodobacter sphaeroides.

PubMed

Pacheco, Josué; Niks, Dimitri; Hille, Russ

2018-03-01

We have examined the kinetic and spectroscopic properties of a tungsten-substituted form of DMSO reductase from Rhodobacter sphaeroides, an enzyme that normally possesses molybdenum. Partial reduction with sodium dithionite yields a well-resolved W(V) EPR signal of the so-called "high-g split" type that exhibits markedly greater g-anisotropy than the corresponding Mo(V) signal of the native form of the enzyme, with the g values shifted to higher magnetic field by as much as Δg ave  = 0.056. Deuteration of the enzyme confirms that the coupled proton is solvent-exchangeable, allowing us to accurately simulate the tungsten hyperfine coupling. Global curve-fitting analysis of UV/vis absorption spectra observed in the course of the reaction of the tungsten-substituted enzyme with sodium dithionite affords a well-defined absorption spectrum for the W(V) species. Surprisingly, the absorption spectrum for this species exhibits significantly larger molar extinction coefficients than either the reduced or the oxidized spectrum. This spectrum, in conjunction with those for fully oxidized W(VI) and fully reduced W(IV) enzyme, has been used to deconvolute the absorption spectra seen in the course of turnover, in the which enzyme is reacted with sodium dithionite and DMSO, demonstrating that the W(V) is an authentic catalytic intermediate that accumulates to approximately 50% of the total enzyme in the steady state.

Enzymology of retinoic acid biosynthesis and degradation

PubMed Central

Kedishvili, Natalia Y.

2013-01-01

All-trans-retinoic acid is a biologically active derivative of vitamin A that regulates numerous physiological processes. The concentration of retinoic acid in the cells is tightly regulated, but the exact mechanisms responsible for this regulation are not completely understood, largely because the enzymes involved in the biosynthesis of retinoic acid have not been fully defined. Recent studies using in vitro and in vivo models suggest that several members of the short-chain dehydrogenase/reductase superfamily of proteins are essential for retinoic acid biosynthesis and the maintenance of retinoic acid homeostasis. However, the exact roles of some of these recently identified enzymes are yet to be characterized. The properties of the known contributors to retinoid metabolism have now been better defined and allow for more detailed understanding of their interactions with retinoid-binding proteins and other retinoid enzymes. At the same time, further studies are needed to clarify the interactions between the cytoplasmic and membrane-bound proteins involved in the processing of hydrophobic retinoid metabolites. This review summarizes current knowledge about the roles of various biosynthetic and catabolic enzymes in the regulation of retinoic acid homeostasis and outlines the remaining questions in the field. PMID:23630397

Kinetic modeling of a bi-enzymatic system for efficient conversion of lactose to lactobionic acid.

PubMed

Van Hecke, Wouter; Bhagwat, Aditya; Ludwig, Roland; Dewulf, Jo; Haltrich, Dietmar; Van Langenhove, Herman

2009-04-01

A model has been developed to describe the interaction between two enzymes and an intermediary redox mediator. In this bi-enzymatic process, the enzyme cellobiose dehydrogenase oxidizes lactose at the C-1 position of the reducing sugar moiety to lactobionolactone, which spontaneously hydrolyzes to lactobionic acid. 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt is used as electron acceptor and is continuously regenerated by laccase. Oxygen is the terminal electron acceptor and is fully reduced to water by laccase, a copper-containing oxidase. Oxygen is added to the system by means of bubble-free oxygenation. Using the model, the productivity of the process is investigated by simultaneous solution of the rate equations for varying enzyme quantities and redox mediator concentrations, solved with the aid of a numerical solution. The isocharts developed in this work provide an easy-to-use graphical tool to determine optimal process conditions. The model allows the optimization of the employed activities of the two enzymes and the redox mediator concentration for a given overall oxygen mass transfer coefficient by using the isocharts. Model predictions are well in agreement with the experimental data.

Understanding sugar yield loss and enzyme inhibition due to oligosaccharides accumulation during high solids-loading enzymatic hydrolisis.

USDA-ARS?s Scientific Manuscript database

During enzymatic hydrolysis of biomass, polysaccharides are cleaved by glycosyl hydrolases to soluble oligosaccharides and further hydrolyzed by ß-glucosidase, ß-xylosidase and other enzymes to monomeric sugars. However, not all oligosaccharides can be fully hydrolyzed and they may accumulate to 18-...

Caffeic and chlorogenic acids inhibit key enzymes linked to type 2 diabetes (in vitro): a comparative study.

PubMed

Oboh, Ganiyu; Agunloye, Odunayo M; Adefegha, Stephen A; Akinyemi, Ayodele J; Ademiluyi, Adedayo O

2015-03-01

Chlorogenic acid is a major phenolic compound that forms a substantial part of plant foods and is an ester of caffeic acid and quinic acid. However, the effect of the structures of both chlorogenic and caffeic acids on their antioxidant and antidiabetic potentials have not been fully understood. Thus, this study sought to investigate and compare the interaction of caffeic acid and chlorogenic acid with α-amylase and α-glucosidase (key enzymes linked to type 2 diabetes) activities in vitro. The inhibitory effect of the phenolic acids on α-amylase and α-glucosidase activities was evaluated. Thereafter, their antioxidant activities as typified by their 1,1-diphenyl-2 picrylhydrazyl radical scavenging ability and ferric reducing antioxidant properties were determined. The results revealed that both phenolic acids inhibited α-amylase and α-glucosidase activities in a dose-dependent manner (2-8 μg/mL). However, caffeic acid had a significantly (p<0.05) higher inhibitory effect on α-amylase [IC50 (concentration of sample causing 50% enzyme inhibition)=3.68 μg/mL] and α-glucosidase (IC50=4.98 μg/mL) activities than chlorogenic acid (α-amylase IC50=9.10 μg/mL and α-glucosidase IC50=9.24 μg/mL). Furthermore, both phenolic acids exhibited high antioxidant properties, with caffeic acid showing higher effects. The esterification of caffeic acid with quinic acid, producing chlorogenic acid, reduces their ability to inhibit α-amylase and α-glucosidase activities. Thus, the inhibition of α-amylase and α-glucosidase activities by the phenolic acids could be part of the possible mechanism by which the phenolic acids exert their antidiabetic effects.

Correction of mouse ornithine transcarbamylase deficiency by gene transfer into the germ line.

PubMed Central

Cavard, C; Grimber, G; Dubois, N; Chasse, J F; Bennoun, M; Minet-Thuriaux, M; Kamoun, P; Briand, P

1988-01-01

The sparse fur with abnormal skin and hair (Spf-ash) mouse is a model for the human X-linked hereditary disorder, ornithine transcarbamylase (OTC) deficiency. In Spf-ash mice, both OTC mRNA and enzyme activity are 5% of control values resulting in hyperammonemia, pronounced orotic aciduria and an abnormal phenotype characterized by growth retardation and sparse fur. Using microinjection, we introduced a construction containing rat OTC cDNA linked to the SV40 early promoter into fertilized eggs of Spf-ash mice. The expression of the transgene resulted in the development of a transgenic mouse whose phenotype and orotic acid excretion are fully normalized. Thus, the possibility of correcting hereditary enzymatic defect by gene transfer of heterologous cDNA coding for the normal enzyme has been demonstrated. Images PMID:3162766

Bacterial molybdoenzymes: old enzymes for new purposes.

PubMed

Leimkühler, Silke; Iobbi-Nivol, Chantal

2016-01-01

Molybdoenzymes are widespread in eukaryotic and prokaryotic organisms where they play crucial functions in detoxification reactions in the metabolism of humans and bacteria, in nitrate assimilation in plants and in anaerobic respiration in bacteria. To be fully active, these enzymes require complex molybdenum-containing cofactors, which are inserted into the apoenzymes after folding. For almost all the bacterial molybdoenzymes, molybdenum cofactor insertion requires the involvement of specific chaperones. In this review, an overview on the molybdenum cofactor biosynthetic pathway is given together with the role of specific chaperones dedicated for molybdenum cofactor insertion and maturation. Many bacteria are involved in geochemical cycles on earth and therefore have an environmental impact. The roles of molybdoenzymes in bioremediation and for environmental applications are presented. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Bg10: A Novel Metagenomics Alcohol-Tolerant and Glucose-Stimulated GH1 ß-Glucosidase Suitable for Lactose-Free Milk Preparation

PubMed Central

Gomes-Pepe, Elisângela Soares; Machado Sierra, Elwi Guillermo; Pereira, Mariana Rangel; Castellane, Tereza Cristina Luque

2016-01-01

New ß-glucosidases with product (glucose) or ethanol tolerances are greatly desired to make industrial processes more marketable and efficient. Therefore, this report describes the in silico/vitro characterization of Bg10, a metagenomically derived homodimeric ß-glucosidase that exhibited a Vmax of 10.81 ± 0.43 μM min-1, Kcat of 175.1± 6.91 min-1, and Km of 0.49 ± 0.12 mM at a neutral pH and 37°C when pNP-ß-D-glucopyranoside was used as the substrate, and the enzyme retained greater than 80% activity within the respective pH and temperature ranges of 6.5 to 8.0 and 35 to 40°C. The enzyme was stimulated by its product, glucose; consequently, the Bg10 activity against 50 and 100 mM of glucose were increased by 36.8% and 22%, respectively, while half of the activity was retained at 350 mM. Moreover, the Bg10 was able to hydrolyse 55% (milk sample) and 100% (purified sugar) of the lactose at low (6°C) and optimum (37°C) temperatures, respectively, suggesting the possibility of further optimization of the reaction for lactose-free dairy production. In addition, the enzyme was able to fully hydrolyse 40 mM of cellobiose at one hour and was tolerant to ethanol up to concentrations of 500 mM (86% of activity), while a 1 M concentration still resulted in 41% residual activity, which could be interesting for biofuel production. PMID:28002476

Stress enhances the gene expression and enzyme activity of phenylalanine ammonia-lyase and the endogenous content of salicylic acid to induce flowering in pharbitis.

PubMed

Wada, Kaede C; Mizuuchi, Kaori; Koshio, Aya; Kaneko, Kentaro; Mitsui, Toshiaki; Takeno, Kiyotoshi

2014-07-01

The involvement of salicylic acid (SA) in the regulation of stress-induced flowering in the short-day plant pharbitis (also called Japanese morning glory) Ipomoea nil (formerly Pharbitis nil) was studied. Pharbitis cv. Violet was induced to flower when grown in 1/100-strength mineral nutrient solution under non-inductive long-day conditions. All fully expanded true leaves were removed from seedlings, leaving only the cotyledons, and flowering was induced under poor-nutrition stress conditions. This indicates that cotyledons can play a role in the regulation of poor-nutrition stress-induced flowering. The expression of the pharbitis homolog of PHENYLALANINE AMMONIA-LYASE, the enzyme activity of phenylalanine ammonia-lyase (PAL; E.C. 4.3.1.5) and the content of SA in the cotyledons were all up-regulated by the stress treatment. The Violet was also induced to flower by low-temperature stress, DNA demethylation and short-day treatment. Low-temperature stress enhanced PAL activity, whereas non-stress factors such as DNA demethylation and short-day treatment decreased the activity. The PAL enzyme activity was also examined in another cultivar, Tendan, obtaining similar results to Violet. The exogenously applied SA did not induce flowering under non-stress conditions but did promote flowering under weak stress conditions in both cultivars. These results suggest that stress-induced flowering in pharbitis is induced, at least partly, by SA, and the synthesis of SA is promoted by PAL. Copyright © 2014 Elsevier GmbH. All rights reserved.

Automated assay for screening the enzymatic release of reducing sugars from micronized biomass.

PubMed

Navarro, David; Couturier, Marie; da Silva, Gabriela Ghizzi Damasceno; Berrin, Jean-Guy; Rouau, Xavier; Asther, Marcel; Bignon, Christophe

2010-07-16

To reduce the production cost of bioethanol obtained from fermentation of the sugars provided by degradation of lignocellulosic biomass (i.e., second generation bioethanol), it is necessary to screen for new enzymes endowed with more efficient biomass degrading properties. This demands the set-up of high-throughput screening methods. Several methods have been devised all using microplates in the industrial SBS format. Although this size reduction and standardization has greatly improved the screening process, the published methods comprise one or more manual steps that seriously decrease throughput. Therefore, we worked to devise a screening method devoid of any manual steps. We describe a fully automated assay for measuring the amount of reducing sugars released by biomass-degrading enzymes from wheat-straw and spruce. The method comprises two independent and automated steps. The first step is the making of "substrate plates". It consists of filling 96-well microplates with slurry suspensions of micronized substrate which are then stored frozen until use. The second step is an enzymatic activity assay. After thawing, the substrate plates are supplemented by the robot with cell-wall degrading enzymes where necessary, and the whole process from addition of enzymes to quantification of released sugars is autonomously performed by the robot. We describe how critical parameters (amount of substrate, amount of enzyme, incubation duration and temperature) were selected to fit with our specific use. The ability of this automated small-scale assay to discriminate among different enzymatic activities was validated using a set of commercial enzymes. Using an automatic microplate sealer solved three main problems generally encountered during the set-up of methods for measuring the sugar-releasing activity of plant cell wall-degrading enzymes: throughput, automation, and evaporation losses. In its present set-up, the robot can autonomously process 120 triplicate wheat-straw samples per day. This throughput can be doubled if the incubation time is reduced from 24 h to 4 h (for initial rates measurements, for instance). This method can potentially be used with any insoluble substrate that is micronizable. A video illustrating the method can be seen at the following URL: http://www.youtube.com/watch?v=NFg6TxjuMWU.

Partial characterization of an atypical family I inorganic pyrophosphatase from cattle tick Rhipicephalus (Boophilus) microplus.

PubMed

Costa, Evenilton P; Campos, Eldo; de Andrade, Caroline P; Façanha, Arnoldo R; Saramago, Luiz; Masuda, Aoi; Vaz, Itabajara da Silva; Fernandez, Jorge H; Moraes, Jorge; Logullo, Carlos

2012-03-23

The present paper presents the partial characterization of a family I inorganic pyrophosphatase from the hard tick Rhipicephalus (Boophilus) microplus (BmPPase). The BmPPase gene was cloned from the tick embryo and sequenced. The deduced amino acid sequence shared high similarity with other eukaryotic PPases, on the other hand, BmPPase presented some cysteine residues non-conserved in other groups. This pyrophosphatase is inhibited by Ca(2+), and the inhibition is antagonized by Mg(2+), suggesting that the balance between free Ca(2+) and free Mg(2+) in the eggs could be involved in BmPPase activity control. We observed that the BmPPase transcripts are present in the fat body, midgut and ovary of ticks, in two developmental stages (partially and fully engorged females). However, higher transcription amounts were found in ovary from fully engorged females. BmPPase activity was considerably abolished by the thiol reagent dithionitrobenzoic acid (DTNB), suggesting that cysteine residues are exposed in its structure. Therefore, these cysteine residues play a critical role in the structural stability of BmPPase. Molecular dynamics simulation analysis indicates that BmPPase is the first Family I PPase that could promote disulfide bonds between cysteine residues 138-339 and 167-295. Finally, we believe that these cysteine residues exposed in the BmPPase structure can play an important controlling role regarding enzyme activity, which would be an interesting mechanism of redox control. The results presented here also indicate that this enzyme can be involved in embryogenesis of this arthropod, and may be useful as a target in the development of new tick control strategies. Published by Elsevier B.V.

Lack of complex I activity in human cells carrying a mutation in MtDNA-encoded ND4 subunit is corrected by the Saccharomyces cerevisiae NADH-quinone oxidoreductase (NDI1) gene.

PubMed

Bai, Y; Hájek, P; Chomyn, A; Chan, E; Seo, B B; Matsuno-Yagi, A; Yagi, T; Attardi, G

2001-10-19

The gene for the single subunit, rotenone-insensitive, and flavone-sensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1) can completely restore the NADH dehydrogenase activity in mutant human cells that lack the essential mitochondrial DNA (mtDNA)-encoded subunit ND4. In particular, the NDI1 gene was introduced into the nuclear genome of the human 143B.TK(-) cell line derivative C4T, which carries a homoplasmic frameshift mutation in the ND4 gene. Two transformants with a low or high level of expression of the exogenous gene were chosen for a detailed analysis. In these cells the corresponding protein is localized in mitochondria, its NADH-binding site faces the matrix compartment as in yeast mitochondria, and in perfect correlation with its abundance restores partially or fully NADH-dependent respiration that is rotenone-insensitive, flavone-sensitive, and antimycin A-sensitive. Thus the yeast enzyme has become coupled to the downstream portion of the human respiratory chain. Furthermore, the P:O ratio with malate/glutamate-dependent respiration in the transformants is approximately two-thirds of that of the wild-type 143B.TK(-) cells, as expected from the lack of proton pumping activity in the yeast enzyme. Finally, whereas the original mutant cell line C4T fails to grow in medium containing galactose instead of glucose, the high NDI1-expressing transformant has a fully restored capacity to grow in galactose medium. The present observations substantially expand the potential of the yeast NDI1 gene for the therapy of mitochondrial diseases involving complex I deficiency.

Two-electron Reduction versus One-electron Oxidation of the Type 3 Pair in the Multicopper Oxidases

PubMed Central

Kjaergaard, Christian H.; Jones, Stephen M.; Gounel, Sébastien; Mano, Nicolas; Solomon, Edward I.

2015-01-01

Multicopper Oxidases (MCOs) utilize an electron shuttling Type 1 Cu (T1) site in conjunction with a mononuclear Type 2 (T2) and a binuclear Type 3 (T3) site, arranged in a trinuclear copper cluster (TNC), to reduce O2 to H2O. Reduction of O2 occurs with limited overpotential indicating that all the coppers in the active site can be reduced via high-potential electron donors. Two forms of the resting enzyme have been observed in MCOs: the Alternative Resting form (AR), where only one of the three TNC Cu’s is oxidized, and the Resting Oxidized form (RO), where all three TNC Cu’s are oxidized. In contrast to the AR form, we show that in the RO form of a high-potential MCO, the binuclear T3 Cu(II) site can be reduced via the 700 mV T1 Cu. Systematic spectroscopic evaluation reveals that this proceeds by a two-electron process, where delivery of the first electron, forming a high energy, meta-stable half reduced T3 state, is followed by the rapid delivery of a second energetically favorable electron to fully reduce the T3 site. Alternatively, when this fully reduced binuclear T3 site is oxidized via the T1 Cu, a different thermodynamically favored half oxidized T3 form, i.e. the AR site, is generated. This behavior is evaluated by DFT calculations, which reveal that the protein backbone plays a significant role in controlling the environment of the active site coppers. This allows for the formation of the meta-stable, half reduced state and thus the complete reductive activation of the enzyme for catalysis. PMID:26075678

Exogenous thyroid hormones regulate the activity of citrate synthase and cytochrome c oxidase in warm- but not cold-acclimated lake whitefish (Coregonus clupeaformis)

USGS Publications Warehouse

Zak, Megan A.; Regish, Amy M.; McCormick, Stephen; Manzon, Richard G.

2017-01-01

Thermal acclimation is known to elicit metabolic adjustments in ectotherms, but the cellular mechanisms and endocrine control of these shifts have not been fully elucidated. Here we examined the relationship between thermal acclimation, thyroid hormones and oxidative metabolism in juvenile lake whitefish. Impacts of thermal acclimation above (19 °C) or below (8 °C) the thermal optimum (13 °C) and exposure to exogenous thyroid hormone (60 µg T4/g body weight) were assessed by quantifying citrate synthase and cytochrome c oxidase activities in liver, red muscle, white muscle and heart. Warm acclimation decreased citrate synthase activity in liver and elevated both citrate synthase and cytochrome c oxidase activities in red muscle. In contrast, induction of hyperthyroidism in warm-acclimated fish stimulated a significant increase in liver citrate synthase and heart cytochrome c oxidase activities, and a decrease in the activity of both enzymes in red muscle. No change in citrate synthase or cytochrome c oxidase activities was observed following cold acclimation in either the presence or absence of exogenous thyroid hormones. Collectively, our results indicate that thyroid hormones influence the activity of oxidative enzymes more strongly in warm-acclimated than in cold-acclimated lake whitefish, and they may play a role in mediating metabolic adjustments observed during thermal acclimation.

Exogenous thyroid hormones regulate the activity of citrate synthase and cytochrome c oxidase in warm- but not cold-acclimated lake whitefish (Coregonus clupeaformis).

PubMed

Zak, Megan A; Regish, Amy M; McCormick, Stephen D; Manzon, Richard G

2017-06-01

Thermal acclimation is known to elicit metabolic adjustments in ectotherms, but the cellular mechanisms and endocrine control of these shifts have not been fully elucidated. Here we examined the relationship between thermal acclimation, thyroid hormones and oxidative metabolism in juvenile lake whitefish. Impacts of thermal acclimation above (19°C) or below (8°C) the thermal optimum (13°C) and exposure to exogenous thyroid hormone (60µg T 4 /g body weight) were assessed by quantifying citrate synthase and cytochrome c oxidase activities in liver, red muscle, white muscle and heart. Warm acclimation decreased citrate synthase activity in liver and elevated both citrate synthase and cytochrome c oxidase activities in red muscle. In contrast, induction of hyperthyroidism in warm-acclimated fish stimulated a significant increase in liver citrate synthase and heart cytochrome c oxidase activities, and a decrease in the activity of both enzymes in red muscle. No change in citrate synthase or cytochrome c oxidase activities was observed following cold acclimation in either the presence or absence of exogenous thyroid hormones. Collectively, our results indicate that thyroid hormones influence the activity of oxidative enzymes more strongly in warm-acclimated than in cold-acclimated lake whitefish, and they may play a role in mediating metabolic adjustments observed during thermal acclimation. Copyright © 2017 Elsevier Inc. All rights reserved.

Endocannabinoids Stimulate Human Melanogenesis via Type-1 Cannabinoid Receptor*

PubMed Central

Pucci, Mariangela; Pasquariello, Nicoletta; Battista, Natalia; Di Tommaso, Monia; Rapino, Cinzia; Fezza, Filomena; Zuccolo, Michela; Jourdain, Roland; Finazzi Agrò, Alessandro; Breton, Lionel; Maccarrone, Mauro

2012-01-01

We show that a fully functional endocannabinoid system is present in primary human melanocytes (normal human epidermal melanocyte cells), including anandamide (AEA), 2-arachidonoylglycerol, the respective target receptors (CB1, CB2, and TRPV1), and their metabolic enzymes. We also show that at higher concentrations AEA induces normal human epidermal melanocyte apoptosis (∼3-fold over controls at 5 μm) through a TRPV1-mediated pathway that increases DNA fragmentation and p53 expression. However, at lower concentrations, AEA and other CB1-binding endocannabinoids dose-dependently stimulate melanin synthesis and enhance tyrosinase gene expression and activity (∼3- and ∼2-fold over controls at 1 μm). This CB1-dependent activity was fully abolished by the selective CB1 antagonist SR141716 or by RNA interference of the receptor. CB1 signaling engaged p38 and p42/44 mitogen-activated protein kinases, which in turn activated the cyclic AMP response element-binding protein and the microphthalmia-associated transcription factor. Silencing of tyrosinase or microphthalmia-associated transcription factor further demonstrated the involvement of these proteins in AEA-induced melanogenesis. In addition, CB1 activation did not engage the key regulator of skin pigmentation, cyclic AMP, showing a major difference compared with the regulation of melanogenesis by α-melanocyte-stimulating hormone through melanocortin 1 receptor. PMID:22431736

Pre-steady-state Kinetics Reveal the Substrate Specificity and Mechanism of Halide Oxidation of Truncated Human Peroxidasin 1*

PubMed Central

Paumann-Page, Martina; Katz, Romy-Sophie; Bellei, Marzia; Schwartz, Irene; Edenhofer, Eva; Sevcnikar, Benjamin; Soudi, Monika

2017-01-01

Human peroxidasin 1 is a homotrimeric multidomain peroxidase that is secreted to the extracellular matrix. The heme enzyme was shown to release hypobromous acid that mediates the formation of specific covalent sulfilimine bonds to reinforce collagen IV in basement membranes. Maturation by proteolytic cleavage is known to activate the enzyme. Here, we present the first multimixing stopped-flow study on a fully functional truncated variant of human peroxidasin 1 comprising four immunoglobulin-like domains and the catalytically active peroxidase domain. The kinetic data unravel the so far unknown substrate specificity and mechanism of halide oxidation of human peroxidasin 1. The heme enzyme is shown to follow the halogenation cycle that is induced by the rapid H2O2-mediated oxidation of the ferric enzyme to the redox intermediate compound I. We demonstrate that chloride cannot act as a two-electron donor of compound I, whereas thiocyanate, iodide, and bromide efficiently restore the ferric resting state. We present all relevant apparent bimolecular rate constants, the spectral signatures of the redox intermediates, and the standard reduction potential of the Fe(III)/Fe(II) couple, and we demonstrate that the prosthetic heme group is post-translationally modified and cross-linked with the protein. These structural features provide the basis of human peroxidasin 1 to act as an effective generator of hypobromous acid, which mediates the formation of covalent cross-links in collagen IV. PMID:28154175

Molybdenum Incorporation in Tungsten Aldehyde Oxidoreductase Enzymes from Pyrococcus furiosus▿ †

PubMed Central

Sevcenco, Ana-Maria; Bevers, Loes E.; Pinkse, Martijn W. H.; Krijger, Gerard C.; Wolterbeek, Hubert T.; Verhaert, Peter D. E. M.; Hagen, Wilfred R.; Hagedoorn, Peter-Leon

2010-01-01

The hyperthermophilic archaeon Pyrococcus furiosus expresses five aldehyde oxidoreductase (AOR) enzymes, all containing a tungsto-bispterin cofactor. The growth of this organism is fully dependent on the presence of tungsten in the growth medium. Previous studies have suggested that molybdenum is not incorporated in the active site of these enzymes. Application of the radioisotope 99Mo in metal isotope native radioautography in gel electrophoresis (MIRAGE) technology to P. furiosus shows that molybdenum can in fact be incorporated in all five AOR enzymes. Mo(V) signals characteristic for molybdopterin were observed in formaldehyde oxidoreductase (FOR) in electron paramagnetic resonance (EPR)-monitored redox titrations. Our finding that the aldehyde oxidation activity of FOR and WOR5 (W-containing oxidoreductase 5) correlates only with the residual tungsten content suggests that the Mo-containing AORs are most likely inactive. An observed W/Mo antagonism is indicative of tungstate-dependent negative feedback of the expression of the tungstate/molybdate ABC transporter. An intracellular selection mechanism for tungstate and molybdate processing has to be present, since tungsten was found to be preferentially incorporated into the AORs even under conditions with comparable intracellular concentrations of tungstate and molybdate. Under the employed growth conditions of starch as the main carbon source in a rich medium, no tungsten- and/or molybdenum-associated proteins are detected in P. furiosus other than the high-affinity transporter, the proteins of the metallopterin insertion machinery, and the five W-AORs. PMID:20562313

Pre-steady-state Kinetics Reveal the Substrate Specificity and Mechanism of Halide Oxidation of Truncated Human Peroxidasin 1.

PubMed

Paumann-Page, Martina; Katz, Romy-Sophie; Bellei, Marzia; Schwartz, Irene; Edenhofer, Eva; Sevcnikar, Benjamin; Soudi, Monika; Hofbauer, Stefan; Battistuzzi, Gianantonio; Furtmüller, Paul G; Obinger, Christian

2017-03-17

Human peroxidasin 1 is a homotrimeric multidomain peroxidase that is secreted to the extracellular matrix. The heme enzyme was shown to release hypobromous acid that mediates the formation of specific covalent sulfilimine bonds to reinforce collagen IV in basement membranes. Maturation by proteolytic cleavage is known to activate the enzyme. Here, we present the first multimixing stopped-flow study on a fully functional truncated variant of human peroxidasin 1 comprising four immunoglobulin-like domains and the catalytically active peroxidase domain. The kinetic data unravel the so far unknown substrate specificity and mechanism of halide oxidation of human peroxidasin 1. The heme enzyme is shown to follow the halogenation cycle that is induced by the rapid H 2 O 2 -mediated oxidation of the ferric enzyme to the redox intermediate compound I. We demonstrate that chloride cannot act as a two-electron donor of compound I, whereas thiocyanate, iodide, and bromide efficiently restore the ferric resting state. We present all relevant apparent bimolecular rate constants, the spectral signatures of the redox intermediates, and the standard reduction potential of the Fe(III)/Fe(II) couple, and we demonstrate that the prosthetic heme group is post-translationally modified and cross-linked with the protein. These structural features provide the basis of human peroxidasin 1 to act as an effective generator of hypobromous acid, which mediates the formation of covalent cross-links in collagen IV. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Oriented and selective enzyme immobilization on functionalized silica carrier using the cationic binding module Z basic2: design of a heterogeneous D-amino acid oxidase catalyst on porous glass.

PubMed

Bolivar, Juan M; Nidetzky, Bernd

2012-06-01

D-amino acid oxidase from Trigonopsis variabilis (TvDAO) is applied in industry for the synthesis of pharmaceutical intermediates. Because free TvDAO is extremely sensitive to exposure to gas-liquid interfaces, biocatalytic processing is usually performed with enzyme immobilizates that offer enhanced stability under bubble aeration. We herein present an "Immobilization by Design" approach that exploits engineered charge complementarity between enzyme and carrier to optimize key features of the immobilization of TvDAO. A fusion protein between TvDAO and the positively charged module Z(basic2) was generated, and a corresponding oppositely charged carrier was obtained by derivatization of mesoporous glass with 3-(trihydroxysilyl)-1-propane-sulfonic acid. Using 250 mM NaCl for charge screening at pH 7.0, the Z(basic2) fusion of TvDAO was immobilized directly from E. coli cell extract with almost absolute selectivity and full retention of catalytic effectiveness of the isolated enzyme in solution. Attachment of the homodimeric enzyme to the carrier was quasi-permanent in low-salt buffer but fully reversible upon elution with 5 M NaCl. Immobilized TvDAO was not sensitive to bubble aeration and received substantial (≥ tenfold) stabilization of the activity at 45°C as compared to free enzyme, suggesting immobilization via multisubunit oriented interaction of enzyme with the insoluble carrier. The Z(basic2) enzyme immobilizate was demonstrated to serve as re-usable heterogeneous catalyst for D-amino acid oxidation. Z(basic2) -mediated binding on a sulfonic acid group-containing glass carrier constitutes a generally useful strategy of enzyme immobilization that supports transition from case-specific empirical development to rational design. Copyright © 2012 Wiley Periodicals, Inc.

MicroRNAs, polyamines, and the activities antioxidant enzymes are associated with in vitro rooting in white pine (Pinus strobus L.).

PubMed

Fei, Yunjun; Xiao, Bo; Yang, Man; Ding, Qiong; Tang, Wei

2016-01-01

Molecular mechanism of in vitro rooting in conifer is not fully understood. After establishment of a regeneration procedure in eastern white pine (Pinus strobus L.) using mature embryos as explants to induce shoot formation on medium containing 3 μM IAA, 6 μM BA and 6 μM TDZ and induce root formation on medium containing 0.001-0.05 μM IAA, 0.001-0.05 μM IBA, 0.001-0.05 μM TDZ, we have investigated the changes of polyamine content and the activities of antioxidant enzymes during in vitro rooting in P. strobus. Our results demonstrated that putrescine (Put), spermidine (Spd), and spermine (Spm) did not increase in P. strobus during the first week of rooting on medium supplemented with 0.01 μM indole-3-acetic acid (IAA), whereas the levels of Put, Spd, and Spm increased during the 1st-3rd week of culture on medium with IAA, and then decreased on medium with IAA. No such a change in Put, Spd, and Spm was observed on medium without IAA. Measurement of antioxidant enzyme activity demonstrated that the activities of polyphenol oxidase, catalase, and peroxidase slightly increased in the first week of culture and reached to the highest peak in the 3rd-5th week of culture. Quantitative RT-PCR results indicated that miR160 was increased on the 7th day, miR162, miR397, and miR408 was increased from the 21th to 35th day, miR857 was increased on the 35th day, and miR827 was increased on the 49th day. These results demonstrated that enhanced polyamine biosynthesis, antioxidant enzyme activity, and microRNAs are correlated with the root induction and formation in P. strobus.

Metabolic and enzymatic changes associated with carbon mobilization, utilization and replenishment triggered in grain amaranth (Amaranthus cruentus) in response to partial defoliation by mechanical injury or insect herbivory

PubMed Central

2012-01-01

Background Amaranthus cruentus and A. hypochondriacus are crop plants grown for grain production in subtropical countries. Recently, the generation of large-scale transcriptomic data opened the possibility to study representative genes of primary metabolism to gain a better understanding of the biochemical mechanisms underlying tolerance to defoliation in these species. A multi-level approach was followed involving gene expression analysis, enzyme activity and metabolite measurements. Results Defoliation by insect herbivory (HD) or mechanical damage (MD) led to a rapid and transient reduction of non-structural carbohydrates (NSC) in all tissues examined. This correlated with a short-term induction of foliar sucrolytic activity, differential gene expression of a vacuolar invertase and its inhibitor, and induction of a sucrose transporter gene. Leaf starch in defoliated plants correlated negatively with amylolytic activity and expression of a β-amylase-1 gene and positively with a soluble starch synthase gene. Fatty-acid accumulation in roots coincided with a high expression of a phosphoenolpyruvate/phosphate transporter gene. In all tissues there was a long-term replenishment of most metabolite pools, which allowed damaged plants to maintain unaltered growth and grain yield. Promoter analysis of ADP-glucose pyrophosphorylase and vacuolar invertase genes indicated the presence of cis-regulatory elements that supported their responsiveness to defoliation. HD and MD had differential effects on transcripts, enzyme activities and metabolites. However, the correlation between transcript abundance and enzymatic activities was very limited. A better correlation was found between enzymes, metabolite levels and growth and reproductive parameters. Conclusions It is concluded that a rapid reduction of NSC reserves in leaves, stems and roots followed by their long-term recovery underlies tolerance to defoliation in grain amaranth. This requires the coordinate action of genes/enzymes that are differentially affected by the way leaf damage is performed. Defoliation tolerance in grain is a complex process that can’t be fully explained at the transcriptomic level only. PMID:22966837

Molecular cloning and functional expression of bovine spleen ecto-NAD+ glycohydrolase: structural identity with human CD38.

PubMed Central

Augustin, A; Muller-Steffner, H; Schuber, F

2000-01-01

Bovine spleen ecto-NAD(+) glycohydrolase, an archetypal member of the mammalian membrane-associated NAD(P)(+) glycohydrolase enzyme family (EC 3.2.2.6), displays catalytic features similar to those of CD38, i.e. a protein originally described as a lymphocyte differentiation marker involved in the metabolism of cyclic ADP-ribose and signal transduction. Using amino acid sequence information obtained from NAD(+) glycohydrolase and from a truncated and hydrosoluble form of the enzyme (hNADase) purified to homogeneity, a full-length cDNA clone was obtained. The deduced sequence indicates a protein of 278 residues with a molecular mass of 31.5 kDa. It predicts that bovine ecto-NAD(+) glycohydrolase is a type II transmembrane protein, with a very short intracellular tail. The bulk of the enzyme, which is extracellular and contains two potential N-glycosylation sites, yields the fully catalytically active hNADase which is truncated by 71 residues. Transfection of HeLa cells with the full-length cDNA resulted in the expression of the expected NAD(+) glycohydrolase, ADP-ribosyl cyclase and GDP-ribosyl cyclase activities at the surface of the cells. The bovine enzyme, which is the first 'classical' NAD(P)(+) glycohydrolase whose structure has been established, presents a particularly high sequence identity with CD38, including the presence of 10 strictly conserved cysteine residues in the ectodomain and putative catalytic residues. However, it lacks two otherwise conserved cysteine residues near its C-terminus. Thus hNADase, the truncated protein of 207 amino acids, represents the smallest functional domain endowed with all the catalytic activities of CD38/NAD(+) glycohydrolases so far identified. Altogether, our data strongly suggest that the cloned bovine spleen ecto-NAD(+) glycohydrolase is the bovine equivalent of CD38. PMID:10600637

Metabolism of the reserve polysaccharide of Streptococcus mitior (mitis): is there a second alpha-1,4-glucan phosphorylase?

PubMed Central

Pulkownik, A; Walker, G J

1976-01-01

The alpha-1,4-glucan phosphorylase (alpha-1,4-glucan: orthophosphate glucosyltransferase; EC 2.4.1.1) associated with the particulate cell fraction of Streptococcus mitior strain S3 was compared with the soluble maltodextrin phosphorylase that had been previously isolated from the same organism (Walker et al., 1969). The particulate enzyme was more sensitive to the glycogen content of the cell than the soluble euzyme; its activity was highest when the cells were grown under conditions favoring high glycogen storage. Substrate specificities of the two high activity towards endogenous glycogen, whereas low-molecular-weight maltodextrins were the preferred substrates for the soluble phosphorylase. The purification of the particulate phosphorylase included incubation of the particulate fraction in 160 mM sodium phosphate-10 mM sodium citrate-0.1% (wt/vol) Triton X-100 buffer (pH 6.7) and ion-exchange chromatography on diethylamino-ethyl- Sephadex A-50. The purified enzyme was fully soluble. The value for the purification factor was variable and depended on (i) the substrate used and (ii) whether the synthetic or the degradative reaction was being measured. The solubilization resulted in considerable changes in the properties of the phosphorylase: the pH optimum for activity was raised from 6.0 to 7.0-7.5 and the substrate specificity was altered. Consequently, the purified enzyme bore greater similarity to the soluble maltodextrin phosphorylase. The reported results are best explained in terms of a single phosphorylase, the specificity which is determind by its binding state in the cell. The enzyme acts as a glycogen phosphorylase in the particulate state and as a maltodextrin phosphorylase when soluble. The equilibrium between the two forms is related to the glycogen content of the cells. PMID:6434

Crystal structure of a chimaeric bacterial glutamate dehydrogenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oliveira, Tânia; Sharkey, Michael A.; Engel, Paul C.

2016-05-23

Glutamate dehydrogenases (EC 1.4.1.2–4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate using NAD(P) +as a cofactor. The bacterial enzymes are hexameric, arranged with 32 symmetry, and each polypeptide consists of an N-terminal substrate-binding segment (domain I) followed by a C-terminal cofactor-binding segment (domain II). The catalytic reaction takes place in the cleft formed at the junction of the two domains. Distinct signature sequences in the nucleotide-binding domain have been linked to the binding of NAD +versusNADP +, but they are not unambiguous predictors of cofactor preference. In the absence of substrate, the two domains move apart as rigid bodies,more » as shown by the apo structure of glutamate dehydrogenase fromClostridium symbiosum. Here, the crystal structure of a chimaeric clostridial/Escherichia colienzyme has been determined in the apo state. The enzyme is fully functional and reveals possible determinants of interdomain flexibility at a hinge region following the pivot helix. The enzyme retains the preference for NADP +cofactor from the parentE. colidomain II, although there are subtle differences in catalytic activity.« less

A null mutation in the first enzyme of flavonoid biosynthesis does not affect male fertility in Arabidopsis.

PubMed Central

Burbulis, I E; Iacobucci, M; Shirley, B W

1996-01-01

Flavonoids are a major class of secondary metabolites that serves a multitude of functions in higher plants, including a recently discovered role in male fertility. Surprisingly, Arabidopsis plants deficient in flavonoid biosynthesis appear to be fully fertile. Using RNA gel blot analysis and polymerase chain reaction-based assays, we have shown that a mutation at the 3' splice acceptor site in the Arabidopsis chalcone synthase gene completely disrupts synthesis of the active form of the enzyme. We also confirmed that this enzyme, which catalyzes the first step of flavonoid biosynthesis, is encoded by a single-copy gene. HPLC analysis of whole flowers and stamens was used to show that plants homozygous for the splice site mutation are completely devoid of flavonoids. This work provides compelling evidence that despite the high levels of these compounds in the pollen of most plant species, flavonoids are not universally required for fertility. The role of flavonoids in plant reproduction may therefore offer an example of convergent functional evolution in secondary metabolism. PMID:8672888

Post-transcriptional regulation tends to attenuate the mRNA noise and to increase the mRNA gain

NASA Astrophysics Data System (ADS)

Shi, Changhong; Wang, Shuqiang; Zhou, Tianshou; Jiang, Yiguo

2015-10-01

Post-transcriptional regulation is ubiquitous in prokaryotic and eukaryotic cells, but how it impacts gene expression remains to be fully explored. Here, we analyze a simple gene model in which we assume that mRNAs are produced in a constitutive manner but are regulated post-transcriptionally by a decapping enzyme that switches between the active state and the inactive state. We derive the analytical mRNA distribution governed by a chemical master equation, which can be well used to analyze the mechanism of how post-transcription regulation influences the mRNA expression level including the mRNA noise. We demonstrate that the mean mRNA level in the stochastic case is always higher than that in the deterministic case due to the stochastic effect of the enzyme, but the size of the increased part depends mainly on the switching rates between two enzyme states. More interesting is that we find that in contrast to transcriptional regulation, post-transcriptional regulation tends to attenuate noise in mRNA. Our results provide insight into the role of post-transcriptional regulation in controlling the transcriptional noise.

Phase II enzyme induction by a carotenoid, lutein, in a PC12D neuronal cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miyake, Seiji; Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582; Wakasa Seikatsu Co., Ltd., 134 Chudoujiminami-cho, Shimogyo-ku, Kyoto 600-8813

Highlights: • Lutein reduced ROS levels in a PC12D neuronal cell line. • Lutein induced mRNAs of phase II antioxidative enzymes in PC12D neuronal cells. • Lutein increased protein levels of HO-1, SOD2, and NQO-1 in PC12D neuronal cells. • Lutein had no effect on intranuclear Nrf2 levels in PC12D neuronal cells. • Lutein did not activate potential upstream Nrf2 nuclear translocation pathways. - Abstract: The mechanism by which lutein, a carotenoid, acts as an antioxidant in retinal cells is still not fully understood. Here, lutein treatment of a neuronal cell line (PC12D) immediately resulted in reduced intracellular ROS levels,more » implying that it has a direct role in ROS scavenging. Significantly, lutein treatment also induced phase II antioxidative enzyme expression, probably via a nuclear factor-like 2 (Nrf2) independent pathway. This latter mechanism could explain why lutein acts diversely to protect against oxidative/cytotoxic stress, and why it is physiologically involved in the human neural tissue, such as the retina.« less

Succinyl-CoA:(R)-Benzylsuccinate CoA-Transferase: an Enzyme of the Anaerobic Toluene Catabolic Pathway in Denitrifying Bacteria†

PubMed Central

Leutwein, Christina; Heider, Johann

2001-01-01

Anaerobic microbial toluene catabolism is initiated by addition of fumarate to the methyl group of toluene, yielding (R)-benzylsuccinate as first intermediate, which is further metabolized via β-oxidation to benzoyl-coenzyme A (CoA) and succinyl-CoA. A specific succinyl-CoA:(R)-benzylsuccinate CoA-transferase activating (R)-benzylsuccinate to the CoA-thioester was purified and characterized from Thauera aromatica. The enzyme is fully reversible and forms exclusively the 2-(R)-benzylsuccinyl-CoA isomer. Only some close chemical analogs of the substrates are accepted by the enzyme: succinate was partially replaced by maleate or methylsuccinate, and (R)-benzylsuccinate was replaced by methylsuccinate, benzylmalonate, or phenylsuccinate. In contrast to all other known CoA-transferases, the enzyme consists of two subunits of similar amino acid sequences and similar sizes (44 and 45 kDa) in an α2β2 conformation. Identity of the subunits with the products of the previously identified toluene-induced bbsEF genes was confirmed by determination of the exact masses via electrospray-mass spectrometry. The deduced amino acid sequences resemble those of only two other characterized CoA-transferases, oxalyl-CoA:formate CoA-transferase and (E)-cinnamoyl-CoA:(R)-phenyllactate CoA-transferase, which represent a new family of CoA-transferases. As suggested by kinetic analysis, the reaction mechanism of enzymes of this family apparently involves formation of a ternary complex between the enzyme and the two substrates. PMID:11418570

Unexpected Presence of Graminan- and Levan-Type Fructans in the Evergreen Frost-Hardy Eudicot Pachysandra terminalis (Buxaceae): Purification, Cloning, and Functional Analysis of a 6-SST/6-SFT Enzyme1[W

PubMed Central

Van den Ende, Wim; Coopman, Marlies; Clerens, Stefan; Vergauwen, Rudy; Le Roy, Katrien; Lammens, Willem; Van Laere, André

2011-01-01

About 15% of flowering plants accumulate fructans. Inulin-type fructans with β(2,1) fructosyl linkages typically accumulate in the core eudicot families (e.g. Asteraceae), while levan-type fructans with β(2,6) linkages and branched, graminan-type fructans with mixed linkages predominate in monocot families. Here, we describe the unexpected finding that graminan- and levan-type fructans, as typically occurring in wheat (Triticum aestivum) and barley (Hordeum vulgare), also accumulate in Pachysandra terminalis, an evergreen, frost-hardy basal eudicot species. Part of the complex graminan- and levan-type fructans as accumulating in vivo can be produced in vitro by a sucrose:fructan 6-fructosyltransferase (6-SFT) enzyme with inherent sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan 6-exohydrolase side activities. This enzyme produces a series of cereal-like graminan- and levan-type fructans from sucrose as a single substrate. The 6-SST/6-SFT enzyme was fully purified by classic column chromatography. In-gel trypsin digestion led to reverse transcription-polymerase chain reaction-based cDNA cloning. The functionality of the 6-SST/6-SFT cDNA was demonstrated after heterologous expression in Pichia pastoris. Both the recombinant and native enzymes showed rather similar substrate specificity characteristics, including peculiar temperature-dependent inherent 1-SST and fructan 6-exohydrolase side activities. The finding that cereal-type fructans accumulate in a basal eudicot species further confirms the polyphyletic origin of fructan biosynthesis in nature. Our data suggest that the fructan syndrome in P. terminalis can be considered as a recent evolutionary event. Putative connections between abiotic stress and fructans are discussed. PMID:21037113

Characterization of a beta-glycosidase highly active on disaccharides and of a beta-galactosidase from Tenebrio molitor midgut lumen.

PubMed

Ferreira, Alexandre H P; Terra, Walter R; Ferreira, Clélia

2003-02-01

The midgut of the yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae) larvae has four beta-glycosidases. The properties of two of these enzymes (betaGly1 and betaGly2) have been described elsewhere. In this paper, the characterization of the other two glycosidases (betaGly3 and betaGly4) is described. BetaGly3 has one active site, hydrolyzes disaccharides, cellodextrins, synthetic substrates and beta-glucosides produced by plants. The enzyme is inhibited by amygdalin, cellotriose, cellotetraose and cellopentaose in high concentrations, probably due to transglycosylation. betaGly3 hydrolyzes beta 1,4-glycosidic linkages with a catalytic rate independent of the substrate polymerization degree (k(int)) of 11.9 s(-1). Its active site is formed by four subsites, where subsites +1 and -1 bind glucose residues with higher affinity than subsite +2. The main role of betaGly3 seems to be disaccharide hydrolysis. BetaGly4 is a beta-galactosidase, since it has highest activity against beta-galactosides. It can also hydrolyze fucosides, but not glucosides, and has Triton X-100 as a non-essential activator (K(a)=15 microM, pH 4.5). betaGly4 has two active sites that can hydrolyze p-nitrophenyl beta-galactoside (NPbetaGal). The one hydrolyzing NPbetaGal with more efficiency is also active against methylumbellipheryl beta-D-galactoside and lactose. The other active site hydrolyzes NPbetaFucoside and binds NPbetaGal weakly. BetaGly4 hydrolyzes hydrophobic substrates with high catalytical efficiency and is able to bind octyl-beta-thiogalactoside in its active site with high affinity. The betaGly4 physiological role is supposed to be the hydrolysis of galactolipids that are found in membranes from vegetal tissues. As the enzyme has a hydrophobic site where Triton X-100 can bind, it might be activated by membrane lipids, thus becoming fully active only at the surface of cell membranes.

Constitutive expression of active microbial transglutaminase in Escherichia coli and comparative characterization to a known variant.

PubMed

Javitt, Gabe; Ben-Barak-Zelas, Zohar; Jerabek-Willemsen, Moran; Fishman, Ayelet

2017-02-28

Microbial transglutaminase (mTG) is a robust enzyme catalyzing the formation of an isopeptide bond between glutamine and lysine residues. It has found use in food applications, pharmaceuticals, textiles, and biomedicine. Overexpression of soluble and active mTG in E. coli has been limited due to improper protein folding and requirement for proteolytic cleavage of the pro-domain. Furthermore, to integrate mTG more fully industrially and academically, thermostable and solvent-stable variants may be imperative. A novel expression system constitutively producing active mTG was designed. Wild-type (WT) mTG and a S2P variant had similar expression levels, comparable to previous studies. Kinetic constants were determined by a glutamate dehydrogenase-coupled assay, and the S2P variant showed an increased affinity and a doubled enzyme efficiency towards Z-Gln-Gly. The melting temperature (T m ) of the WT was determined by intrinsic fluorescence measurements to be 55.8 ± 0.1 °C and of the S2P variant to be 56.3 ± 0.4 °C and 45.5 ± 0.1 °C, showing a moderately different thermostability profile. Stability in water miscible organic solvents was determined for both the WT and S2P variant. Of the solvents tested, incubation of mTG in isopropanol for 24 h at 4 °C showed the strongest stabilizing effect with mTG retaining 61 and 72% activity for WT and S2P respectively in 70% isopropanol. Both enzymes also showed an increased initial activity in the presence of organic solvents with the highest activity increase in 40% DMSO. Nevertheless, both enzymes were inactivated in 70% of all organic solvents tested. A constitutive expression system of active mTG in E. coli without downstream proteolytic cleavage processing was used for overexpression and characterization. High throughput techniques for testing thermostability and kinetics were useful in streamlining analysis and could be used in the future for quickly identifying beneficial mutants. Hitherto untested thermostability and stability of mTG in organic solvents was evaluated, which can pave the way for use of the enzyme in novel applications and processes.

Energy metabolism of cerebral mitochondria during aging, ischemia and post-ischemic recovery assessed by functional proteomics of enzymes.

PubMed

Villa, Roberto Federico; Gorini, Antonella; Ferrari, Federica; Hoyer, Siegfried

2013-12-01

Stroke is a leading cause of death and disability, but most of the therapeutic approaches failed in clinical trials. The energy metabolism alterations, due to marked ATP decline, are strongly related to stroke and, at present, their physiopathological roles are not fully understood. Thus, the aim of this study was to evaluate the effects of aging on ischemia-induced changes in energy mitochondrial transduction and the consequences on overall brain energy metabolism in an in vivo experimental model of complete cerebral ischemia of 15min duration and during post-ischemic recirculation after 1, 24, 48, 72 and 96h, in 1year "adult" and 2year-old "aged" rats. The maximum rate (Vmax) of citrate synthase, malate dehydrogenase, succinate dehydrogenase for Krebs' cycle; NADH-cytochrome c reductase and cytochrome oxidase for electron transfer chain (ETC) were assayed in non-synaptic "free" mitochondria and in two populations of intra-synaptic mitochondria, i.e., "light" and "heavy" mitochondria. The catalytic activities of enzymes markedly differ according to: (a) mitochondrial type (non-synaptic, intra-synaptic), (b) age, (c) acute effects of ischemia and (d) post-ischemic recirculation at different times. Enzyme activities changes are injury maturation events and strictly reflect the bioenergetic state of the tissue in each specific experimental condition respect to the energy demand, as shown by the comparative evaluation of the energy-linked metabolites and substrates content. Remarkably, recovery of mitochondrial function was more difficult for intra-synaptic mitochondria in "aged" rats, but enzyme activities of energy metabolism tended to normalize in all mitochondrial populations after 96h of recirculation. This observation is relevant for Therapy, indicating that mitochondrial enzymes may be important metabolic factors for the responsiveness of ischemic penumbra towards the restore of cerebral functions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Evidence for communality in the primary determinants of CYP74 catalysis and of structural similarities between CYP74 and classical mammalian P450 enzymes.

PubMed

Hughes, Richard K; Yousafzai, Faridoon K; Ashton, Ruth; Chechetkin, Ivan R; Fairhurst, Shirley A; Hamberg, Mats; Casey, Rod

2008-09-01

In silico structural analysis of CYP74C3, a membrane-associated P450 enzyme from the plant Medicago truncatula (barrel medic) with hydroperoxide lyase (HPL) specificity, showed that it had strong similarities to the structural folds of the classical microsomal P450 enzyme from rabbits (CYP2C5). It was not only the secondary structure predictions that supported the analysis but site directed mutagenesis of the substrate interacting residues was also consistent with it. This led us to develop a substrate-binding model of CYP74C3 which predicted three amino acid residues, N285, F287, and G288 located in the putative I-helix and distal haem pocket of CYP74C3 to be in close proximity to the preferred substrate 13-HPOTE. These residues were judged to be in equivalent positions to those identified in SRS-4 of CYP2C5. Significance of the residues and their relevance to the model were further assessed by site directed mutagenesis of the three residues followed by EPR spectroscopic and detailed kinetic investigations of the mutated proteins in the presence and absence of detergent. Although point mutation of the residues had no effect on the haem content of the mutated proteins, significant effects on the spin state equilibrium of the haem iron were noted. Kinetic effects of the mutations, which were investigated using three different substrates, were dramatic in nature. In the presence of detergent with the preferred substrate (13-HPOTE), the catalytic center activities and substrate binding affinities of the mutant proteins were reduced by a factor of 8-32 and 4-12, respectively, compared with wild-type--a two orders of magnitude reduction in catalytic efficiencies. We believe this is the first report where primary determinants of catalysis for any CYP74 enzyme, which are fully consistent with our model, have been identified. Our working model predicts that N285 is close enough to suggest that a hydrogen bond with the peroxy group of the enzyme substrate 13-HPOTE is warranted, whereas significance of F287 may arise from a strong hydrophobic interaction between the alkyl group(s) of the substrate and the phenyl ring of F287. We believe that G288 is crucial because of its size. Any other residue with a relatively bulky side chain will hinder the access of substrate to the active site. The effects of the mutations suggests that subtle protein conformational changes in the putative substrate-binding pocket regulate the formation of a fully active monomer-micelle complex with low-spin haem iron and that structural communication exists between the substrate- and micelle-binding sites of CYP74C3. Conservation in CYP74 sequence alignments suggests that N285, F287, and G288 in CYP74C3 and the equivalent residues at positions in other CYP74 enzymes are likely to be critical to catalysis. To support this we show that G324 in CYP74D4 (Arabidopsis AOS), equivalent to G288 in CYP74C3, is a primary determinant of positional specificity. We suggest that the overall structure of CYP74 enzymes is likely to be very similar to those described for classical P450 monooxygenase enzymes. 2008 Wiley-Liss, Inc.

Design of Stomach Acid-Stable and Mucin-Binding Enzyme Polymer Conjugates.

PubMed

Cummings, Chad S; Campbell, Alan S; Baker, Stefanie L; Carmali, Sheiliza; Murata, Hironobu; Russell, Alan J

2017-02-13

The reduced immunogenicity and increased stability of protein-polymer conjugates has made their use in therapeutic applications particularly attractive. However, the physicochemical interactions between polymer and protein, as well as the effect of this interaction on protein activity and stability, are still not fully understood. In this work, polymer-based protein engineering was used to examine the role of polymer physicochemical properties on the activity and stability of the chymotrypsin-polymer conjugates and their degree of binding to intestinal mucin. Four different chymotrypsin-polymer conjugates, each with the same polymer density, were synthesized using "grafting-from" atom transfer radical polymerization. The influence of polymer charge on chymotrypsin-polymer conjugate mucin binding, bioactivity, and stability in stomach acid was determined. Cationic polymers covalently attached to chymotrypsin showed high mucin binding, while zwitterionic, uncharged, and anionic polymers showed no mucin binding. Cationic polymers also increased chymotrypsin activity from pH 6-8, while zwitterionic polymers had no effect, and uncharged and anionic polymers decreased enzyme activity. Lastly, cationic polymers decreased the tendency of chymotrypsin to structurally unfold at extremely low pH, while uncharged and anionic polymers induced unfolding more quickly. We hypothesized that when polymers are covalently attached to the surface of a protein, the degree to which those polymers interact with the protein surface is the predominant determinant of whether the polymer will stabilize or inactivate the protein. Preferential interactions between the polymer and the protein lead to removal of water from the surface of the protein, and this, we believe, inactivates the enzyme.

Increased microglial catalase activity in multiple sclerosis grey matter.

PubMed

Gray, Elizabeth; Kemp, Kevin; Hares, Kelly; Redondo, Julianna; Rice, Claire; Scolding, Neil; Wilkins, Alastair

2014-04-22

Chronic demyelination, on-going inflammation, axonal loss and grey matter neuronal injury are likely pathological processes that contribute to disease progression in multiple sclerosis (MS). Although the precise contribution of each process and their aetiological substrates is not fully known, recent evidence has implicated oxidative damage as a major cause of tissue injury in MS. The degree of tissue injury caused by oxidative molecules, such as reactive oxygen species (ROS), is balanced by endogenous anti-oxidant enzymes which detoxify ROS. Understanding endogenous mechanisms which protect the brain against oxidative injury in MS is important, since enhancing anti-oxidant responses is a major therapeutic strategy for preventing irreversible tissue injury in the disease. Our aims were to determine expression and activity levels of the hydrogen peroxide-reducing enzyme catalase in MS grey matter (GM). In MS GM, a catalase enzyme activity was elevated compared to control GM. We measured catalase protein expression by immune dot-blotting and catalase mRNA by a real-time polymerase chain reaction (RT-PCR). Protein analysis studies showed a strong positive correlation between catalase and microglial marker IBA-1 in MS GM. In addition, calibration of catalase mRNA level with reference to the microglial-specific transcript AIF-1 revealed an increase in this transcript in MS. This was reflected by the extent of HLA-DR immunolabeling in MS GM which was significantly elevated compared to control GM. Collectively, these observations provide evidence that microglial catalase activity is elevated in MS grey matter and may be an important endogenous anti-oxidant defence mechanism in MS. Copyright © 2014 Elsevier B.V. All rights reserved.

Characterization of Paenibacillus sp. MBT213 Isolated from Raw Milk and Its Ability to Convert Ginsenoside Rb1 into Ginsenoside Rd from Panax ginseng

PubMed Central

Cho, Soo Hyun; Park, Young W; Song, Gyu Yong

2017-01-01

This study was conducted to isolate and characterize Paenibacillus sp. MBT213 possessing β-glucosidase activity from raw milk, and examine the enzymatic capacity on the hydrolysis of a major ginsenoside (Rb1). Strain MBT213 was found to have a high hydrolytic ability on ginsenoside Rb1 by Esculin Iron Agar test. 16S rDNA analysis revealed that MBT213 was Paenibacillu sp. Crude enzyme of MBT213 strain exhibited high conversion capacity on ginsenoside Rb1 into ginsenoside Rd proven by TLC and HPLC analyses. The API ZYM kit confirmed that Paenibacillu sp. MBT213 exerted higher β-glucosidase and β-galactosidase activity than other strains. Optimum pH and temperature for crude enzyme were found at 7.0 and 35°C in hydrolysis of ginsenoside Rb1. After 10 d of optimal reaction conditions for the crude enzyme, ginsenoside Rb1 fully converted to ginsenoside Rd. Ginseng roots (20%) were fermented for 14 d, and analyzed by HPLC showed that amount of ginsenoside Rb1 significantly decreased, while that of ginsenoside Rd was significantly increased. The study confirmed that the β-glucosidase produced by Paenibacillus sp. MBT213 can hydrolyze the major ginsenoside Rb1 and convert to Rd during fermentation of the ginseng. The β-glucosidase activity of this novel Paenibacillus sp. MBT213 strain may be utilized in development of variety of health foods, dairy foods and pharmaceutical products. PMID:29147097

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Xiangyu; Qin, Xiangjing; Chen, Lei

Glycyl-tRNA synthetase (GlyRS) is the enzyme that covalently links glycine to cognate tRNA for translation. It is of great interest because of its nonconserved quaternary structures, unique species-specific aminoacylation properties, and noncanonical functions in neurological diseases, but none of these is fully understood. We report two crystal structures of human GlyRS variants, in the free form and in complex with tRNA Gly respectively, and reveal new aspects of the glycylation mechanism. We discover that insertion 3 differs considerably in conformation in catalysis and that it acts like a "switch" and fully opens to allow tRNA to bind in a cross-subunitmore » fashion. The flexibility of the protein is supported by molecular dynamics simulation, as well as enzymatic activity assays. The biophysical and biochemical studies suggest that human GlyRS may utilize its flexibility for both the traditional function (regulate tRNA binding) and alternative functions (roles in diseases).« less

Alliinase and cysteine synthase transcription in developing garlic (Allium sativum L.) over time.

PubMed

Mitrová, Katarina; Svoboda, Pavel; Milella, Luigi; Ovesná, Jaroslava

2018-06-15

Garlic is a valuable source of healthy compounds, including secondary metabolites rich in sulphur such as cysteine sulphoxides (CSOs). Here, we present new qRT-PCR assays analysing the transcription of two genes encoding key enzymes in CSO biosynthetic pathways (cysteine synthase and alliinase) in developing garlic. We also identified a set of genes (ACT I, GAPDH, and TUB) to use as transcription normalisation controls. We showed that the (normalised) transcription of both enzymes was highest during sprouting and decreased significantly in fully developed leaves, which are the major CSO-producing organs. Transcriptional activity further declined at the end of the growing season. Different cultivars show similar sulphur metabolism gene expression when European garlics were compared to Chinese and American genotypes. The qRT-PCR assays presented are also suitable for investigating the effects of agricultural practices on CSO formation in garlic to satisfy consumer demands. Copyright © 2017. Published by Elsevier Ltd.

Cloning, expression, and characterization of bacterial L-arabinose 1-dehydrogenase involved in an alternative pathway of L-arabinose metabolism.

PubMed

Watanabe, Seiya; Kodaki, Tsutomu; Kodak, Tsutomu; Makino, Keisuke

2006-02-03

Azospirillum brasiliense converts L-arabinose to alpha-ketoglutarate via five hypothetical enzymatic steps. We purified and characterized L-arabinose 1-dehydrogenase (EC 1.1.1.46), catalyzing the conversion of L-arabinose to L-arabino-gamma-lactone as an enzyme responsible for the first step of this alternative pathway of L-arabinose metabolism. The purified enzyme preferred NADP+ to NAD+ as a coenzyme. Kinetic analysis revealed that the enzyme had high catalytic efficiency for both L-arabinose and D-galactose. The gene encoding L-arabinose 1-dehydrogenase was cloned using a partial peptide sequence of the purified enzyme and was overexpressed in Escherichia coli as a fully active enzyme. The enzyme consists of 308 amino acids and has a calculated molecular mass of 33,663.92 Da. The deduced amino acid sequence had some similarity to glucose-fructose oxidoreductase, D-xylose 1-dehydrogenase, and D-galactose 1-dehydrogenase. Site-directed mutagenesis revealed that the enzyme possesses unique catalytic amino acid residues. Northern blot analysis showed that this gene was induced by L-arabinose but not by D-galactose. Furthermore, a disruptant of the L-arabinose 1-dehydrogenase gene did not grow on L-arabinose but grew on D-galactose at the same growth rate as the wild-type strain. There was a partial gene for L-arabinose transport in the flanking region of the L-arabinose 1-dehydrogenase gene. These results indicated that the enzyme is involved in the metabolism of L-arabinose but not D-galactose. This is the first identification of a gene involved in an alternative pathway of L-arabinose metabolism in bacterium.

Iminosugar inhibitors of carbohydrate-active enzymes that underpin cereal grain germination and endosperm metabolism

PubMed Central

Andriotis, Vasilios M. E.; Rejzek, Martin; Rugen, Michael D.; Svensson, Birte; Smith, Alison M.; Field, Robert A.

2016-01-01

Starch is a major energy store in plants. It provides most of the calories in the human diet and, as a bulk commodity, it is used across broad industry sectors. Starch synthesis and degradation are not fully understood, owing to challenging biochemistry at the liquid/solid interface and relatively limited knowledge about the nature and control of starch degradation in plants. Increased societal and commercial demand for enhanced yield and quality in starch crops requires a better understanding of starch metabolism as a whole. Here we review recent advances in understanding the roles of carbohydrate-active enzymes in starch degradation in cereal grains through complementary chemical and molecular genetics. These approaches have allowed us to start dissecting aspects of starch degradation and the interplay with cell-wall polysaccharide hydrolysis during germination. With a view to improving and diversifying the properties and uses of cereal grains, it is possible that starch degradation may be amenable to manipulation through genetic or chemical intervention at the level of cell wall metabolism, rather than simply in the starch degradation pathway per se. PMID:26862201

Production of fibrolytic enzymes by Aspergillus japonicus C03 using agro-industrial residues with potential application as additives in animal feed.

PubMed

Facchini, Fernanda Dell Antonio; Vici, Ana Claudia; Reis, Victor Ricardo Amin; Jorge, João Atilio; Terenzi, Héctor Francisco; Reis, Ricardo Andrade; Polizeli, Maria de Lourdes Teixeira de Moraes

2011-03-01

Solid-state fermentation obtained from different and low-cost carbon sources was evaluated to endocellulases and endoxylanases production by Aspergillus japonicus C03. Regarding the enzymatic production the highest levels were observed at 30 °C, using soy bran added to crushed corncob or wheat bran added to sugarcane bagasse, humidified with salt solutions, and incubated for 3 days (xylanase) or 6 days (cellulase) with 70% relative humidity. Peptone improved the xylanase and cellulase activities in 12 and 29%, respectively. The optimum temperature corresponded to 60 °C and 50-55 °C for xylanase and cellulase, respectively, both having 4.0 as optimum pH. Xylanase was fully stable up to 40 °C, which is close to the rumen temperature. The enzymes were stable in pH 4.0-7.0. Cu++ and Mn++ increased xylanase and cellulase activities by 10 and 64%, respectively. A. japonicus C03 xylanase was greatly stable in goat rumen fluid for 4 h during in vivo and in vitro experiments.

Identification and characterization of a triacylglycerol lipase in Arabidopsis homologous to mammalian acid lipases.

PubMed

El-Kouhen, Karim; Blangy, Stéphanie; Ortiz, Emilia; Gardies, Anne-Marie; Ferté, Natalie; Arondel, Vincent

2005-11-07

Triacylglycerol (TAG) lipases have been thoroughly characterized in mammals and microorganisms. By contrast, very little is known on plant TAG lipases. An Arabidopsis cDNA called AtLip1 (At2g15230), which exhibits strong homology to lysosomal acid lipase, was found to drive the synthesis of an active TAG lipase when expressed in the baculovirus system. The lipase had a maximal activity at pH 6 and the specific activity was estimated to be about 45 micromol min(-1) mg(-1) protein using triolein as a substrate. Knock-out mutant analysis showed no phenotype during germination indicating that this enzyme is fully dispensable for TAG storage breakdown during germination. Northern blot analyses indicated that the transcript is present in all tissues tested.

Biochemical characterization and comparison of aspartylglucosaminidases secreted in venom of the parasitoid wasps Asobara tabida and Leptopilina heterotoma

PubMed Central

Coulette, Quentin; Lemauf, Séverine; Colinet, Dominique; Prévost, Geneviève; Anselme, Caroline; Poirié, Marylène

2017-01-01

Aspartylglucosaminidase (AGA) is a low-abundance intracellular enzyme that plays a key role in the last stage of glycoproteins degradation, and whose deficiency leads to human aspartylglucosaminuria, a lysosomal storage disease. Surprisingly, high amounts of AGA-like proteins are secreted in the venom of two phylogenetically distant hymenopteran parasitoid wasp species, Asobara tabida (Braconidae) and Leptopilina heterotoma (Cynipidae). These venom AGAs have a similar domain organization as mammalian AGAs. They share with them key residues for autocatalysis and activity, and the mature α- and β-subunits also form an (αβ)2 structure in solution. Interestingly, only one of these AGAs subunits (α for AtAGA and β for LhAGA) is glycosylated instead of the two subunits for lysosomal human AGA (hAGA), and these glycosylations are partially resistant to PGNase F treatment. The two venom AGAs are secreted as fully activated enzymes, they have a similar aspartylglucosaminidase activity and are both also efficient asparaginases. Once AGAs are injected into the larvae of the Drosophila melanogaster host, the asparaginase activity may play a role in modulating their physiology. Altogether, our data provide new elements for a better understanding of the secretion and the role of venom AGAs as virulence factors in the parasitoid wasps’ success. PMID:28742131

Effects of continuous hypoxia on energy metabolism in cultured cerebro-cortical neurons.

PubMed

Malthankar-Phatak, Gauri H; Patel, Anant B; Xia, Ying; Hong, Soonsun; Chowdhury, Golam M I; Behar, Kevin L; Orina, Isaac A; Lai, James C K

2008-09-10

Mechanisms underlying hypoxia-induced neuronal adaptation have not been fully elucidated. In the present study we investigated glucose metabolism and the activities of glycolytic and TCA cycle enzymes in cerebro-cortical neurons exposed to hypoxia (3 days in 1% of O2) or normoxia (room air). Hypoxia led to increased activities of LDH (194%), PK (90%), and HK (24%) and decreased activities of CS (15%) and GDH (34%). Neurons were incubated with [1-(13)C]glucose for 45 and 120 min under normoxic or hypoxic (120 min only) conditions and 13C enrichment determined in the medium and cell extract using 1H-{13C}-NMR. In hypoxia-treated neurons [3-(13)C]lactate release into the medium was 428% greater than in normoxia-treated controls (45-min normoxic incubation) and total flux through lactate was increased by 425%. In contrast glucose oxidation was reduced significantly in hypoxia-treated neurons, even when expressed relative to total cellular protein, which correlated with the reduced activities of the measured mitochondrial enzymes. The results suggest that surviving neurons adapt to prolonged hypoxia by up-regulation of glycolysis and down-regulation of oxidative energy metabolism, similar to certain other cell types. The factors leading to adaptation and survival for some neurons but not others remain to be determined.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gilbert, Nathaniel C.; Rui, Zhe; Neau, David B.

The enzyme 5-lipoxygenase (5-LOX) initiates biosynthesis of the proinflammatory leukotriene lipid mediators and, together with 15-LOX, is also required for synthesis of the anti-inflammatory lipoxins. The catalytic activity of 5-LOX is regulated through multiple mechanisms, including Ca{sup 2+}-targeted membrane binding and phosphorylation at specific serine residues. To investigate the consequences of phosphorylation at S663, we mutated the residue to the phosphorylation mimic Asp, providing a homogenous preparation suitable for catalytic and structural studies. The S663D enzyme exhibits robust 15-LOX activity, as determined by spectrophotometric and HPLC analyses, with only traces of 5-LOX activity remaining; synthesis of the anti-inflammatory lipoxin A4more » from arachidonic acid is also detected. The crystal structure of the S663D mutant in the absence and presence of arachidonic acid (in the context of the previously reported Stable-5-LOX) reveals substantial remodeling of helices that define the active site so that the once fully encapsulated catalytic machinery is solvent accessible. Our results suggest that phosphorylation of 5-LOX at S663 could not only down-regulate leukotriene synthesis but also stimulate lipoxin production in inflammatory cells that do not express 15-LOX, thus redirecting lipid mediator biosynthesis to the production of proresolving mediators of inflammation.« less

Agonistic Human Antibodies Binding to Lecithin-Cholesterol Acyltransferase Modulate High Density Lipoprotein Metabolism*

PubMed Central

Gunawardane, Ruwanthi N.; Fordstrom, Preston; Piper, Derek E.; Masterman, Stephanie; Siu, Sophia; Liu, Dongming; Brown, Mike; Lu, Mei; Tang, Jie; Zhang, Richard; Cheng, Janet; Gates, Andrew; Meininger, David; Chan, Joyce; Carlson, Tim; Walker, Nigel; Schwarz, Margrit; Delaney, John; Zhou, Mingyue

2016-01-01

Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouseTM platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease. PMID:26644477

Structural and Kinetic Insights Reveal That the Amino Acid Pair Gln-228/Asn-254 Modulates the Transfructosylating Specificity of Schwanniomyces occidentalis β-Fructofuranosidase, an Enzyme That Produces Prebiotics*

PubMed Central

Álvaro-Benito, Miguel; Sainz-Polo, M. Angela; González-Pérez, David; González, Beatriz; Plou, Francisco J.; Fernández-Lobato, María; Sanz-Aparicio, Julia

2012-01-01

Schwanniomyces occidentalis β-fructofuranosidase (Ffase) is a GH32 dimeric enzyme that releases fructose from the nonreducing end of various oligosaccharides and essential storage fructans such as inulin. It also catalyzes the transfer of a fructosyl unit to an acceptor producing 6-kestose and 1-kestose, prebiotics that stimulate the growth of bacteria beneficial for human health. We report here the crystal structure of inactivated Ffase complexed with fructosylnystose and inulin, which shows the intricate net of interactions keeping the substrate tightly bound at the active site. Up to five subsites were observed, the sugar unit located at subsite +3 being recognized by interaction with the β-sandwich domain of the adjacent subunit within the dimer. This explains the high activity observed against long substrates, giving the first experimental evidence of the direct role of a GH32 β-sandwich domain in substrate binding. Crucial residues were mutated and their hydrolase/transferase (H/T) activities were fully characterized, showing the involvement of the Gln-228/Asn-254 pair in modulating the H/T ratio and the type β(2–1)/β(2–6) linkage formation. We generated Ffase mutants with new transferase activity; among them, Q228V gives almost specifically 6-kestose, whereas N254T produces a broader spectrum product including also neokestose. A model for the mechanism of the Ffase transfructosylation reaction is proposed. The results contribute to an understanding of the molecular basis regulating specificity among GH-J clan members, which represent an interesting target for rational design of enzymes, showing redesigned activities to produce tailor-made fructooligosaccharides. PMID:22511773

Cloning of a phenol oxidase gene from Acremonium murorum and its expression in Aspergillus awamori.

PubMed

Gouka, R J; van der Heiden, M; Swarthoff, T; Verrips, C T

2001-06-01

Fungal multicopper oxidases have many potential industrial applications, since they perform reactions under mild conditions. We isolated a phenol oxidase from the fungus Acremonium murorum var. murorum that was capable of decolorizing plant chromophores (such as anthocyanins). This enzyme is of interest in laundry-cleaning products because of its broad specificity for chromophores. We expressed an A. murorum cDNA library in Saccharomyces cerevisiae and subsequently identified enzyme-producing yeast colonies based on their ability to decolor a plant chromophore. The cDNA sequence contained an open reading frame of 1,806 bp encoding an enzyme of 602 amino acids. The phenol oxidase was overproduced by Aspergillus awamori as a fusion protein with glucoamylase, cleaved in vivo, and purified from the culture broth by hydrophobic-interaction chromatography. The phenol oxidase is active at alkaline pH (the optimum for syringaldazine is pH 9) and high temperature (optimum, 60 degrees C) and is fully stable for at least 1 h at 60 degrees C under alkaline conditions. These characteristics and the high production level of 0.6 g of phenol oxidase per liter in shake flasks, which is equimolar with the glucoamylase protein levels, make this enzyme suitable for use in processes that occur under alkaline conditions, such as laundry cleaning.

Cloning of a Phenol Oxidase Gene from Acremonium murorum and Its Expression in Aspergillus awamori

PubMed Central

Gouka, Robin J.; van der Heiden, Monique; Swarthoff, Ton; Verrips, C. Theo

2001-01-01

Fungal multicopper oxidases have many potential industrial applications, since they perform reactions under mild conditions. We isolated a phenol oxidase from the fungus Acremonium murorum var. murorum that was capable of decolorizing plant chromophores (such as anthocyanins). This enzyme is of interest in laundry-cleaning products because of its broad specificity for chromophores. We expressed an A. murorum cDNA library in Saccharomyces cerevisiae and subsequently identified enzyme-producing yeast colonies based on their ability to decolor a plant chromophore. The cDNA sequence contained an open reading frame of 1,806 bp encoding an enzyme of 602 amino acids. The phenol oxidase was overproduced by Aspergillus awamori as a fusion protein with glucoamylase, cleaved in vivo, and purified from the culture broth by hydrophobic-interaction chromatography. The phenol oxidase is active at alkaline pH (the optimum for syringaldazine is pH 9) and high temperature (optimum, 60°C) and is fully stable for at least 1 h at 60°C under alkaline conditions. These characteristics and the high production level of 0.6 g of phenol oxidase per liter in shake flasks, which is equimolar with the glucoamylase protein levels, make this enzyme suitable for use in processes that occur under alkaline conditions, such as laundry cleaning. PMID:11375170

Synthesis and in silico studies of novel sulfonamides having oxadiazole ring: As β-glucuronidase inhibitors.

PubMed

Taha, Muhammad; Baharudin, Mohd Syukri; Ismail, Nor Hadiani; Selvaraj, Manikandan; Salar, Uzma; Alkadi, Khaled A A; Khan, Khalid Mohammed

2017-04-01

Novel sulfonamides having oxadiazole ring were synthesized by multistep reaction and evaluated to check in vitro β-glucuronidase inhibitory activity. Luckily, except compound 13, all compounds were found to demonstrate good inhibitory activity in the range of IC 50 =2.40±0.01-58.06±1.60μM when compared to the standard d-saccharic acid 1,4-lactone (IC 50 =48.4±1.25μM). Structure activity relationship was also presented. However, in order to ensure the SAR as well as the molecular interactions of compounds with the active site of enzyme, molecular docking studies on most active compounds 19, 16, 4 and 6 was carried out. All derivatives were fully characterized by 1 H NMR, 13 C NMR and EI-MS spectroscopic techniques. CHN analysis was also presented. Copyright © 2017 Elsevier Inc. All rights reserved.

Positively charged mini-protein Zbasic2 as a highly efficient silica binding module: opportunities for enzyme immobilization on unmodified silica supports.

PubMed

Bolivar, Juan M; Nidetzky, Bernd

2012-07-03

Silica is a highly attractive support material for protein immobilization in a wide range of biotechnological and biomedical-analytical applications. Without suitable derivatization, however, the silica surface is not generally usable for attachment of proteins. We show here that Z(basic2) (a three α-helix bundle mini-protein of 7 kDa size that exposes clustered positive charges from multiple arginine residues on one side) functions as highly efficient silica binding module (SBM), allowing chimeras of target protein with SBM to become very tightly attached to underivatized glass at physiological pH conditions. We used two enzymes, d-amino acid oxidase and sucrose phosphorylase, to demonstrate direct immobilization of Z(basic2) protein from complex biological samples with extremely high selectivity. Immobilized enzymes displayed full biological activity, suggesting that their binding to the glass surface had occurred in a preferred orientation via the SBM. We also show that charge complementarity was the main principle of affinity between SBM and glass surface, and Z(basic2) proteins were bound in a very strong, yet fully reversible manner, presumably through multipoint noncovalent interactions. Z(basic2) proteins were immobilized on porous glass in a loading of 30 mg protein/g support or higher, showing that attachment via the SBM combines excellent binding selectivity with a technically useful binding capacity. Therefore, Z(basic2) and silica constitute a fully orthogonal pair of binding module and insoluble support for oriented protein immobilization, and this opens up new opportunities for the application of silica-based materials in the development of supported heterogeneous biocatalysts.

Improved analysis of C4 and C3 photosynthesis via refined in vitro assays of their carbon fixation biochemistry

PubMed Central

Sharwood, Robert E.; Sonawane, Balasaheb V.; Ghannoum, Oula; Whitney, Spencer M.

2016-01-01

Plants operating C3 and C4 photosynthetic pathways exhibit differences in leaf anatomy and photosynthetic carbon fixation biochemistry. Fully understanding this underpinning biochemical variation is requisite to identifying solutions for improving photosynthetic efficiency and growth. Here we refine assay methods for accurately measuring the carboxylase and decarboxylase activities in C3 and C4 plant soluble protein. We show that differences in plant extract preparation and assay conditions are required to measure NADP-malic enzyme and phosphoenolpyruvate carboxylase (pH 8, Mg2+, 22 °C) and phosphoenolpyruvate carboxykinase (pH 7, >2mM Mn2+, no Mg2+) maximal activities accurately. We validate how the omission of MgCl2 during leaf protein extraction, lengthy (>1min) centrifugation times, and the use of non-pure ribulose-1,5-bisphosphate (RuBP) significantly underestimate Rubisco activation status. We show how Rubisco activation status varies with leaf ontogeny and is generally lower in mature C4 monocot leaves (45–60% activation) relative to C3 monocots (55–90% activation). Consistent with their >3-fold lower Rubisco contents, full Rubisco activation in soluble protein from C4 leaves (<5min) was faster than in C3 plant samples (<10min), with addition of Rubisco activase not required for full activation. We conclude that Rubisco inactivation in illuminated leaves primarily stems from RuBP binding to non-carbamylated enzyme, a state readily reversible by dilution during cellular protein extraction. PMID:27122573

Structural and computational dissection of the catalytic mechanism of the inorganic pyrophosphatase from Mycobacterium tuberculosis.

PubMed

Pratt, Andrew C; Dewage, Sajeewa W; Pang, Allan H; Biswas, Tapan; Barnard-Britson, Sandra; Cisneros, G Andrés; Tsodikov, Oleg V

2015-10-01

Family I inorganic pyrophosphatases (PPiases) are ubiquitous enzymes that are critical for phosphate metabolism in all domains of life. The detailed catalytic mechanism of these enzymes, including the identity of the general base, is not fully understood. We determined a series of crystal structures of the PPiase from Mycobacterium tuberculosis (Mtb PPiase) bound to catalytic metals, inorganic pyrophosphate (PPi; the reaction substrate) and to one or two inorganic phosphate ions (Pi; the reaction product), ranging in resolution from 1.85 to 3.30Å. These structures represent a set of major kinetic intermediates in the catalytic turnover pathway for this enzyme and suggest an order of association and dissociation of the divalent metals, the substrate and the two products during the catalytic turnover. The active site of Mtb PPiase exhibits significant structural differences from the well characterized Escherichia coli PPiase in the vicinity of the bound PPi substrate. Prompted by these differences, quantum mechanics/molecular mechanics (QM/MM) analysis yielded an atomic description of the hydrolysis step for Mtb PPiase and, unexpectedly, indicated that Asp89, rather than Asp54 that was proposed for E. coli PPiase, can abstract a proton from a water molecule to activate it for a nucleophilic attack on the PPi substrate. Mutagenesis studies of the key Asp residues of Mtb PPiase supported this mechanism. This combination of structural and computational analyses clarifies our understanding of the mechanism of family I PPiases and has potential utility for rational development of drugs targeting this enzyme. Copyright © 2015 Elsevier Inc. All rights reserved.

Reaching for mechanistic consensus across life kingdoms: structure and insights into catalysis of the myo-inositol-1-phosphate synthase (mIPS) from Archaeoglobus fulgidus.

PubMed

Stieglitz, Kimberly A; Yang, Hongying; Roberts, Mary F; Stec, Boguslaw

2005-01-11

myo-Inositol-1-phosphate synthase (mIPS) catalyzes the first step in the synthesis of l-myo-inositol-1-phosphate. We have solved and refined the structure of the mIPS from the hyperthermophilic sulfate reducer Archaeoglobus fulgidus at 1.9 A resolution. The enzyme crystallized from poly(ethylene glycol) in the P1 space group with one tetramer in the asymmetric unit and provided a view of the entire biologically active oligomer. Despite significant changes in sequence length and amino acid composition, the general architecture of the archaeal enzyme is similar to that of the eukaryotic mIPS from Saccharomyces cerevisiae and bacterial mIPS from Mycobacterium tuberculosis. The enhanced thermostability of the archaeal enzyme as compared to that from yeast is consistent with deletion of a number of surface loops that results in a significantly smaller protein. In the structure of the A. fulgidus mIPS, the active sites of all four subunits were fully ordered and contained NAD(+) and inorganic phosphate. The structure also contained a single metal ion (identified as K(+)) in two of the four subunits. The analysis of the electrostatic potential maps of the protein suggested the presence of a second metal-ion-binding site in close proximity to the first metal ion and NAD(+). The modeling of the substrate and known inhibitors suggests a critical role for the second metal ion in catalysis and provides insights into the common elements of the catalytic cycle in enzymes from different life kingdoms.

Development of a fully integrated falling film microreactor for gas-liquid-solid biotransformation with surface immobilized O2 -dependent enzyme.

PubMed

Bolivar, Juan M; Krämer, Christina E M; Ungerböck, Birgit; Mayr, Torsten; Nidetzky, Bernd

2016-09-01

Microstructured flow reactors are powerful tools for the development of multiphase biocatalytic transformations. To expand their current application also to O2 -dependent enzymatic conversions, we have implemented a fully integrated falling film microreactor that provides controllable countercurrent gas-liquid phase contacting in a multi-channel microstructured reaction plate. Advanced non-invasive optical sensing is applied to measure liquid-phase oxygen concentrations in both in- and out-flow as well as directly in the microchannels (width: 600 μm; depth: 200 μm). Protein-surface interactions are designed for direct immobilization of catalyst on microchannel walls. Target enzyme (here: d-amino acid oxidase) is fused to the positively charged mini-protein Zbasic2 and the channel surface contains a negatively charged γ-Al2 O3 wash-coat layer. Non-covalent wall attachment of the chimeric Zbasic2 _oxidase resulted in fully reversible enzyme immobilization with fairly uniform surface coverage and near complete retention of biological activity. The falling film at different gas and liquid flow rates as well as reactor inclination angles was shown to be mostly wavy laminar. The calculated film thickness was in the range 0.5-1.3 × 10(-4)  m. Direct O2 concentration measurements at the channel surface demonstrated that the liquid side mass transfer coefficient (KL ) for O2 governed the overall gas/liquid/solid mass transfer and that the O2 transfer rate (≥0.75 mM · s(-1) ) vastly exceeded the maximum enzymatic reaction rate in a wide range of conditions. A value of 7.5 (±0.5) s(-1) was determined for the overall mass transfer coefficient KL a, comprising a KL of about 7 × 10(-5)  m · s(-1) and a specific surface area of up to 10(5)  m(-1) . Biotechnol. Bioeng. 2016;113: 1862-1872. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Trichoderma harzianum T-78 supplementation of compost stimulates the antioxidant defence system in melon plants.

PubMed

Bernal-Vicente, Agustina; Pascual, José A; Tittarelli, Fabio; Hernández, José A; Diaz-Vivancos, Pedro

2015-08-30

Compost is emerging as an alternative plant growing medium in efforts to achieve more sustainable agriculture. The addition of specific microorganisms such as Trichoderma harzianum to plant growth substrates increases yields and reduces plant diseases, but the mechanisms of such biostimulants and the biocontrol effects are not yet fully understood. In this work we investigated how the addition of citrus and vineyard composts, either alone or in combination with T. harzianum T-78, affects the antioxidant defence system in melon plants under nursery conditions. Compost application and/or Trichoderma inoculation modulated the antioxidant defence system in melon plants. The combination of citrus compost and Trichoderma showed a biostimulant effect that correlated with an increase in ascorbate recycling enzymes (monodehydroascorbate reductase, dehydroascorbate reductase) and peroxidase. Moreover, the inoculation of both composts with Trichoderma increased the activity of antioxidant enzymes, especially those involved in ascorbate recycling. Based on the long-established relationship between ascorbic acid and plant defence responses as well as plant growth and development, it can be suggested that ascorbate recycling activities play a major role in the protection provided by Trichoderma and its biostimulant effect and that these outcomes are linked to increases in antioxidant enzymes. We can conclude that the combination of citrus compost and T. harzianum T-78 constitutes a viable, environmentally friendly strategy for improving melon plant production. © 2014 Society of Chemical Industry.

Enzyme dynamics and engineering: one step at a time.

PubMed

Tokuriki, Nobuhiko; Jackson, Colin J

2014-10-23

Although protein dynamics are accepted as being essential for enzyme function, their effects are not fully understood. In this issue of Chemistry and Biology, Gobeil and coworkers describe how engineered changes in the millisecond motions of a mutant TEM-1 β-lactamase do not significantly affect substrate turnover. This mutational robustness has implications for protein engineering and design strategies.

Phospholipase D function in Saccharomyces cerevisiae.

PubMed

Mendonsa, Rima; Engebrecht, JoAnne

2009-09-01

Phosphatidylinositol 4,5-bisphosphate-regulated phosphatidylcholine-specific phospholipase D is conserved from yeast to man. The essential role of this enzyme in yeast is to mediate the fusion of Golgi and endosome-derived vesicles to generate the prospore membrane during the developmental program of sporulation, through the production of the fusogenic lipid phosphatidic acid. In addition to recruiting proteins required for fusion, phosphatidic acid is believed to lower the energy barrier to stimulate membrane curvature. During mitotic growth, phospholipase D activity is dispensable unless the major phosphatidylinositol/phosphatidylcholine transfer protein is absent; it also appears to play a nonessential role in the mating signal transduction pathway. The regulation of phospholipase D activity during both sporulation and mitotic growth is still not fully understood and awaits further characterization.

Metal organic frameworks for enzyme immobilization in biofuel cells

NASA Astrophysics Data System (ADS)

Bodell, JaDee

Interest in biofuel cells has been rapidly expanding as an ever-growing segment of the population gains access to electronic devices. The largest areas of growth for new populations using electronic devices are often in communities without electrical infrastructure. This lack of infrastructure in remote environments is one of the key driving factors behind the development of biofuel cells. Biofuel cells employ biological catalysts such as enzymes to catalyze oxidation and reduction reactions of select fuels to generate power. There are several benefits to using enzymes to catalyze reactions as compared to traditional fuel cells which use metal catalysts. First, enzymes are able to catalyze reactions at or near room temperature, whereas traditional metal catalysts are only efficient at very high temperatures. Second, biofuel cells can operate under mild pH conditions which is important for the eventual design of safe, commercially viable devices. Also, biofuel cells allow for implantable and flexible technologies. Finally, enzymes exhibit high selectivity and can be combined to fully oxidize or reduce the fuel which can generate several electrons from a single molecule of fuel, increasing the overall device efficiency. One of the main challenges which persist in biofuel cells is the instability of enzymes over time which tend to denature after hours or days. For a viable commercial biofuel cell to be produced, the stability of enzymes must be extended to months or years. Enzymes have been shown to have improved stability after being immobilized. The focus of this research was to find a metal organic framework (MOF) structure which could successfully immobilize enzymes while still allowing for electron transport to occur between the catalytic center of the enzyme and the electrode surface within a biofuel cell for power generation. Four MOF structures were successfully synthesized and were subsequently tested to determine the MOF's ability to immobilize the following enzymes: nicotinamide adenine dinucleotide (NAD)-dependent alcohol and aldehyde dehydrogenases, and pyrroloquinoline quinone (PQQ)-dependent alcohol and aldehyde dehydrogenases, as well as flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase. Tb-meso MOF was shown to immobilize PQQ-dependent enzymes through ? stacking interactions of the heme in the enzyme and the triazine molecules in the ligand of the MOF. However, the PQQ-dependent dehydrogenases did not have enough catalytic activity present to be measured electrochemically. Finally, ZIF-90 was synthesized under aqueous conditions in the presence of FAD-dependent glucose dehydrogenase (GDH) which led to size selective sheltering of FAD-GDH. FAD-GDH had activity an order of magnitude larger than any of the alcohol dehydrogenases, which provided sufficient catalytic activity to measure electrochemically. The FAD-GDH bound within ZIF-90 was used to build a full biofuel cell resulting in an open circuit voltage of 708 +/- 16 mV and a maximum power density of 2.75 +/- 0.40 microW/cm2.

Automated assay for screening the enzymatic release of reducing sugars from micronized biomass

PubMed Central

2010-01-01

Background To reduce the production cost of bioethanol obtained from fermentation of the sugars provided by degradation of lignocellulosic biomass (i.e., second generation bioethanol), it is necessary to screen for new enzymes endowed with more efficient biomass degrading properties. This demands the set-up of high-throughput screening methods. Several methods have been devised all using microplates in the industrial SBS format. Although this size reduction and standardization has greatly improved the screening process, the published methods comprise one or more manual steps that seriously decrease throughput. Therefore, we worked to devise a screening method devoid of any manual steps. Results We describe a fully automated assay for measuring the amount of reducing sugars released by biomass-degrading enzymes from wheat-straw and spruce. The method comprises two independent and automated steps. The first step is the making of "substrate plates". It consists of filling 96-well microplates with slurry suspensions of micronized substrate which are then stored frozen until use. The second step is an enzymatic activity assay. After thawing, the substrate plates are supplemented by the robot with cell-wall degrading enzymes where necessary, and the whole process from addition of enzymes to quantification of released sugars is autonomously performed by the robot. We describe how critical parameters (amount of substrate, amount of enzyme, incubation duration and temperature) were selected to fit with our specific use. The ability of this automated small-scale assay to discriminate among different enzymatic activities was validated using a set of commercial enzymes. Conclusions Using an automatic microplate sealer solved three main problems generally encountered during the set-up of methods for measuring the sugar-releasing activity of plant cell wall-degrading enzymes: throughput, automation, and evaporation losses. In its present set-up, the robot can autonomously process 120 triplicate wheat-straw samples per day. This throughput can be doubled if the incubation time is reduced from 24 h to 4 h (for initial rates measurements, for instance). This method can potentially be used with any insoluble substrate that is micronizable. A video illustrating the method can be seen at the following URL: http://www.youtube.com/watch?v=NFg6TxjuMWU PMID:20637080

The pentose phosphate pathway of glucose metabolism. Hormonal and dietary control of the oxidative and non-oxidative reactions of the cycle in liver

PubMed Central

Novello, F.; Gumaa, J. A.; McLean, Patricia

1969-01-01

1. Measurements were made of the non-oxidative reactions of the pentose phosphate cycle in liver (transketolase, transaldolase, ribulose 5-phosphate epimerase and ribose 5-phosphate isomerase activities) in a variety of hormonal and nutritional conditions. In addition, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were measured for comparison with the oxidative reactions of the cycle; hexokinase, glucokinase and phosphoglucose isomerase activities were also included. Starvation for 2 days caused significant lowering of activity of all the enzymes of the pentose phosphate cycle based on activity in the whole liver. Re-feeding with a high-carbohydrate diet restored all the enzyme activities to the range of the control values with the exception of that of glucose 6-phosphate dehydrogenase, which showed the well-known `overshoot' effect. Re-feeding with a high-fat diet also restored the activities of all the enzymes of the pentose phosphate cycle and of hexokinase; glucokinase activity alone remained unchanged. Expressed as units/g. of liver or units/mg. of protein hexokinase, glucose 6-phosphate dehydrogenase, transketolase and pentose phosphate isomerase activities were unchanged by starvation; both 6-phosphogluconate dehydrogenase and ribulose 5-phosphate epimerase activities decreased faster than the liver weight or protein content. 2. Alloxan-diabetes resulted in a decrease of approx. 30–40% in the activities of 6-phosphogluconate dehydrogenase, ribose 5-phosphate isomerase, ribulose 5-phosphate epimerase and transketolase; in contrast with this glucose 6-phosphate dehydrogenase, transaldolase and phosphoglucose isomerase activities were unchanged. Treatment of alloxan-diabetic rats with protamine–zinc–insulin for 3 days caused a very marked increase to above normal levels of activity in all the enzymes of the pentose phosphate pathway except ribulose 5-phosphate epimerase, which was restored to the control value. Hexokinase activity was also raised by this treatment. After 7 days treatment of alloxan-diabetic rats with protamine–zinc–insulin the enzyme activities returned towards the control values. 3. In adrenalectomized rats the two most important changes were the rise in hexokinase activity and the fall in transketolase activity; in addition, ribulose 5-phosphate epimerase activity was also decreased. These effects were reversed by cortisone treatment. In addition, in cortisone-treated adrenalectomized rats glucokinase activity was significantly lower than the control value. 4. In thyroidectomized rats both ribose 5-phosphate isomerase and transketolase activities were decreased; in contrast with this transaldolase activity did not change significantly. Hypophysectomy caused a 50% fall in transketolase activity that was partially reversed by treatment with thyroxine and almost fully reversed by treatment with growth hormone for 8 days. 5. The results are discussed in relation to the hormonal control of the non-oxidative reactions of the pentose phosphate cycle, the marked changes in transketolase activity being particularly outstanding. PMID:5791534

Influence of sulfur oxyanions on reductive dehalogenation activities in Desulfomonile tiedjei.

PubMed Central

Townsend, G T; Suflita, J M

1997-01-01

The inhibition of aryl reductive dehalogenation reactions by sulfur oxyanions has been demonstrated in environmental samples, dehalogenating enrichments, and the sulfate-reducing bacterium Desulfomonile tiedjei; however, this phenomenon is not well understood. We examined the effects of sulfate, sulfite, and thiosulfate on reductive dehalogenation in the model microorganism D. tiedjei and found separate mechanisms of inhibition due to these oxyanions under growth versus nongrowth conditions. Dehalogenation activity was greatly reduced in extracts of cells grown in the presence of both 3-chlorobenzoate, the substrate or inducer for the aryl dehalogenation activity, and either sulfate, sulfite, or thiosulfate, indicating that sulfur oxyanions repress the requisite enzymes. In extracts of fully induced cells, thiosulfate and sulfite, but not sulfate, were potent inhibitors of aryl dehalogenation activity even in membrane fractions lacking the cytoplasmically located sulfur oxyanion reductase. These results suggest that under growth conditions, sulfur oxyanions serve as preferred electron acceptors and negatively influence dehalogenation activity in D. tiedjei by regulating the amount of active aryl dehalogenase in cells. Additionally, in vitro inhibition by sulfur oxyanions is due to the interaction of the reactive species with enzymes involved in dehalogenation and need not involve competition between two respiratory processes for reducing equivalents. Sulfur oxyanions also inhibited tetrachloroethylene dehalogenation by the same mechanisms, further indicating that chloroethylenes are fortuitously dehalogenated by the aryl dehalogenase. The commonly observed inhibition of reductive dehalogenation reactions under sulfate-reducing conditions may be due to similar regulation mechanisms in other dehalogenating microorganisms that contain multiple respiratory activities. PMID:9293011

Synthesis, crystal structures, fluorescence and xanthine oxidase inhibitory activity of pyrazole-based 1,3,4-oxadiazole derivatives

NASA Astrophysics Data System (ADS)

Qi, De-Qiang; Yu, Chuan-Ming; You, Jin-Zong; Yang, Guang-Hui; Wang, Xue-Jie; Zhang, Yi-Ping

2015-11-01

A series of pyrazole-based 1,3,4-oxadiazole derivatives were rationally designed and synthesized in good yields by following a convenient route. All the newly synthesized molecules were fully characterized by IR, 1H NMR and elemental analysis. Eight compounds were structurally determined by single crystal X-ray diffraction analysis. The fluorescence properties of all the compounds were investigated in dimethyl sulfoxide media. In addition, these newly synthesized compounds were evaluated for in vitro inhibitory activity against commercial enzyme xanthine oxidase (XO) by measuring the formation of uric acid from xanthine. Among the compounds synthesized and tested, 3d and 3e were found to be moderate inhibitory activity against commercial XO with IC50 = 72.4 μM and 75.6 μM. The studies gave a new insight in further optimization of pyrazole-based 1,3,4-oxadiazole derivatives with excellent fluorescence properties and XO inhibitory activity.

Preparation and biophysical characterization of recombinant Pseudomonas aeruginosa phosphorylcholine phosphatase.

PubMed

Beassoni, Paola R; Berti, Federico Pérez de; Otero, Lisandro H; Risso, Valeria A; Ferreyra, Raul G; Lisa, Angela T; Domenech, Carlos E; Ermácora, Mario R

2010-06-01

Pseudomonas aeruginosa infections constitute a widespread health problem with high economical and social impact, and the phosphorylcholine phosphatase (PchP) of this bacterium is a potential target for antimicrobial treatment. However, drug design requires high-resolution structural information and detailed biophysical knowledge not available for PchP. An obstacle in the study of PchP is that current methods for its expression and purification are suboptimal and allowed only a preliminary kinetic characterization of the enzyme. Herein, we describe a new procedure for the efficient preparation of recombinant PchP overexpressed in Escherichia coli. The enzyme is purified from urea solubilized inclusion bodies and refolded by dialysis. The product of PchP refolding is a mixture of native PchP and a kinetically-trapped, alternatively-folded aggregate that is very slowly converted into the native state. The properly folded and fully active enzyme is isolated from the refolding mixture by size-exclusion chromatography. PchP prepared by the new procedure was subjected to chemical and biophysical characterization, and its basic optical, hydrodynamic, metal-binding, and catalytic properties are reported. The unfolding of the enzyme was also investigated, and its thermal stability was determined. The obtained information should help to compare PchP with other phosphatases and to obtain a better understanding of its catalytic mechanism. In addition, preliminary trials showed that PchP prepared by the new protocol is suitable for crystallization, opening the way for high-resolution studies of the enzyme structure.

Bothrops jararaca envenomation: Pathogenesis of hemostatic disturbances and intravascular hemolysis.

PubMed

Senise, Luana V; Yamashita, Karine M; Santoro, Marcelo L

2015-11-01

To attain fully functional biological activity, vitamin-K dependent coagulation factors (VKDCF) are γ-carboxylated prior to secretion from liver. Warfarin impairs the γ-carboxylation, and consequently their physiological function. Bothrops jararaca snake venom (BjV) contains several activators of blood coagulation, especially procoagulant enzymes (prothrombin and factor X activators) and thrombin-like enzymes. In order to clarify the relative contribution of prothrombin and factor X activators to the hemostatic disturbances occurring during experimental B. jararaca envenomation, warfarin was used to deplete VKDCF, prior to BjV administration. Male Wistar rats were pretreated with saline (Sal) or warfarin (War) and inoculated subsequently with BjV or saline, thus forming four groups: Sal + Sal (negative control), Sal + BjV (positive control), War + Sal (warfarinization control), and War + BjV. Three hours after inoculation, prothrombin and factor X levels fell 40% and 50%, respectively; levels of both factors decreased more than 97% in the War + Sal and War + BjV groups. Platelet counts dropped 93% and 76% in Sal + BjV and War + BjV, respectively, and plasma fibrinogen levels decreased 86% exclusively in Sal + BjV. After 6 and 24 h, platelet counts and fibrinogen levels increased progressively. A dramatic augmentation in plasma hemoglobin levels and the presence of schizocytes and microcytes in the Sal + BjV group indicated the development of intravascular hemolysis, which was prevented by warfarin pretreatment. Our findings show that intravascular thrombin generation has the foremost role in the pathogenesis of coagulopathy and intravascular hemolysis, but not in the development of thrombocytopenia, in B. jararaca envenomation in rats; in addition, fibrinogenases (metalloproteinases) may contribute to coagulopathy more than thrombin-like enzymes. © 2015 by the Society for Experimental Biology and Medicine.

Distributional shift of urea production site from the extraembryonic yolk sac membrane to the embryonic liver during the development of cloudy catshark (Scyliorhinus torazame).

PubMed

Takagi, Wataru; Kajimura, Makiko; Tanaka, Hironori; Hasegawa, Kumi; Ogawa, Shuntaro; Hyodo, Susumu

2017-09-01

Urea is an essential osmolyte for marine cartilaginous fishes. Adult elasmobranchs and holocephalans are known to actively produce urea in the liver, muscle and other extrahepatic organs; however, osmoregulatory mechanisms in the developing cartilaginous fish embryo with an undeveloped urea-producing organ are poorly understood. We recently described the contribution of extraembryonic yolk sac membranes (YSM) to embryonic urea synthesis during the early developmental period of the oviparous holocephalan elephant fish (Callorhinchus milii). In the present study, to test whether urea production in the YSM is a general phenomenon among oviparous Chondrichthyes, we investigated gene expression and activities of ornithine urea cycle (OUC) enzymes together with urea concentrations in embryos of the elasmobranch cloudy catshark (Scyliorhinus torazame). The intracapsular fluid, in which the catshark embryo develops, had a similar osmolality to seawater, and embryos maintained a high concentration of urea at levels similar to that of adult plasma throughout development. Relative mRNA expressions and activities of catshark OUC enzymes were significantly higher in YSM than in embryos until stage 32. Concomitant with the development of the embryonic liver, the expression levels and activities of OUC enzymes were markedly increased in the embryo from stage 33, while those of the YSM decreased from stage 32. The present study provides further evidence that the YSM contributes to embryonic urea homeostasis until the liver and other extrahepatic organs become fully functional, and that urea-producing tissue shifts from the YSM to the embryonic liver in the late developmental period of oviparous marine cartilaginous fishes. Copyright © 2017 Elsevier Inc. All rights reserved.

Defective Cytochrome P450-Catalysed Drug Metabolism in Niemann-Pick Type C Disease

PubMed Central

Wassif, Christopher A.; Gray, James; Burkert, Kathryn R.; Smith, David A.; Morris, Lauren; Cologna, Stephanie M.; Peer, Cody J.; Sissung, Tristan M.; Uscatu, Constantin-Daniel; Figg, William D.; Pavan, William J.; Vite, Charles H.; Porter, Forbes D.; Platt, Frances M.

2016-01-01

Niemann-Pick type C (NPC) disease is a neurodegenerative lysosomal storage disease caused by mutations in either the NPC1 or NPC2 gene. NPC is characterised by storage of multiple lipids in the late endosomal/lysosomal compartment, resulting in cellular and organ system dysfunction. The underlying molecular mechanisms that lead to the range of clinical presentations in NPC are not fully understood. While evaluating potential small molecule therapies in Npc1-/- mice, we observed a consistent pattern of toxicity associated with drugs metabolised by the cytochrome P450 system, suggesting a potential drug metabolism defect in NPC1 disease. Investigation of the P450 system in the context of NPC1 dysfunction revealed significant changes in the gene expression of many P450 associated genes across the full lifespan of Npc1-/- mice, decreased activity of cytochrome P450 reductase, and a global decrease of multiple cytochrome P450 catalysed dealkylation reactions. In vivo drug metabolism studies using a prototypic P450 metabolised drug, midazolam, confirmed dysfunction in drug clearance in the Npc1-/- mouse. Expression of the Phase II enzyme uridinediphosphate-glucuronosyltransferase (UGT) was also significantly reduced in Npc1-/- mice. Interestingly, reduced activity within the P450 system was also observed in heterozygous Npc1+/- mice. The reduced activity of P450 enzymes may be the result of bile acid deficiency/imbalance in Npc1-/- mice, as bile acid treatment significantly rescued P450 enzyme activity in Npc1-/- mice and has the potential to be an adjunctive therapy for NPC disease patients. The dysfunction in the cytochrome P450 system were recapitulated in the NPC1 feline model. Additionally, we present the first evidence that there are alterations in the P450 system in NPC1 patients. PMID:27019000

Enzyme-substrate and enzyme-inhibitor complexes of triose phosphate isomerase studied by 31P nuclear magnetic resonance.

PubMed Central

Campbell, I D; Jones, R B; Kiener, P A; Waley, S G

1979-01-01

The complex formed between the enzyme triose phosphate isomerase (EC 5.3.1.1.), from rabbit and chicken muscle, and its substrate dihydroxyacetone phosphate was studied by 31P n.m.r. Two other enzyme-ligant complexes examined were those formed by glycerol 3-phosphate (a substrate analogue) and by 2-phosphoglycollate (potential transition-state analogue). Separate resonances were observed in the 31P n.m.r. spectrum for free and bound 2-phosphoglycollate, and this sets an upper limit to the rate constant for dissociation of the enzyme-inhibitor complex; the linewidth of the resonance assigned to the bound inhibitor provided further kinetic information. The position of this resonance did not vary with pH but remained close to that of the fully ionized form of the free 2-phosphoglycollate. It is the fully ionized form of this ligand that binds to the enzyme. The proton uptake that accompanies binding shows protonation of a group on the enzyme. On the basis of chemical and crystallographic information [Hartman (1971) Biochemistry 10, 146--154; Miller & Waley (1971) Biochem. J. 123, 163--170; De la Mare, Coulson, Knowles, Priddle & Offord )1972) Biochem. J. 129, 321--331; Phillips, Rivers, Sternberg, Thornton & Wilson (1977) Biochem. Soc. Trans. 5, 642--647] this group is believed to be glutamate-165. On the other hand, the position of the resonance of D-glycerol 3 phosphate (sn-glycerol 1-phosphate) in the enzyme-ligand complex changes with pH, and both monoanion and dianon of the ligand bind, although dianion binds better. The substrate, dihydroxyacetone phosphate, behaves essentially like glycerol 3-phosphate. The experiments with dihydroxy-acetone phosphate and triose phosphate isomerase have to be carried out at 1 degree C because at 37 degrees C there is conversion into methyl glyoxal and orthophosphate. The mechanismof the enzymic reaction and the reasons for rate-enhancement are considered, and aspects of the pH-dependence are discussed in an Appendix. PMID:38777

Substrate Binding and Catalytic Mechanism of Human Choline Acetyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim,A.; Rylett, J.; Shilton, B.

2006-01-01

Choline acetyltransferase (ChAT) catalyzes the synthesis of the neurotransmitter acetylcholine from choline and acetyl-CoA, and its presence is a defining feature of cholinergic neurons. We report the structure of human ChAT to a resolution of 2.2 {angstrom} along with structures for binary complexes of ChAT with choline, CoA, and a nonhydrolyzable acetyl-CoA analogue, S-(2-oxopropyl)-CoA. The ChAT-choline complex shows which features of choline are important for binding and explains how modifications of the choline trimethylammonium group can be tolerated by the enzyme. A detailed model of the ternary Michaelis complex fully supports the direct transfer of the acetyl group from acetyl-CoAmore » to choline through a mechanism similar to that seen in the serine hydrolases for the formation of an acyl-enzyme intermediate. Domain movements accompany CoA binding, and a surface loop, which is disordered in the unliganded enzyme, becomes localized and binds directly to the phosphates of CoA, stabilizing the complex. Interactions between this surface loop and CoA may function to lower the K{sub M} for CoA and could be important for phosphorylation-dependent regulation of ChAT activity.« less

Gene therapy augments the efficacy of hematopoietic cell transplantation and fully corrects mucopolysaccharidosis type I phenotype in the mouse model

PubMed Central

Visigalli, Ilaria; Delai, Stefania; Politi, Letterio S.; Di Domenico, Carmela; Cerri, Federica; Mrak, Emanuela; D'Isa, Raffaele; Ungaro, Daniela; Stok, Merel; Sanvito, Francesca; Mariani, Elisabetta; Staszewsky, Lidia; Godi, Claudia; Russo, Ilaria; Cecere, Francesca; del Carro, Ubaldo; Rubinacci, Alessandro; Brambilla, Riccardo; Quattrini, Angelo; Di Natale, Paola; Ponder, Katherine; Naldini, Luigi

2010-01-01

Type I mucopolysaccharidosis (MPS I) is a lysosomal storage disorder caused by the deficiency of α-L-iduronidase, which results in glycosaminoglycan accumulation in tissues. Clinical manifestations include skeletal dysplasia, joint stiffness, visual and auditory defects, cardiac insufficiency, hepatosplenomegaly, and mental retardation (the last being present exclusively in the severe Hurler variant). The available treatments, enzyme-replacement therapy and hematopoietic stem cell (HSC) transplantation, can ameliorate most disease manifestations, but their outcome on skeletal and brain disease could be further improved. We demonstrate here that HSC gene therapy, based on lentiviral vectors, completely corrects disease manifestations in the mouse model. Of note, the therapeutic benefit provided by gene therapy on critical MPS I manifestations, such as neurologic and skeletal disease, greatly exceeds that exerted by HSC transplantation, the standard of care treatment for Hurler patients. Interestingly, therapeutic efficacy of HSC gene therapy is strictly dependent on the achievement of supranormal enzyme activity in the hematopoietic system of transplanted mice, which allows enzyme delivery to the brain and skeleton for disease correction. Overall, our data provide evidence of an efficacious treatment for MPS I Hurler patients, warranting future development toward clinical testing. PMID:20847202

Biology, ecology, and biotechnological applications of anaerobic bacteria adapted to environmental stresses in temperature, pH, salinity, or substrates.

PubMed Central

Lowe, S E; Jain, M K; Zeikus, J G

1993-01-01

Anaerobic bacteria include diverse species that can grow at environmental extremes of temperature, pH, salinity, substrate toxicity, or available free energy. The first evolved archaebacterial and eubacterial species appear to have been anaerobes adapted to high temperatures. Thermoanaerobes and their stable enzymes have served as model systems for basic and applied studies of microbial cellulose and starch degradation, methanogenesis, ethanologenesis, acetogenesis, autotrophic CO2 fixation, saccharidases, hydrogenases, and alcohol dehydrogenases. Anaerobes, unlike aerobes, appear to have evolved more energy-conserving mechanisms for physiological adaptation to environmental stresses such as novel enzyme activities and stabilities and novel membrane lipid compositions and functions. Anaerobic syntrophs do not have similar aerobic bacterial counterparts. The metabolic end products of syntrophs are potent thermodynamic inhibitors of energy conservation mechanisms, and they require coordinated consumption by a second partner organism for species growth. Anaerobes adapted to environmental stresses and their enzymes have biotechnological applications in organic waste treatment systems and chemical and fuel production systems based on biomass-derived substrates or syngas. These kinds of anaerobes have only recently been examined by biologists, and considerably more study is required before they are fully appreciated by science and technology. Images PMID:8336675

A literature review of MTHFR (C677T and A1298C polymorphisms) and cancer risk.

PubMed

Izmirli, Muzeyyen

2013-01-01

5,10-Methlenetetrahydrofolate reductase (MTHFR) is one of the most important enzymes for folate metabolism. This enzyme is mapped on chromosome 1, which is located at the end of the short arm (1p36.3). The C677T and A1298C are MTHFR polymorphisms that decrease in vitro MTHFR enzyme activity. Folate metabolism plays a key role in cell metabolism. These reactions are associated with purine-pyrimidine synthesis: DNA, RNA, and protein methylation. Polymorphism is also a factor in biodiversity, and be affected by ethnic heritage and geographic locale. In the case of unknown outcomes, not only should all geographical regions be investigated to ascertain biodiversity, but all populations as well to fully understand the variations in the effect. PUBMED was searched from January 2006 to December 2011 to develop an investigatory pursuit strategy. MTHFR, cancer, C677T, A1298C, and polymorphisms were key words used to focus the search. The literature review included all published relevant cancer types and MTHFR polymorphisms for that 5 years period. All selected polymorphisms data for cancer types was listed in tables for easy access and retrieval.

Hydrogel delivery of lysostaphin eliminates orthopedic implant infection by Staphylococcus aureus and supports fracture healing

PubMed Central

Johnson, Christopher T.; Wroe, James A.; Agarwal, Rachit; Martin, Karen E.; Guldberg, Robert E.; Donlan, Rodney M.; Westblade, Lars F.; García, Andrés J.

2018-01-01

Orthopedic implant infections are a significant clinical problem, with current therapies limited to surgical debridement and systemic antibiotic regimens. Lysostaphin is a bacteriolytic enzyme with high antistaphylococcal activity. We engineered a lysostaphin-delivering injectable PEG hydrogel to treat Staphylococcus aureus infections in bone fractures. The injectable hydrogel formulation adheres to exposed tissue and fracture surfaces, ensuring efficient, local delivery of lysostaphin. Lysostaphin encapsulation within this synthetic hydrogel maintained enzyme stability and activity. Lysostaphin-delivering hydrogels exhibited enhanced antibiofilm activity compared with soluble lysostaphin. Lysostaphin-delivering hydrogels eradicated S. aureus infection and outperformed prophylactic antibiotic and soluble lysostaphin therapy in a murine model of femur fracture. Analysis of the local inflammatory response to infections treated with lysostaphin-delivering hydrogels revealed indistinguishable differences in cytokine secretion profiles compared with uninfected fractures, demonstrating clearance of bacteria and associated inflammation. Importantly, infected fractures treated with lysostaphin-delivering hydrogels fully healed by 5 wk with bone formation and mechanical properties equivalent to those of uninfected fractures, whereas fractures treated without the hydrogel carrier were equivalent to untreated infections. Finally, lysostaphin-delivering hydrogels eliminate methicillin-resistant S. aureus infections, supporting this therapy as an alternative to antibiotics. These results indicate that lysostaphin-delivering hydrogels effectively eliminate orthopedic S. aureus infections while simultaneously supporting fracture repair. PMID:29760099

Guiding Principles of Hydrogenase Catalysis Instigated and Clarified by Protein Film Electrochemistry.

PubMed

Armstrong, Fraser A; Evans, Rhiannon M; Hexter, Suzannah V; Murphy, Bonnie J; Roessler, Maxie M; Wulff, Philip

2016-05-17

Protein film electrochemistry (PFE) is providing cutting-edge insight into the chemical principles underpinning biological hydrogen. Attached to an electrode, many enzymes exhibit "reversible" electrocatalytic behavior, meaning that a catalyzed redox reaction appears reversible or quasi-reversible when viewed by cyclic voltammetry. This efficiency is most relevant for enzymes that are inspiring advances in renewable energy, such as hydrogen-activating and CO2-reducing enzymes. Exploiting the rich repertoire of available instrumental methods, PFE experiments yield both a general snapshot and fine detail, all from tiny samples of enzyme. The dynamic electrochemical investigations blaze new trails and add exquisite detail to the information gained from structural and spectroscopic studies. This Account describes recent investigations of hydrogenases carried out in Oxford, including ideas initiated with PFE and followed through with complementary techniques, all contributing to an eventual complete picture of fast and efficient H2 activation without Pt. By immobilization of an enzyme on an electrode, catalytic electron flow and the chemistry controlling it can be addressed at the touch of a button. The buried nature of the active site means that structures that have been determined by crystallography or spectroscopy are likely to be protected, retained, and fully relevant in a PFE experiment. An electrocatalysis model formulated for the PFE of immobilized enzymes predicts interesting behavior and gives insight into why some hydrogenases are H2 producers and others are H2 oxidizers. Immobilization also allows for easy addition and removal of inhibitors along with precise potential control, one interesting outcome being that formaldehyde forms a reversible complex with reduced [FeFe]-hydrogenases, thereby providing insight into the order of electron and proton transfers. Experiments on O2-tolerant [NiFe]-hydrogenases show that O2 behaves like a reversible inhibitor: it is also a substrate, and implicit in the description of some hydrogenases as "H2/O2 oxidoreductases" is the hypothesis that fast and efficient multielectron transfer is a key to O2 tolerance because it promotes complete reduction of O2 to harmless water. Not only is a novel [4Fe-3S] cluster (able to transfer two electrons consecutively) an important component, but connections to additional electron sources (other Fe-S clusters, an electrode, another quaternary structure unit, or the physiological membrane itself) ensure that H2 oxidation can be sustained in the presence of O2, as demonstrated with enzyme fuel cells able to operate on a H2/air mixture. Manipulating the H-H bond in the active site is the simplest proton-coupled electron-transfer reaction to be catalyzed by an enzyme. Unlike small molecular catalysts or the surfaces of materials, metalloenzymes are far better suited to engineering the all-important outer-coordination shell. Hence, recent successful site-directed mutagenesis of the conserved outer-shell "canopy" residues in a [NiFe]-hydrogenase opens up new opportunities for understanding the mechanism of H2 activation beyond the role of the inner coordination shell.

Mechanisms of lead-induced poisoning.

PubMed

Nemsadze, K; Sanikidze, T; Ratiani, L; Gabunia, L; Sharashenidze, T

2009-01-01

Lead is a ubiquitous environmental toxin that is capable of causing numerous acute and chronic circulatory, neurological, hematological, gastrointestinal, reproductive and immunological pathologies. The mechanism of lead induced toxicity is not fully understood. The prime targets to lead toxicity are the heme synthesis enzymes, thiol-containing antioxidants and enzymes (superoxide dismutase, catalase, glutathione peroxidase, glucose 6-phosphate dehydrogenase and antioxidant molecules like GSH). The low blood lead levels are sufficient to inhibit the activity of these enzymes and induce generation of reactive oxygen species and intensification oxidative stress. Oxidative stress plays important role in pathogenesis of lead-induced toxicity and pathogenesis of coupled disease. The primary target of lead toxicity is the central nervous system. There are different cellular, intracellular and molecular mechanisms of lead neurotoxicity: such as induction of oxidative stress, intensification of apoptosis of neurocites, interfering with Ca(2+) dependent enzyme like nitric oxide synthase. Population studies have demonstrated a link between lead exposure and subsequent development of hypertension and cardiovascular disease. The vascular endothelium is now regarded as the main target organ for the toxic effect of lead. Lead affects the vasoactive function of endothelium through the increased production of reactive oxygen species, inactivation of endogenous nitric oxide and downregulation of soluble guanylate cyclase by reactive oxygen species, leading to a limiting nitric oxide availability, impairing nitric oxide signaling. This review summarizes recent findings of the mechanism of the lead-induced toxicity and possibilities of its prevention.

sup 1 H and sup 51 V NMR studies of the interaction of vanadate and 2-vanadio-3-phosphoglycerate with phosphoglycerate mutase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, S.; Gresser, M.J.; Tracey, A.S.

1992-03-17

The formation of complexes of vanadate with 2-phosphoglycerate and 3-phosphoglycerate have been studied using {sup 51}V nuclear magnetic resonance spectroscopy. Signals attributed to two 2,3-diphosphoglycerate analogues, 2-vanadio-3-phosphoglycerate and 2-phospho-3-vanadioglycerate, were detected but were not fully resolved from signals of inorganic vanadate and the anhydride formed between vanadate and the phosphate ester moieties of the individual phosphoglycerates. Equilibrium constants for formation of the two 2,3-bisphosphate analogues were estimated as 2.5 M{sup {minus}1} for 2-vanadio-3-phosphoglycerate and 0.2 M{sup {minus}1} for 2-phospho-3-vanadioglycerate. The results of the binding study are fully consistent with noncooperativity in the binding of vanadiophosphoglycerate to the two active sitesmore » of phosphoglycerate mutase (PGM). The results obtained here are in accord with these vanadate-phosphoglycerate complexes being much more potent inhibitors of phosphoglycerate mutase than either monomeric or dimeric vanadate. These results strongly support the view that phosphoryl transfer in this enzyme involves a pentacoordinate phosphate intermediate and suggests that the two active sites operate independently of each other.« less

Fully Integrated Lignocellulosic Biorefinery with Onsite Production of Enzymes and Yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2010-06-14

Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitutionmore » of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

RETINOIC ACID SYNTHESIS AND DEGRADATION

PubMed Central

Kedishvili, Natalia Y.

2017-01-01

Retinoic acid was identified as the biologically active form of vitamin A almost 70 years ago, but the exact enzymes and control mechanisms that regulate its biosynthesis and degradation are yet to be fully defined. The currently accepted model postulates that RA is produced in two sequential oxidative steps: first, retinol is oxidized reversibly to retinaldehyde, and then retinaldehyde is oxidized irreversibly to RA, which is inactivated by conversion to hydroxylated derivatives. This chapter describes the history, development and recent advances in our understanding of the enzymatic pathways and mechanisms that control the rate of RA production and degradation. Gene knockout studies provided strong evidence that the members of the short chain dehydrogenase reductase superfamily of proteins play indispensable roles in retinoic acid biosynthesis during development. Furthermore, recent finding that two of these proteins regulate the rate of retinoic acid biosynthesis by mutually activating each other provided a novel insight into the mechanism of this regulation. Despite significant progress made since the middle of the 20th century many unanswered questions still remain, and there is much to be learned, especially about trafficking of the hydrophobic retinoid substrates between membrane bound and cytosolic enzymes and the roles of the retinoid binding proteins. PMID:27830503

Conditional knock-out of lipoic acid protein ligase 1 reveals redundancy pathway for lipoic acid metabolism in Plasmodium berghei malaria parasite.

PubMed

Wang, Min; Wang, Qiong; Gao, Xiang; Su, Zhong

2017-06-27

Lipoic acid is a cofactor for α-keto acid dehydrogenase system that is involved in the central energy metabolism. In the apicomplexan parasite, Plasmodium, lipoic acid protein ligase 1 (LplA1) and LplA2 catalyse the ligation of acquired lipoic acid to the dehydrogenase complexes in the mitochondrion. The enzymes LipB and LipA mediate lipoic acid synthesis and ligation to the enzymes in the apicoplast. These enzymes in the lipoic acid metabolism machinery have been shown to play important roles in the biology of Plasmodium parasites, but the relationship between the enzymes is not fully elucidated. We used an anhydrotetracycline (ATc)-inducible transcription system to generate transgenic P. berghei parasites in which the lplA1 gene was conditionally knocked out (LplA1-cKO). Phenotypic changes and the lplA1 and lplA2 gene expression profiles of cloned LplA1-cKO parasites were analysed. LplA1-cKO parasites showed severely impaired growth in vivo in the first 8 days of infection, and retarded blood-stage development in vitro, in the absence of ATc. However, these parasites resumed viability in the late stage of infection and mounted high levels of parasitemia leading to the death of the hosts. Although lplA1 mRNA expression was regulated tightly by ATc during the whole course of infection, lplA2 mRNA expression was significantly increased in the late stage of infection only in the LplA1-cKO parasites that were not exposed to ATc. The lplA2 gene can be activated as an alternative pathway to compensate for the loss of LplA1 activity and to maintain lipoic acid metabolism.

Elucidation of interactions of Alzheimer amyloid beta peptides (Abeta40 and Abeta42) with insulin degrading enzyme: a molecular dynamics study.

PubMed

Bora, Ram Prasad; Prabhakar, Rajeev

2010-05-11

In this study, interactions of the two full-length Alzheimer amyloid beta peptides (Abeta40 and Abeta42) with the fully active form of insulin degrading enzyme (IDE) through unrestrained, all-atom MD simulations have been investigated. This enzyme is a Zn-containing metallopeptidase that catalyzes the degradation of the monomeric forms of these peptides, and this process is critical for preventing the progression of Alzheimer's disease (AD). The available X-ray structures of the free and small fragment-bound (Asp1-Glu3 and Lys16-Asp23 of Abeta40 and Asp1-Glu3 and Lys16-Glu22 of Abeta42) mutated forms of IDE and NMR structures of the full-length Abeta40 and Abeta42 have been used to build the starting structures for these simulations. The most representative structures derived from the Abeta40-IDE and Abeta42-IDE simulations accurately reproduced the locations of the active site Zn(2+) metal and small fragments of the substrates and their interactions with the enzyme from the X-ray structures. The remaining fragments of both the substrates were found to interact with IDE through several hydrogen bonding, pi-pi, CH-pi, and NH-pi interactions. In comparison to Abeta40, Abeta42 is more flexible and interacts through a smaller number (17-22) of hydrogen bonds in the catalytic chamber of IDE. Both the substrates adopted more beta-sheet character in the IDE environment, an observation that is in line with experiments. Their structural characteristics inside IDE are significantly different than the ones observed in aqueous solution. The atomistic level details provided by these simulations can help in the elucidation of binding and degrading mechanisms of the Abeta peptides by IDE.

Exhaustive exercise--a near death experience for skeletal muscle cells?

PubMed

Behringer, Michael; Montag, Johannes; Franz, Alexander; McCourt, Molly L; Mester, Joachim; Nosaka, Kazunori Ken

2014-12-01

In sports medicine, muscle enzymes in the blood are frequently used as an indicator of muscle damage. It is commonly assumed that mechanical stress disrupts plasma membrane to an extent that allows large molecules, such as enzymes, to leak into the extracellular space. However, this does not appear to fully explain changes in muscle enzyme activity in the blood after exercise. Apart from this mechanically induced membrane damage, we hypothesize that, under critical metabolic conditions, ATP consuming enzymes like creatine kinase (CK) are "volitionally" expulsed by muscle cells in order to prevent cell death. This would put themselves into a situation comparable to that of CK deficient muscle fibers, which have been shown in animal experiments to be virtually infatigable at the expense of muscle strength. Additionally we expand on this hypothesis with the idea that membrane blebbing is a way for the muscle fibers to store CK in fringe areas of the muscle fiber or to expulse CK from the cytosol by detaching the blebs from the plasma membrane. The blebbing has been shown to occur in heart muscle cells under ischaemic conditions and has been speculated to be an alternative pathway for the expulsion of troponin. The blebbing has also been seen skeletal muscle cells when intracellular calcium concentration increases. Cytoskeletal damage, induced by reactive oxygen species (ROS) or by calcium activated proteases in concert with increasing intracellular pressure, seems to provoke this type of membrane reaction. If these hypotheses are confirmed by future investigations, our current understanding of CK as a blood muscle damage marker will be fundamentally affected. Copyright © 2014 Elsevier Ltd. All rights reserved.

Structural-Functional Analysis Reveals a Specific Domain Organization in Family GH20 Hexosaminidases.

PubMed

Val-Cid, Cristina; Biarnés, Xevi; Faijes, Magda; Planas, Antoni

2015-01-01

Hexosaminidases are involved in important biological processes catalyzing the hydrolysis of N-acetyl-hexosaminyl residues in glycosaminoglycans and glycoconjugates. The GH20 enzymes present diverse domain organizations for which we propose two minimal model architectures: Model A containing at least a non-catalytic GH20b domain and the catalytic one (GH20) always accompanied with an extra α-helix (GH20b-GH20-α), and Model B with only the catalytic GH20 domain. The large Bifidobacterium bifidum lacto-N-biosidase was used as a model protein to evaluate the minimal functional unit due to its interest and structural complexity. By expressing different truncated forms of this enzyme, we show that Model A architectures cannot be reduced to Model B. In particular, there are two structural requirements general to GH20 enzymes with Model A architecture. First, the non-catalytic domain GH20b at the N-terminus of the catalytic GH20 domain is required for expression and seems to stabilize it. Second, the substrate-binding cavity at the GH20 domain always involves a remote element provided by a long loop from the catalytic domain itself or, when this loop is short, by an element from another domain of the multidomain structure or from the dimeric partner. Particularly, the lacto-N-biosidase requires GH20b and the lectin-like domain at the N- and C-termini of the catalytic GH20 domain to be fully soluble and functional. The lectin domain provides this remote element to the active site. We demonstrate restoration of activity of the inactive GH20b-GH20-α construct (model A architecture) by a complementation assay with the lectin-like domain. The engineering of minimal functional units of multidomain GH20 enzymes must consider these structural requirements.

[Vitamin E and experimental caries in rats fed a cariogenic diet and zinc].

PubMed

Rapisarda, E; Longo, A

1981-01-01

A cariogenic diet with zinc and vitamin E administered to rats for 90 days led to a reduction in caries of 21.87% by comparison with animals fed with the cariogenic diet only, and 3.12% by comparison with those that received the diet plus zinc. Although the details of the mechanism of action of vitamin E are not fully known, it is felt that its demonstrated cariostatic effect depends on its antioxidant activity and its protection of the sulphydryl groups of some enzyme system, together with its direct intervention in cell respiration. Since both vitamin E and zinc activate NAD-dependent LDH, their simultaneous administration enhances their individual cariostatic effects by bringing about a lower accumulation of lactic acid in the bacterial plaque.

Large Conformational Changes of Insertion 3 in Human Glycyl-tRNA Synthetase (hGlyRS) during Catalysis

DOE PAGES

Deng, Xiangyu; Qin, Xiangjing; Chen, Lei; ...

2016-01-21

Glycyl-tRNA synthetase (GlyRS) is the enzyme that covalently links glycine to cognate tRNA for translation. It is of great interest because of its nonconserved quaternary structures, unique species-specific aminoacylation properties, and noncanonical functions in neurological diseases, but none of these is fully understood. We report two crystal structures of human GlyRS variants, in the free form and in complex with tRNA Gly respectively, and reveal new aspects of the glycylation mechanism. We discover that insertion 3 differs considerably in conformation in catalysis and that it acts like a "switch" and fully opens to allow tRNA to bind in a cross-subunitmore » fashion. The flexibility of the protein is supported by molecular dynamics simulation, as well as enzymatic activity assays. The biophysical and biochemical studies suggest that human GlyRS may utilize its flexibility for both the traditional function (regulate tRNA binding) and alternative functions (roles in diseases).« less

NADPH oxidase-derived H2O2 subverts pathogen signaling by oxidative phosphotyrosine conversion to PB-DOPA

PubMed Central

Alvarez, Luis A.; Kovačič, Lidija; Rodríguez, Javier; Gosemann, Jan-Hendrik; Kubica, Malgorzata; Pircalabioru, Gratiela G.; Friedmacher, Florian; Cean, Ada; Ghişe, Alina; Sărăndan, Mihai B.; Puri, Prem; Daff, Simon; Plettner, Erika; von Kriegsheim, Alex; Bourke, Billy; Knaus, Ulla G.

2016-01-01

Strengthening the host immune system to fully exploit its potential as antimicrobial defense is vital in countering antibiotic resistance. Chemical compounds released during bidirectional host–pathogen cross-talk, which follows a sensing-response paradigm, can serve as protective mediators. A potent, diffusible messenger is hydrogen peroxide (H2O2), but its consequences on extracellular pathogens are unknown. Here we show that H2O2, released by the host on pathogen contact, subverts the tyrosine signaling network of a number of bacteria accustomed to low-oxygen environments. This defense mechanism uses heme-containing bacterial enzymes with peroxidase-like activity to facilitate phosphotyrosine (p-Tyr) oxidation. An intrabacterial reaction converts p-Tyr to protein-bound dopa (PB-DOPA) via a tyrosinyl radical intermediate, thereby altering antioxidant defense and inactivating enzymes involved in polysaccharide biosynthesis and metabolism. Disruption of bacterial signaling by DOPA modification reveals an infection containment strategy that weakens bacterial fitness and could be a blueprint for antivirulence approaches. PMID:27562167

Synthesis and kinetic evaluation of cyclophostin and cyclipostins phosphonate analogs as selective and potent inhibitors of microbial lipases.

PubMed

Point, Vanessa; Malla, Raj K; Diomande, Sadia; Martin, Benjamin P; Delorme, Vincent; Carriere, Frederic; Canaan, Stephane; Rath, Nigam P; Spilling, Christopher D; Cavalier, Jean-François

2012-11-26

A new series of customizable diastereomeric cis- and trans-monocyclic enol-phosphonate analogs to Cyclophostin and Cyclipostins were synthesized. Their potencies and mechanisms of inhibition toward six representative lipolytic enzymes belonging to distinct lipase families were examined. With mammalian gastric and pancreatic lipases no inhibition occurred with any of the compounds tested. Conversely, Fusarium solani Cutinase and lipases from Mycobacterium tuberculosis (Rv0183 and LipY) were all fully inactivated. The best inhibitors displayed a cis conformation (H and OMe) and exhibited higher inhibitory activities than the lipase inhibitor Orlistat toward the same enzymes. Our results have revealed that chemical group at the γ-carbon of the phosphonate ring strongly impacts the inhibitory efficiency, leading to a significant improvement in selectivity toward a target lipase over another. The powerful and selective inhibition of microbial (fungal and mycobacterial) lipases suggests that these seven-membered monocyclic enol-phosphonates should provide useful leads for the development of novel and highly selective antimicrobial agents.

Synthesis and kinetic evaluation of Cyclophostin and Cyclipostins phosphonate analogs as selective and potent inhibitors of microbial lipases

PubMed Central

Point, Vanessa; Malla, Raj K.; Diomande, Sadia; Martin, Benjamin P.; Delorme, Vincent; Carriere, Frederic; Canaan, Stephane; Rath, Nigam P.; Spilling, Christopher D.; Cavalier, Jean-François

2012-01-01

New series of customizable diastereomeric cis- and trans-monocyclic enol-phosphonate analogs to Cyclophostin and Cyclipostins were synthesized. Their potencies and mechanisms of inhibition toward six representative lipolytic enzymes belonging to distinct lipase families were examined. With mammalian gastric and pancreatic lipases no inhibition occurred with any of the compounds tested. Conversely, Fusarium solani Cutinase and lipases from Mycobacterium tuberculosis (Rv0183 and LipY) were all fully inactivated. Best inhibitors displayed a cis conformation (H and OMe) and exhibited higher inhibitory activities than the lipase inhibitor Orlistat towards same enzymes. Our results have revealed that chemical group at the γ-carbon of the phosphonate ring strongly impacts the inhibitory efficiency, leading to a significant improvement in selectivity toward a target lipase over another. The powerful and selective inhibition of microbial (fungal and mycobacterial) lipases suggests that these 7-membered monocyclic enol-phosphonates should provide useful leads for the development of novel and highly selective antimicrobial agents. PMID:23095026

NADPH oxidase-derived H2O2 subverts pathogen signaling by oxidative phosphotyrosine conversion to PB-DOPA.

PubMed

Alvarez, Luis A; Kovačič, Lidija; Rodríguez, Javier; Gosemann, Jan-Hendrik; Kubica, Malgorzata; Pircalabioru, Gratiela G; Friedmacher, Florian; Cean, Ada; Ghişe, Alina; Sărăndan, Mihai B; Puri, Prem; Daff, Simon; Plettner, Erika; von Kriegsheim, Alex; Bourke, Billy; Knaus, Ulla G

2016-09-13

Strengthening the host immune system to fully exploit its potential as antimicrobial defense is vital in countering antibiotic resistance. Chemical compounds released during bidirectional host-pathogen cross-talk, which follows a sensing-response paradigm, can serve as protective mediators. A potent, diffusible messenger is hydrogen peroxide (H2O2), but its consequences on extracellular pathogens are unknown. Here we show that H2O2, released by the host on pathogen contact, subverts the tyrosine signaling network of a number of bacteria accustomed to low-oxygen environments. This defense mechanism uses heme-containing bacterial enzymes with peroxidase-like activity to facilitate phosphotyrosine (p-Tyr) oxidation. An intrabacterial reaction converts p-Tyr to protein-bound dopa (PB-DOPA) via a tyrosinyl radical intermediate, thereby altering antioxidant defense and inactivating enzymes involved in polysaccharide biosynthesis and metabolism. Disruption of bacterial signaling by DOPA modification reveals an infection containment strategy that weakens bacterial fitness and could be a blueprint for antivirulence approaches.

Cryptic indole hydroxylation by a non-canonical terpenoid cyclase parallels bacterial xenobiotic detoxification

NASA Astrophysics Data System (ADS)

Kugel, Susann; Baunach, Martin; Baer, Philipp; Ishida-Ito, Mie; Sundaram, Srividhya; Xu, Zhongli; Groll, Michael; Hertweck, Christian

2017-06-01

Terpenoid natural products comprise a wide range of molecular architectures that typically result from C-C bond formations catalysed by classical type I/II terpene cyclases. However, the molecular diversity of biologically active terpenoids is substantially increased by fully unrelated, non-canonical terpenoid cyclases. Their evolutionary origin has remained enigmatic. Here we report the in vitro reconstitution of an unusual flavin-dependent bacterial indoloterpenoid cyclase, XiaF, together with a designated flavoenzyme-reductase (XiaP) that mediates a key step in xiamycin biosynthesis. The crystal structure of XiaF with bound FADH2 (at 2.4 Å resolution) and phylogenetic analyses reveal that XiaF is, surprisingly, most closely related to xenobiotic-degrading enzymes. Biotransformation assays show that XiaF is a designated indole hydroxylase that can be used for the production of indigo and indirubin. We unveil a cryptic hydroxylation step that sets the basis for terpenoid cyclization and suggest that the cyclase has evolved from xenobiotics detoxification enzymes.

Bilirubin oxidase-like proteins from Podospora anserina: promising thermostable enzymes for application in transformation of plant biomass.

PubMed

Xie, Ning; Ruprich-Robert, Gwenaël; Silar, Philippe; Chapeland-Leclerc, Florence

2015-03-01

Plant biomass degradation by fungi is a critical step for production of biofuels, and laccases are common ligninolytic enzymes envisioned for ligninolysis. Bilirubin oxidases (BODs)-like are related to laccases, but their roles during lignocellulose degradation have not yet been fully investigated. The two BODs of the ascomycete fungus Podospora anserina were characterized by targeted gene deletions. Enzymatic assay revealed that the bod1(Δ) and bod2(Δ) mutants lost partly a thermostable laccase activity. A triple mutant inactivated for bod1, bod2 and mco, a previously investigated multicopper oxidase gene distantly related to laccases, had no thermostable laccase activity. The pattern of fruiting body production in the bod1(Δ) bod2(Δ) double mutant was changed. The bod1(Δ) and bod2(Δ) mutants were reduced in their ability to grow on ligneous and cellulosic materials. Furthermore, bod1(Δ) and bod2(Δ) mutants were defective towards resistance to phenolic substrates and H2 O2 , which may also impact lignocellulose breakdown. Double and triple mutants were more affected than single mutants, evidencing redundancy of function among BODs and mco. Overall, the data show that bod1, bod2 and mco code for non-canonical thermostable laccases that participate in the degradation of lignocellulose. Thanks to their thermal stability, these enzymes may be more promising candidate for biotechnological application than canonical laccases. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Soluble expression of an amebic cysteine protease in the cytoplasm of Escherichia coli SHuffle Express cells and purification of active enzyme.

PubMed

Jalomo-Khayrova, Ekaterina; Mares, Rosa E; Muñoz, Patricia L A; Meléndez-López, Samuel G; Rivero, Ignacio A; Ramos, Marco A

2018-04-03

Recombinant production of amebic cysteine proteases using Escherichia coli cells as the bacterial system has become a challenging effort, with protein insolubility being the most common issue. Since many of these enzymes need a native conformation stabilized by disulfide bonds, an elaborate process of oxidative folding is usually demanded to get a functional protein. The cytoplasm of E. coli SHuffle Express cells owns an enhanced ability to properly fold proteins with disulfide bonds. Because of this cellular feature, it was possible to assume that this strain represents a reliable expression system and worthwhile been considered as an efficient bacterial host for the recombinant production of amebic cysteine proteases. Using E. coli SHuffle Express cells as the bacterial system, we efficiently produce soluble recombinant EhCP1protein. Enzymatic and inhibition analyses revealed that it exhibits proper catalytic abilities, proceeds effectively over the substrate (following an apparent Michaelis-Menten kinetics), and displays a typical inhibition profile. We report the first feasibility study of the recombinant production of amebic cysteine proteases using E. coli SHuffle Express as the bacterial host. We present a simple protocol for the recombinant expression and purification of fully soluble and active EhCP1 enzyme. We confirm the suitability of recombinant EhCP1 as a therapeutic target. We propose an approachable bacterial system for the recombinant production of amebic proteins, particularly for those with a need for proper oxidative folding.

Human Topoisomerase I C-Terminal Domain Fragment Containing the Active Site Tyrosine is a Molten Globule: Implication for the Formation of Competent Productive Complex

PubMed Central

Punchihewa, Chandanamali; Dai, Jixun; Carver, Megan; Yang, Danzhou

2007-01-01

Human topoisomerase I (topo I) is an essential cellular enzyme that relaxes DNA supercoiling. The 6.3 kDa C-terminal domain of topo I contains the active site tyrosine (Tyr723) but lacks enzymatic activity by itself. Activity can be fully reconstituted when the C-terminal is associated with the 56 kDa core domain. Even though several crystal structures of topo I/DNA complexes are available, crystal structures of the free topo I protein or its individual domain fragments have been difficult to obtain. In this report we analyze the human topo I C-terminal domain structure using a variety of biophysical methods. Our results indicate that this fragment protein (topo6.3) appears to be in a molten globule state. It appears to have a native-like tertiary fold that contains a large population of α-helix secondary structure and extensive surface hydrophobic regions. Topo6.3 is known to be readily activated with the association of the topo I core domain, and the molten globule state of topo6.3 is likely to be an energy-favorable conformation for the free topo I C-terminal domain protein. The structural fluctuation and plasticity may represent an efficient mechanism in the topo I functional pathway, where the flexibility aids in the complementary association with the core domain and in the formation of a fully productive topo I complex. PMID:17434318

Repellents Inhibit P450 Enzymes in Stegomyia (Aedes) aegypti

PubMed Central

Jaramillo Ramirez, Gloria Isabel; Logan, James G.; Loza-Reyes, Elisa; Stashenko, Elena; Moores, Graham D.

2012-01-01

The primary defence against mosquitoes and other disease vectors is often the application of a repellent. Despite their common use, the mechanism(s) underlying the activity of repellents is not fully understood, with even the mode of action of DEET having been reported to be via different mechanisms; e.g. interference with olfactory receptor neurones or actively detected by olfactory receptor neurones on the antennae or maxillary palps. In this study, we discuss a novel mechanism for repellence, one of P450 inhibition. Thirteen essential oil extracts from Colombian plants were assayed for potency as P450 inhibitors, using a kinetic fluorometric assay, and for repellency using a modified World Health Organisation Pesticide Evaluations Scheme (WHOPES) arm-in cage assay with Stegomyia (Aedes) aegypti mosquitoes. Bootstrap analysis on the inhibition analysis revealed a significant correlation between P450-inhibition and repellent activity of the oils. PMID:23152795

Structural Basis for Catalytic Activation of a Serine Recombinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Keenholtz, Ross A.; Rowland, Sally-J.; Boocock, Martin R.

2014-10-02

Sin resolvase is a site-specific serine recombinase that is normally controlled by a complex regulatory mechanism. A single mutation, Q115R, allows the enzyme to bypass the entire regulatory apparatus, such that no accessory proteins or DNA sites are required. Here, we present a 1.86 {angstrom} crystal structure of the Sin Q115R catalytic domain, in a tetrameric arrangement stabilized by an interaction between Arg115 residues on neighboring subunits. The subunits have undergone significant conformational changes from the inactive dimeric state previously reported. The structure provides a new high-resolution view of a serine recombinase active site that is apparently fully assembled, suggestingmore » roles for the conserved active site residues. The structure also suggests how the dimer-tetramer transition is coupled to assembly of the active site. The tetramer is captured in a different rotational substate than that seen in previous hyperactive serine recombinase structures, and unbroken crossover site DNA can be readily modeled into its active sites.« less

Alternative mitochondrial respiratory chains from two crustaceans: Artemia franciscana nauplii and the white shrimp, Litopenaeus vannamei.

PubMed

Rodriguez-Armenta, Chrystian; Uribe-Carvajal, Salvador; Rosas-Lemus, Monica; Chiquete-Felix, Natalia; Huerta-Ocampo, Jose Angel; Muhlia-Almazan, Adriana

2018-04-01

Mitochondrial ATP is synthesized by coupling between the electron transport chain and complex V. In contrast, physiological uncoupling of these processes allows mitochondria to consume oxygen at high rates without ATP synthesis. Such uncoupling mechanisms prevent reactive oxygen species overproduction. One of these mechanisms are the alternative redox enzymes from the mitochondrial respiratory chain, which may help cells to maintain homeostasis under stress independently of ATP synthesis. To date, no reports have been published on alternative redox enzymes in crustaceans mitochondria. Specific inhibitors were used to identify alternative redox enzymes in mitochondria isolated from Artemia franciscana nauplii, and the white shrimp, Litopenaeus vannamei. We report the presence of two alternative redox enzymes in the respiratory chain of A. franciscana nauplii, whose isolated mitochondria used glycerol-3-phosphate as a substrate, suggesting the existence of a glycerol-3-phosphate dehydrogenase. In addition, cyanide and octyl-gallate were necessary to fully inhibit this species' mitochondrial oxygen consumption, suggesting an alternative oxidase is present. The in-gel activity analysis confirmed that additional mitochondrial redox proteins exist in A. franciscana. A mitochondrial glycerol-3-phosphate dehydrogenase oxidase was identified by protein sequencing as part of a branched respiratory chain, and an alternative oxidase was also identified in this species by western blot. These results indicate different adaptive mechanisms from artemia to face environmental challenges related to the changing levels of oxygen concentration in seawater through their life cycles. No alternative redox enzymes were found in shrimp mitochondria, further efforts will determine the existence of an uncoupling mechanism such as uncoupling proteins.

Hydrolysis of VX and related compounds by organophosphorus hydrolase. Final report, Februray-December 1993

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kolakowski, J.E.; DeFrank, J.J.; Lai, K.

1995-11-01

Organophosphorus Hydrolase (OPH) is a fully characterized and cloned enzyme, derived from Pseudomonas diminuta, consisting of 365 amino acids with a total molecular weight of 38,0(X). The enzyme has a leader sequence of 29 amino acids which has been removed in the construction used in this study. OPH was evaluated for its effectiveness in catalyzing the S-(2-diisopwpylaminoethyl) methylphosphonothioate (VX) and its analogs.

Inhibition of choline acetyltransferase as a mechanism for cholinergic dysfunction induced by amyloid-β peptide oligomers.

PubMed

Nunes-Tavares, Nilson; Santos, Luís Eduardo; Stutz, Bernardo; Brito-Moreira, Jordano; Klein, William L; Ferreira, Sérgio T; de Mello, Fernando G

2012-06-01

Dysregulated cholinergic signaling is an early hallmark of Alzheimer disease (AD), usually ascribed to degeneration of cholinergic neurons induced by the amyloid-β peptide (Aβ). It is now generally accepted that neuronal dysfunction and memory deficits in the early stages of AD are caused by the neuronal impact of soluble Aβ oligomers (AβOs). AβOs build up in AD brain and specifically attach to excitatory synapses, leading to synapse dysfunction. Here, we have investigated the possibility that AβOs could impact cholinergic signaling. The activity of choline acetyltransferase (ChAT, the enzyme that carries out ACh production) was inhibited by ~50% in cultured cholinergic neurons exposed to low nanomolar concentrations of AβOs. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, lactate dehydrogenase release, and [(3)H]choline uptake assays showed no evidence of neuronal damage or loss of viability that could account for reduced ChAT activity under these conditions. Glutamate receptor antagonists fully blocked ChAT inhibition and oxidative stress induced by AβOs. Antioxidant polyunsaturated fatty acids had similar effects, indicating that oxidative damage may be involved in ChAT inhibition. Treatment with insulin, previously shown to down-regulate neuronal AβO binding sites, fully prevented AβO-induced inhibition of ChAT. Interestingly, we found that AβOs selectively bind to ~50% of cultured cholinergic neurons, suggesting that ChAT is fully inhibited in AβO-targeted neurons. Reduction in ChAT activity instigated by AβOs may thus be a relevant event in early stage AD pathology, preceding the loss of cholinergic neurons commonly observed in AD brains.

What is the role of the second "structural" NADP+-binding site in human glucose 6-phosphate dehydrogenase?

PubMed

Wang, Xiao-Tao; Chan, Ting Fai; Lam, Veronica M S; Engel, Paul C

2008-08-01

Human glucose 6-phosphate dehydrogenase, purified after overexpression in E. coli, was shown to contain one molecule/subunit of acid-extractable "structural" NADP+ and no NADPH. This tightly bound NADP+ was reduced by G6P, presumably following migration to the catalytic site. Gel-filtration yielded apoenzyme, devoid of bound NADP+ but, surprisingly, still fully active. Mr of the main component of "stripped" enzyme by gel filtration was approximately 100,000, suggesting a dimeric apoenzyme (subunit Mr = 59,000). Holoenzyme also contained tetramer molecules and, at high protein concentration, a dynamic equilibrium gave an apparent intermediate Mr of 150 kDa. Fluorescence titration of the stripped enzyme gave the K d for structural NADP+ as 37 nM, 200-fold lower than for "catalytic" NADP+. Structural NADP+ quenches 91% of protein fluorescence. At 37 degrees C, stripped enzyme, much less stable than holoenzyme, inactivated irreversibly within 2 d. Inactivation at 4 degrees C was partially reversed at room temperature, especially with added NADP+. Apoenzyme was immediately active, without any visible lag, in rapid-reaction studies. Human G6PD thus forms active dimer without structural NADP+. Apparently, the true role of the second, tightly bound NADP+ is to secure long-term stability. This fits the clinical pattern, G6PD deficiency affecting the long-lived non-nucleate erythrocyte. The Kd values for two class I mutants, G488S and G488V, were 273 nM and 480 nM, respectively (seven- and 13-fold elevated), matching the structural prediction of weakened structural NADP+ binding, which would explain decreased stability and consequent disease. Preparation of native apoenzyme and measurement of Kd constant for structural NADP+ will now allow quantitative assessment of this defect in clinical G6PD mutations.

The central active site arginine in sulfite oxidizing enzymes alters kinetic properties by controlling electron transfer and redox interactions.

PubMed

Hsiao, Ju-Chun; McGrath, Aaron P; Kielmann, Linda; Kalimuthu, Palraj; Darain, Farzana; Bernhardt, Paul V; Harmer, Jeffrey; Lee, Mihwa; Meyers, Kimberley; Maher, Megan J; Kappler, Ulrike

2018-01-01

A central conserved arginine, first identified as a clinical mutation leading to sulfite oxidase deficiency, is essential for catalytic competency of sulfite oxidizing molybdoenzymes, but the molecular basis for its effects on turnover and substrate affinity have not been fully elucidated. We have used a bacterial sulfite dehydrogenase, SorT, which lacks an internal heme group, but transfers electrons to an external, electron accepting cytochrome, SorU, to investigate the molecular functions of this arginine residue (Arg78). Assay of the SorT Mo centre catalytic competency in the absence of SorU showed that substitutions in the central arginine (R78Q, R78K and R78M mutations) only moderately altered SorT catalytic properties, except for R78M which caused significant reduction in SorT activity. The substitutions also altered the Mo-centre redox potentials (Mo VI/V potential lowered by ca. 60-80mV). However, all Arg78 mutations significantly impaired the ability of SorT to transfer electrons to SorU, where activities were reduced 17 to 46-fold compared to SorT WT , precluding determination of kinetic parameters. This was accompanied by the observation of conformational changes in both the introduced Gln and Lys residues in the crystal structure of the enzymes. Taking into account data collected by others on related SOE mutations we propose that the formation and maintenance of an electron transfer complex between the Mo centre and electron accepting heme groups is the main function of the central arginine, and that the reduced turnover and increases in K Msulfite are caused by the inefficient operation of the oxidative half reaction of the catalytic cycle in enzymes carrying these mutations. Copyright © 2017 Elsevier B.V. All rights reserved.

Fully Enzymatic Membraneless Glucose|Oxygen Fuel Cell That Provides 0.275 mA cm(-2) in 5 mM Glucose, Operates in Human Physiological Solutions, and Powers Transmission of Sensing Data.

PubMed

Ó Conghaile, Peter; Falk, Magnus; MacAodha, Domhnall; Yakovleva, Maria E; Gonaus, Christoph; Peterbauer, Clemens K; Gorton, Lo; Shleev, Sergey; Leech, Dónal

2016-02-16

Coimmobilization of pyranose dehydrogenase as an enzyme catalyst, osmium redox polymers [Os(4,4'-dimethoxy-2,2'-bipyridine)2(poly(vinylimidazole))10Cl](+) or [Os(4,4'-dimethyl-2,2'-bipyridine)2(poly(vinylimidazole))10Cl](+) as mediators, and carbon nanotube conductive scaffolds in films on graphite electrodes provides enzyme electrodes for glucose oxidation. The recombinant enzyme and a deglycosylated form, both expressed in Pichia pastoris, are investigated and compared as biocatalysts for glucose oxidation using flow injection amperometry and voltammetry. In the presence of 5 mM glucose in phosphate-buffered saline (PBS) (50 mM phosphate buffer solution, pH 7.4, with 150 mM NaCl), higher glucose oxidation current densities, 0.41 mA cm(-2), are obtained from enzyme electrodes containing the deglycosylated form of the enzyme. The optimized glucose-oxidizing anode, prepared using deglycosylated enzyme coimmobilized with [Os(4,4'-dimethyl-2,2'-bipyridine)2(poly(vinylimidazole))10Cl](+) and carbon nanotubes, was coupled with an oxygen-reducing bilirubin oxidase on gold nanoparticle dispersed on gold electrode as a biocathode to provide a membraneless fully enzymatic fuel cell. A maximum power density of 275 μW cm(-2) is obtained in 5 mM glucose in PBS, the highest to date under these conditions, providing sufficient power to enable wireless transmission of a signal to a data logger. When tested in whole human blood and unstimulated human saliva maximum power densities of 73 and 6 μW cm(-2) are obtained for the same fuel cell configuration, respectively.

Exploring substrate binding and discrimination in fructose1, 6-bisphosphate and tagatose 1,6-bisphosphate aldolases.

PubMed

Zgiby, S M; Thomson, G J; Qamar, S; Berry, A

2000-03-01

Fructose 1,6-bisphosphate aldolase catalyses the reversible condensation of glycerone-P and glyceraldehyde 3-phosphate into fructose 1,6-bisphosphate. A recent structure of the Escherichia coli Class II fructose 1,6-bisphosphate aldolase [Hall, D.R., Leonard, G.A., Reed, C.D., Watt, C.I., Berry, A. & Hunter, W.N. (1999) J. Mol. Biol. 287, 383-394] in the presence of the transition state analogue phosphoglycolohydroxamate delineated the roles of individual amino acids in binding glycerone-P and in the initial proton abstraction steps of the mechanism. The X-ray structure has now been used, together with sequence alignments, site-directed mutagenesis and steady-state enzyme kinetics to extend these studies to map important residues in the binding of glyceraldehyde 3-phosphate. From these studies three residues (Asn35, Ser61 and Lys325) have been identified as important in catalysis. We show that mutation of Ser61 to alanine increases the Km value for fructose 1, 6-bisphosphate 16-fold and product inhibition studies indicate that this effect is manifested most strongly in the glyceraldehyde 3-phosphate binding pocket of the active site, demonstrating that Ser61 is involved in binding glyceraldehyde 3-phosphate. In contrast a S61T mutant had no effect on catalysis emphasizing the importance of an hydroxyl group for this role. Mutation of Asn35 (N35A) resulted in an enzyme with only 1.5% of the activity of the wild-type enzyme and different partial reactions indicate that this residue effects the binding of both triose substrates. Finally, mutation of Lys325 has a greater effect on catalysis than on binding, however, given the magnitude of the effects it is likely that it plays an indirect role in maintaining other critical residues in a catalytically competent conformation. Interestingly, despite its proximity to the active site and high sequence conservation, replacement of a fourth residue, Gln59 (Q59A) had no significant effect on the function of the enzyme. In a separate study to characterize the molecular basis of aldolase specificity, the agaY-encoded tagatose 1,6-bisphosphate aldolase of E. coli was cloned, expressed and kinetically characterized. Our studies showed that the two aldolases are highly discriminating between the diastereoisomers fructose bisphosphate and tagatose bisphosphate, each enzyme preferring its cognate substrate by a factor of 300-1500-fold. This produces an overall discrimination factor of almost 5 x 105 between the two enzymes. Using the X-ray structure of the fructose 1,6-bisphosphate aldolase and multiple sequence alignments, several residues were identified, which are highly conserved and are in the vicinity of the active site. These residues might potentially be important in substrate recognition. As a consequence, nine mutations were made in attempts to switch the specificity of the fructose 1,6-bisphosphate aldolase to that of the tagatose 1,6-bisphosphate aldolase and the effect on substrate discrimination was evaluated. Surprisingly, despite making multiple changes in the active site, many of which abolished fructose 1, 6-bisphosphate aldolase activity, no switch in specificity was observed. This highlights the complexity of enzyme catalysis in this family of enzymes, and points to the need for further structural studies before we fully understand the subtleties of the shaping of the active site for complementarity to the cognate substrate.

Bacterial enzymes involved in lignin degradation.

PubMed

de Gonzalo, Gonzalo; Colpa, Dana I; Habib, Mohamed H M; Fraaije, Marco W

2016-10-20

Lignin forms a large part of plant biomass. It is a highly heterogeneous polymer of 4-hydroxyphenylpropanoid units and is embedded within polysaccharide polymers forming lignocellulose. Lignin provides strength and rigidity to plants and is rather resilient towards degradation. To improve the (bio)processing of lignocellulosic feedstocks, more effective degradation methods of lignin are in demand. Nature has found ways to fully degrade lignin through the production of dedicated ligninolytic enzyme systems. While such enzymes have been well thoroughly studied for ligninolytic fungi, only in recent years biochemical studies on bacterial enzymes capable of lignin modification have intensified. This has revealed several types of enzymes available to bacteria that enable them to act on lignin. Two major classes of bacterial lignin-modifying enzymes are DyP-type peroxidases and laccases. Yet, recently also several other bacterial enzymes have been discovered that seem to play a role in lignin modifications. In the present review, we provide an overview of recent advances in the identification and use of bacterial enzymes acting on lignin or lignin-derived products. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

Hydrogel delivery of lysostaphin eliminates orthopedic implant infection by Staphylococcus aureus and supports fracture healing.

PubMed

Johnson, Christopher T; Wroe, James A; Agarwal, Rachit; Martin, Karen E; Guldberg, Robert E; Donlan, Rodney M; Westblade, Lars F; García, Andrés J

2018-05-29

Orthopedic implant infections are a significant clinical problem, with current therapies limited to surgical debridement and systemic antibiotic regimens. Lysostaphin is a bacteriolytic enzyme with high antistaphylococcal activity. We engineered a lysostaphin-delivering injectable PEG hydrogel to treat Staphylococcus aureus infections in bone fractures. The injectable hydrogel formulation adheres to exposed tissue and fracture surfaces, ensuring efficient, local delivery of lysostaphin. Lysostaphin encapsulation within this synthetic hydrogel maintained enzyme stability and activity. Lysostaphin-delivering hydrogels exhibited enhanced antibiofilm activity compared with soluble lysostaphin. Lysostaphin-delivering hydrogels eradicated S. aureus infection and outperformed prophylactic antibiotic and soluble lysostaphin therapy in a murine model of femur fracture. Analysis of the local inflammatory response to infections treated with lysostaphin-delivering hydrogels revealed indistinguishable differences in cytokine secretion profiles compared with uninfected fractures, demonstrating clearance of bacteria and associated inflammation. Importantly, infected fractures treated with lysostaphin-delivering hydrogels fully healed by 5 wk with bone formation and mechanical properties equivalent to those of uninfected fractures, whereas fractures treated without the hydrogel carrier were equivalent to untreated infections. Finally, lysostaphin-delivering hydrogels eliminate methicillin-resistant S. aureus infections, supporting this therapy as an alternative to antibiotics. These results indicate that lysostaphin-delivering hydrogels effectively eliminate orthopedic S. aureus infections while simultaneously supporting fracture repair. Copyright © 2018 the Author(s). Published by PNAS.

Surfactant bilayers maintain transmembrane protein activity.

PubMed

Rayan, Gamal; Adrien, Vladimir; Reffay, Myriam; Picard, Martin; Ducruix, Arnaud; Schmutz, Marc; Urbach, Wladimir; Taulier, Nicolas

2014-09-02

In vitro studies of membrane proteins are of interest only if their structure and function are significantly preserved. One approach is to insert them into the lipid bilayers of highly viscous cubic phases rendering the insertion and manipulation of proteins difficult. Less viscous lipid sponge phases are sometimes used, but their relatively narrow domain of existence can be easily disrupted by protein insertion. We present here a sponge phase consisting of nonionic surfactant bilayers. Its extended domain of existence and its low viscosity allow easy insertion and manipulation of membrane proteins. We show for the first time, to our knowledge, that transmembrane proteins, such as bacteriorhodopsin, sarcoplasmic reticulum Ca(2+)ATPase (SERCA1a), and its associated enzymes, are fully active in a surfactant phase. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Effect of boric acid on antioxidant enzyme activity, lipid peroxidation, and ultrastructure of midgut and fat body of Galleria mellonella.

PubMed

Büyükgüzel, Ender; Büyükgüzel, Kemal; Snela, Milena; Erdem, Meltem; Radtke, Katarzyna; Ziemnicki, Kazimierz; Adamski, Zbigniew

2013-04-01

Boric acid is widely used as an insecticide, acaricide, herbicide, and fungicide and also during various industrial processings. Hence, numerous populations are subjects to this toxic compound. Its action on animals is still not fully known and understood. We examined the effect of boric acid on larvae of greater wax moth (Galleria mellonella). The chemical appeared to be toxic for larvae, usually in a concentration-dependent manner. Exposed groups revealed increased lipid peroxidation and altered activity of catalase, superoxide dismutase, glutathione S-transferase, and glutathione peroxidase. We also observed changes of ultrastructure, which were in tune with biochemical assays. We suggest that boric acid has a broad mode of action, which may affect exposed larvae, and even if sublethal, they may lead to disturbances within exposed populations.

Bacterial Synthesis of Unusual Sulfonamide and Sulfone Antibiotics by Flavoenzyme-Mediated Sulfur Dioxide Capture.

PubMed

Baunach, Martin; Ding, Ling; Willing, Karsten; Hertweck, Christian

2015-11-02

Sulfa drugs, such as sulfonilamide and dapsone, are classical antibiotics that have been in clinical use worldwide. Despite the relatively simple architectures, practically no natural products are known to feature such aromatic sulfonamide or diarylsulfone substructures. We report the unexpected discovery of three fully unprecedented, sulfonyl-bridged alkaloid dimers (sulfadixiamycins A-C) from recombinant Streptomyces species harboring the entire xiamycin biosynthesis gene cluster. Sulfadixiamycins exhibit moderate antimycobacterial activities and potent antibiotic activities even against multidrug-resistant bacteria. Gene inactivation, complementation, and biotransformation experiments revealed that a flavin-dependent enzyme (XiaH) plays a key role in sulfadixiamycin biosynthesis. XiaH mediates a radical-based, three-component reaction involving two equivalents of xiamycin and sulfur dioxide, which is reminiscent of radical styrene/SO2 copolymerization. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure-Activity Relationships of Orotidine-5′-Monophosphate Decarboxylase Inhibitors as Anticancer Agents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bello, A.; Konforte, D; Poduch, E

2009-01-01

A series of 6-substituted and 5-fluoro-6-substituted uridine derivatives were synthesized and evaluated for their potential as anticancer agents. The designed molecules were synthesized from either fully protected uridine or the corresponding 5-fluorouridine derivatives. The mononucleotide derivatives were used for enzyme inhibition investigations against ODCase. Anticancer activities of all the synthesized derivatives were evaluated using the nucleoside forms of the inhibitors. 5-Fluoro-UMP was a very weak inhibitor of ODCase. 6-Azido-5-fluoro and 5-fluoro-6-iodo derivatives are covalent inhibitors of ODCase, and the active site Lys145 residue covalently binds to the ligand after the elimination of the 6-substitution. Among the synthesized nucleoside derivatives, 6-azido-5-fluoro,more » 6-amino-5-fluoro, and 6-carbaldehyde-5-fluoro derivatives showed potent anticancer activities in cell-based assays against various leukemia cell lines. On the basis of the overall profile, 6-azido-5-fluoro and 6-amino-5-fluoro uridine derivatives exhibited potential for further investigations.« less

The C-terminal residue of phage Vp16 PDF, the smallest peptide deformylase, acts as an offset element locking the active conformation.

PubMed

Grzela, Renata; Nusbaum, Julien; Fieulaine, Sonia; Lavecchia, Francesco; Bienvenut, Willy V; Dian, Cyril; Meinnel, Thierry; Giglione, Carmela

2017-09-08

Prokaryotic proteins must be deformylated before the removal of their first methionine. Peptide deformylase (PDF) is indispensable and guarantees this mechanism. Recent metagenomics studies revealed new idiosyncratic PDF forms as the most abundant family of viral sequences. Little is known regarding these viral PDFs, including the capacity of the corresponding encoded proteins to ensure deformylase activity. We provide here the first evidence that viral PDFs, including the shortest PDF identified to date, Vp16 PDF, display deformylase activity in vivo, despite the absence of the key ribosome-interacting C-terminal region. Moreover, characterization of phage Vp16 PDF underscores unexpected structural and molecular features with the C-terminal Isoleucine residue significantly contributing to deformylase activity both in vitro and in vivo. This residue fully compensates for the absence of the usual long C-domain. Taken together, these data elucidate an unexpected mechanism of enzyme natural evolution and adaptation within viral sequences.

Neurotensin effect on Na+, K+-ATPase is CNS area- and membrane-dependent and involves high affinity NT1 receptor.

PubMed

López Ordieres, María Graciela; Rodríguez de Lores Arnaiz, Georgina

2002-11-01

We have previously shown that peptide neurotensin inhibits cerebral cortex synaptosomal membrane Na+, K+-ATPase, an effect fully prevented by blockade of neurotensin NT1 receptor by antagonist SR 48692. The work was extended to analyze neurotensin effect on Na+, K+-ATPase activity present in other synaptosomal membranes and in CNS myelin and mitochondrial fractions. Results indicated that, besides inhibiting cerebral cortex synaptosomal membrane Na+, K+-ATPase, neurotensin likewise decreased enzyme activity in homologous striatal membranes as well as in a commercial preparation obtained from porcine cerebral cortex. However, the peptide failed to alter either Na+, K+-ATPase activity in cerebellar synaptosomal and myelin membranes or ATPase activity in mitochondrial preparations. Whenever an effect was recorded with the peptide, it was blocked by antagonist SR 48692, indicating the involvement of the high affinity neurotensin receptor (NT1), as well as supporting the contention that, through inhibition of ion transport at synaptic membrane level, neurotensin plays a regulatory role in neurotransmission.

Hsp31, a member of the DJ-1 superfamily, is a multitasking stress responder with chaperone activity

PubMed Central

Aslam, Kiran; Hazbun, Tony R.

2016-01-01

ABSTRACT Among different types of protein aggregation, amyloids are a biochemically well characterized state of protein aggregation that are associated with a large number of neurodegenerative diseases including Parkinson's disease, Alzheimer and Creutzfeldt-Jakob disease. Yeast, Saccharomyces cerevisiae is an insightful model to understand the underlying mechanism of protein aggregation. Many yeast molecular chaperones can modulate aggregation and misfolding of proteins including α-Syn and the Sup35 prion. Hsp31 is a homodimeric protein structurally similar to human DJ-1, a Parkinson's disease-linked protein, and both are members of the DJ-1/ThiJ/PfpI superfamily. An emerging view is that Hsp31 and its associated superfamily members each have divergent multitasking functions that have the common theme of responding and managing various types of cellular stress. Hsp31 has several biochemical activities including chaperone and detoxifying enzyme activities that modulate at various points of a stress pathway such as toxicity associated with protein misfolding. However, we have shown the protective role of Hsp31's chaperone activity can operate independent of detoxifying enzyme activities in preventing the early stages of protein aggregate formation and associated cellular toxicities. We provide additional data that collectively supports the multiple functional roles that can be accomplished independent of each other. We present data indicating Hsp31 purified from yeast is more active compared to expression and purification from E. coli suggesting that posttranslational modifications could be important for Hsp31 to be fully active. We also compare the similarities and differences in activities among paralogs of Hsp31 supporting a model in which this protein family has overlapping but diverging roles in responding to various sources of cellular stresses. PMID:27097320

Comparison of melibiose utilizing baker's yeast strains produced by genetic engineering and classical breeding.

PubMed

Vincent, S F; Bell, P J; Bissinger, P; Nevalainen, K M

1999-02-01

Yeast strains currently used in the baking industry cannot fully utilize the trisaccharide raffinose found in beet molasses due to the absence of melibiase (alpha-galactosidase) activity. To overcome this deficiency, the MEL1 gene encoding melibiase enzyme was introduced into baker's yeast by both classical breeding and recombinant DNA technology. Both types of yeast strains were capable of vigorous fermentation in the presence of high levels of sucrose, making them suitable for the rapidly developing Asian markets where high levels of sugar are used in bread manufacture. Melibiase expression appeared to be dosage-dependent, with relatively low expression sufficient for complete melibiose utilization in a model fermentation system.

An S-Oxygenated [NiFe] Complex Modelling Sulfenate Intermediates of an O2 -Tolerant Hydrogenase.

PubMed

Lindenmaier, Nils J; Wahlefeld, Stefan; Bill, Eckhard; Szilvási, Tibor; Eberle, Christopher; Yao, Shenglai; Hildebrandt, Peter; Horch, Marius; Zebger, Ingo; Driess, Matthias

2017-02-13

To understand the molecular details of O 2 -tolerant hydrogen cycling by a soluble NAD + -reducing [NiFe] hydrogenase, we herein present the first bioinspired heterobimetallic S-oxygenated [NiFe] complex as a structural and vibrational spectroscopic model for the oxygen-inhibited [NiFe] active site. This compound and its non-S-oxygenated congener were fully characterized, and their electronic structures were elucidated in a combined experimental and theoretical study with emphasis on the bridging sulfenato moiety. Based on the vibrational spectroscopic properties of these complexes, we also propose novel strategies for exploring S-oxygenated intermediates in hydrogenases and similar enzymes. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

REBASE--a database for DNA restriction and modification: enzymes, genes and genomes.

PubMed

Roberts, Richard J; Vincze, Tamas; Posfai, Janos; Macelis, Dana

2015-01-01

REBASE is a comprehensive and fully curated database of information about the components of restriction-modification (RM) systems. It contains fully referenced information about recognition and cleavage sites for both restriction enzymes and methyltransferases as well as commercial availability, methylation sensitivity, crystal and sequence data. All genomes that are completely sequenced are analyzed for RM system components, and with the advent of PacBio sequencing, the recognition sequences of DNA methyltransferases (MTases) are appearing rapidly. Thus, Type I and Type III systems can now be characterized in terms of recognition specificity merely by DNA sequencing. The contents of REBASE may be browsed from the web http://rebase.neb.com and selected compilations can be downloaded by FTP (ftp.neb.com). Monthly updates are also available via email. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Grapevine Botryosphaeria dieback fungi have specific aggressiveness factor repertory involved in wood decay and stilbene metabolization.

PubMed

Stempien, Elodie; Goddard, Mary-Lorène; Wilhelm, Kim; Tarnus, Céline; Bertsch, Christophe; Chong, Julie

2017-01-01

Grapevine trunk diseases: Eutypa dieback, esca and Botryosphaeria dieback, which incidence has increased recently, are associated with several symptoms finally leading to the plant death. In the absence of efficient treatments, these diseases are a major problem for the viticulture; however, the factors involved in disease progression are not still fully identified. In order to get a better understanding of Botryosphaeria dieback development in grapevine, we have investigated different factors involved in Botryosphaeriaceae fungi aggressiveness. We first evaluated the activity of the wood-degrading enzymes of different isolates of Neofusicoccum parvum and Diplodia seriata, two major fungi associated with Botryosphaeria dieback. We further examinated the ability of these fungi to metabolize major grapevine phytoalexins: resveratrol and δ-viniferin. Our results demonstrate that Botryosphaeriaceae were characterized by differential wood decay enzymatic activities and have the capacity to rapidly degrade stilbenes. N. parvum is able to degrade parietal polysaccharides, whereas D. seriata has a better capacity to degrade lignin. Growth of both fungi exhibited a low sensitivity to resveratrol, whereas δ-viniferin has a fungistatic effect, especially on N. parvum Bourgogne S-116. We further show that Botryosphaeriaceae are able to metabolize rapidly resveratrol and δ-viniferin. The best stilbene metabolizing activity was measured for D. seriata. In conclusion, the different Botryosphaeriaceae isolates are characterized by a specific aggressiveness repertory. Wood and phenolic compound decay enzymatic activities could enable Botryosphaeriaceae to bypass chemical and physical barriers of the grapevine plant. The specific signature of Botryosphaeriaceae aggressiveness factors could explain the importance of fungi complexes in synergistic activity in order to fully colonize the host.

Some pharmacological effects of cinnamon and ginger herbs in obese diabetic rats

PubMed Central

Shalaby, Mostafa Abbas; Saifan, Hamed Yahya

2014-01-01

Aims: The present study was designed to assess some pharmacological effects of cinnamon (CAE) and ginger (GAE) aqueous extracts in obese diabetic rats, and to elucidate the potential mechanisms. Materials and Methods: Forty-two Sprague-Dawley rats were randomized into 6 equal groups. Group 1 was a negative control and the other groups were rendered obese by feeding rats on high-fat diet for 4 weeks. The obese rats were subcutaneously injected with alloxan for 5*days to induce diabetes. Group 2 was a positive control, and Groups 3, 4, 5 and 6 were orally given CAE in doses 200 and 400 mg/kg and GAE in the same doses, respectively for 6 weeks. Blood samples were collected for serum biochemical analyses. Kidneys were dissected out to assay activity of tissue antioxidant enzymes: Superoxide dismutase, glutathione peroxidase and catalase. Results: CAE and GAE significantly reduced body weight and body fat mass; normalized serum levels of liver enzymes; improved lipid profile; decreased blood glucose and leptin and increased insulin serum levels in obese diabetic rats. Both extracts also increased activity of kidney antioxidant enzymes. Conclusion: CAE and GAE exhibit anti-obesity, hepatoprotective, hypolipidemic, antidiabetic and anti-oxidant effects in obese diabetic rats. These results confirm the previous reports on both extracts. The potential mechanisms underlying these effects are fully discussed and clarified. Our results affirm the traditional use of cinnamon and ginger for treating patients suffering from obesity and diabetes. The obese diabetic rat model used in this study is a novel animal model used in pharmacology researches. PMID:26401364

Structural rearrangements occurring upon cofactor binding in the Mycobacterium smegmatis β-ketoacyl-acyl carrier protein reductase MabA.

PubMed

Küssau, Tanja; Flipo, Marion; Van Wyk, Niel; Viljoen, Albertus; Olieric, Vincent; Kremer, Laurent; Blaise, Mickaël

2018-05-01

In mycobacteria, the ketoacyl-acyl carrier protein (ACP) reductase MabA (designated FabG in other bacteria) catalyzes the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products. This first reductive step in the fatty-acid biosynthesis elongation cycle is essential for bacteria, which makes MabA/FabG an interesting drug target. To date, however, very few molecules targeting FabG have been discovered and MabA remains the only enzyme of the mycobacterial type II fatty-acid synthase that lacks specific inhibitors. Despite the existence of several MabA/FabG crystal structures, the structural rearrangement that occurs upon cofactor binding is still not fully understood. Therefore, unlocking this knowledge gap could help in the design of new inhibitors. Here, high-resolution crystal structures of MabA from Mycobacterium smegmatis in its apo, NADP + -bound and NADPH-bound forms are reported. Comparison of these crystal structures reveals the structural reorganization of the lid region covering the active site of the enzyme. The crystal structure of the apo form revealed numerous residues that trigger steric hindrance to the binding of NADPH and substrate. Upon NADPH binding, these residues are pushed away from the active site, allowing the enzyme to adopt an open conformation. The transition from an NADPH-bound to an NADP + -bound form is likely to facilitate release of the product. These results may be useful for subsequent rational drug design and/or for in silico drug-screening approaches targeting MabA/FabG.

Asymmetric bioreduction of acetophenones by Baker's yeast and its cell-free extract encapsulated in sol-gel silica materials

NASA Astrophysics Data System (ADS)

Kato, Katsuya; Nakamura, Hitomi; Nakanishi, Kazuma

2014-02-01

Baker's yeast (BY) encapsulated in silica materials was synthesized using a yeast cell suspension and its cell-free extract during a sol-gel reaction of tetramethoxysilane with nitric acid as a catalyst. The synthesized samples were fully characterized using various methods, such as scanning electron microscopy, nitrogen adsorption-desorption, Fourier transform infrared spectroscopy, thermogravimetry, and differential thermal analysis. The BY cells were easily encapsulated inside silica-gel networks, and the ratio of the cells in the silica gel was approximately 75 wt%, which indicated that a large volume of BY was trapped with a small amount of silica. The enzyme activity (asymmetric reduction of prochiral ketones) of BY and its cell-free extract encapsulated in silica gel was investigated in detail. The activities and enantioselectivities of free and encapsulated BY were similar to those of acetophenone and its fluorine derivatives, which indicated that the conformation structure of BY enzymes inside silica-gel networks did not change. In addition, the encapsulated BY exhibited considerably better solvent (methanol) stability and recyclability compared to free BY solution. We expect that the development of BY encapsulated in sol-gel silica materials will significantly impact the industrial-scale advancement of high-efficiency and low-cost biocatalysts for the synthesis of valuable chiral alcohols.

The role of nonbonded interactions in the conformational dynamics of organophosphorous hydrolase adsorbed onto functionalized mesoporous silica surfaces.

PubMed

Gomes, Diego E B; Lins, Roberto D; Pascutti, Pedro G; Lei, Chenghong; Soares, Thereza A

2010-01-14

The enzyme organophosphorous hydrolase (OPH) catalyzes the hydrolysis of a wide variety of organophosphorous compounds with high catalytic efficiency and broad substrate specificity. The immobilization of OPH in functionalized mesoporous silica (FMS) surfaces increases significantly its catalytic specific activity, as compared to the enzyme in solution, with important applications for the detection and decontamination of insecticides and chemical warfare agents. Experimental measurements of immobilization efficiency as a function of the charge and coverage percentage of different functional groups have been interpreted as electrostatic forces being the predominant interactions underlying the adsorption of OPH onto FMS surfaces. Explicit solvent molecular dynamics simulations have been performed for OPH in bulk solution and adsorbed onto two distinct interaction potential models of the FMS functional groups to investigate the relative contributions of nonbonded interactions to the conformational dynamics and adsorption of the protein. Our results support the conclusion that electrostatic interactions are responsible for the binding of OPH to the FMS surface. However, these results also show that van der Waals forces are detrimental for interfacial adhesion. In addition, it is found that OPH adsorption onto the FMS models favors a protein conformation whose active site is fully accessible to the substrate, in contrast to the unconfined protein.

Pancreatic bicarbonate secretion involves two proton pumps.

PubMed

Novak, Ivana; Wang, Jing; Henriksen, Katrine L; Haanes, Kristian A; Krabbe, Simon; Nitschke, Roland; Hede, Susanne E

2011-01-07

Pancreas secretes fluid rich in digestive enzymes and bicarbonate. The alkaline secretion is important in buffering of acid chyme entering duodenum and for activation of enzymes. This secretion is formed in pancreatic ducts, and studies to date show that plasma membranes of duct epithelium express H(+)/HCO(3)(-) transporters, which depend on gradients created by the Na(+)/K(+)-ATPase. However, the model cannot fully account for high-bicarbonate concentrations, and other active transporters, i.e. pumps, have not been explored. Here we show that pancreatic ducts express functional gastric and non-gastric H(+)-K(+)-ATPases. We measured intracellular pH and secretion in small ducts isolated from rat pancreas and showed their sensitivity to H(+)-K(+) pump inhibitors and ion substitutions. Gastric and non-gastric H(+)-K(+) pumps were demonstrated on RNA and protein levels, and pumps were localized to the plasma membranes of pancreatic ducts. Quantitative analysis of H(+)/HCO(3)(-) and fluid transport shows that the H(+)-K(+) pumps can contribute to pancreatic secretion in several species. Our results call for revision of the bicarbonate transport physiology in pancreas, and most likely other epithelia. Furthermore, because pancreatic ducts play a central role in several pancreatic diseases, it is of high relevance to understand the role of H(+)-K(+) pumps in pathophysiology.

A study of proteases and protease-inhibitor complexes in biological fluids

PubMed Central

Granelli-Piperno, A; Reich, E

1978-01-01

We have (a) screened a variety of cell lines and body fluids for plasminogen activators and (b) studied the activity of proteases bound to α2- macroglobulin after exposing the complexes to partial degradation and/or denaturing procedures to unmask proteolytic activity. The respective results show (a) that the plasminogen activators in urine and cell culture media are generally of lower molecular weight than those in plasma; and (b) that proteases bound to α2-macroglobulin recover the ability to attack macromolecular substrates after exposure to sodium dodecyl sulfate while retaining the electrophoretic mobility of the protease inhibitor complex. This indicates that the protease and inhibitor are probably linked by covalent bonds. In contrast, other complexes formed between proteases and inhibitors of lower molecular weight (such as soybean or Kunitz inhibitors) are fully dissociated by sodium dodecyl sulfate (SDS). The experiments described were based on a new procedure for detecting proteolytic enzyme activity in SDS-polyacrylamide gels. The method relies on solutions of nonionic detergents for extracting SDS, after which the electrophoretic gel is applied to an indicator gel consisting of a fibrin- agar mixture. The method is sensitive, permitting the detection of proteinases in less than 1 μl of fresh plasma, and it is effective for resolving small differences in molecular weight. The procedure can be quantitated and, with minor modifications appropriate to each particular system, it has been applied to a broad spectrum of serine enzymes and proenzymes, including some that function in the pathways of fibrinolysis, coagulation and kinin-generation. Other potential applications appear likely. PMID:78958

P-I class metalloproteinase from Bothrops moojeni venom is a post-proline cleaving peptidase with kininogenase activity: insights into substrate selectivity and kinetic behavior.

PubMed

Okamoto, Débora N; Kondo, Marcia Y; Oliveira, Lilian C G; Honorato, Rodrigo V; Zanphorlin, Leticia M; Coronado, Monika A; Araújo, Mariana S; da Motta, Guacyara; Veronez, Camila L; Andrade, Sheila S; Oliveira, Paulo S L; Arni, Raghuvir K; Cintra, Adelia C O; Sampaio, Suely V; Juliano, Maria A; Juliano, Luiz; Murakami, Mário T; Gouvea, Iuri E

2014-03-01

Snake venom metalloproteinases (SVMPs) belonging to P-I class are able to hydrolyze extracellular matrix proteins and coagulation factors triggering local and systemic reactions by multiple molecular mechanisms that are not fully understood. BmooMPα-I, a P-I class SMVP from Bothrops moojeni venom, was active upon neuro- and vaso-active peptides including angiotensin I, bradykinin, neurotensin, oxytocin and substance P. Interestingly, BmooMPα-I showed a strong bias towards hydrolysis after proline residues, which is unusual for most of characterized peptidases. Moreover, the enzyme showed kininogenase activity similar to that observed in plasma and cells by kallikrein. FRET peptide assays indicated a relative promiscuity at its S2-S'2 subsites, with proline determining the scissile bond. This unusual post-proline cleaving activity was confirmed by the efficient hydrolysis of the synthetic combinatorial library MCA-GXXPXXQ-EDDnp, described as resistant for canonical peptidases, only after Pro residues. Structural analysis of the tripeptide LPL complexed with BmooMPα-I, generated by molecular dynamics simulations, assisted in defining the subsites and provided the structural basis for subsite preferences such as the restriction of basic residues at the S2 subsite due to repulsive electrostatic effects and the steric impediment for large aliphatic or aromatic side chains at the S1 subsite. These new functional and structural findings provided a further understanding of the molecular mechanisms governing the physiological effects of this important class of enzymes in envenomation process. Copyright © 2014 Elsevier B.V. All rights reserved.

Probing the catalytic roles of n2-site glutamate residues in Escherichia coli glutamine synthetase by mutagenesis.

PubMed Central

Witmer, M. R.; Palmieri-Young, D.; Villafranca, J. J.

1994-01-01

The contribution of metal ion ligand type and charge to catalysis and regulation at the lower affinity metal ion site (n2 site) of Escherichia coli glutamine synthetase (GS) was tested by mutagenesis and kinetic analysis. The 2 glutamate residues at the n2 site, E129 and E357, were changed to E129D, E129H, E357H, E357Q, and E357D, representing conservative and nonconservative alterations. Unadenylylated and fully adenylylated enzyme forms were studied. The Mn(2+)-KD values, UV-cis and fluorescence emission properties were similar for all mutants versus WTGS, except E129H. For kinetic determinations with both Mn2+ and Mg2+, nonconservative mutants (E357H, E129H, E357Q) showed lower biosynthetic activities than conservative mutants (E129D, E357D). Relative to WTGS, all the unadenylylated Mn(2+)-activated enzymes showed reduced kcat/Km values for ATP (> 7-fold) and for glutamate (> 10-fold). Of the unadenylylated Mg(2+)-activated enzymes, only E129D showed kinetic parameters competitive with WTGS, and adenylylated E129D was a 20-fold better catalyst than WTGS. We propose the n2-site metal ion activates ADP for departure in the phosphorylation of glutamate by ATP to generate gamma-glutamyl phosphate. Alteration of the charge density at this metal ion alters the transition-state energy for phosphoryl group transfer and may affect ATP binding and/or ADP release. Thus, the steady-state kinetic data suggest that modifying the charge density increases the transition-state energies for chemical steps. Importantly, the data demonstrate that each ligand position has a specialized spatial environment and the charge of the ligand modulates the catalytic steps occurring at the metal ion. The data are discussed in the context of the known X-ray structures of GS. PMID:7849593

Serum protease activity in chronic kidney disease patients: The GANI_MED renal cohort

PubMed Central

Wolke, Carmen; Teumer, Alexander; Endlich, Karlhans; Endlich, Nicole; Rettig, Rainer; Stracke, Sylvia; Fiene, Beate; Aymanns, Simone; Felix, Stephan B; Hannemann, Anke

2016-01-01

Serum or plasma proteases have been associated with various diseases including cancer, inflammation, or reno-cardiovascular diseases. We aimed to investigate whether the enzymatic activities of serum proteases are associated with the estimated glomerular filtration rate (eGFR) in patients with different stages of chronic kidney disease (CKD). Our study population comprised 268 participants of the “Greifswald Approach to Individualized Medicine” (GANI_MED) cohort. Enzymatic activity of aminopeptidase A, aminopeptidase B, alanyl (membrane) aminopeptidase, insulin-regulated aminopeptidase, puromycin-sensitive aminopeptidase, leucine aminopeptidase 3, prolyl-endopeptidase (PEP), dipeptidyl peptidase 4 (DPP4), angiotensin I-converting enzyme, and angiotensin I-converting enzyme 2 (ACE2) proteases was measured in serum. Linear regression of the respective protease was performed on kidney function adjusted for age and sex. Kidney function was modeled either by the continuous Modification of Diet in Renal Disease (MDRD)-based eGFR or dichotomized by eGFR < 15 mL/min/1.73 m2 or <45 mL/min/1.73 m2, respectively. Results with a false discovery rate below 0.05 were deemed statistically significant. Among the 10 proteases investigated, only the activities of ACE2 and DPP4 were correlated with eGFR. Patients with lowest eGFR exhibited highest DPP4 and ACE2 activities. DPP4 and PEP were correlated with age, but all other serum protease activities showed no associations with age or sex. Our data indicate that ACE2 and DPP4 enzymatic activity are associated with the eGFR in patients with CKD. This finding distinguishes ACE2 and DPP4 from other serum peptidases analyzed and clearly indicates that further analyses are warranted to identify the precise role of these serum ectopeptidases in the pathogenesis of CKD and to fully elucidate underlying molecular mechanisms. Impact statement • Renal and cardiac diseases are very common and often occur concomitantly, resulting in increased morbidity and mortality. Understanding of molecular mechanisms linking both diseases is limited, available fragmentary data point to a role of the renin–angiotensin system (RAS) and, in particular, Ras-related peptidases. • Here, a comprehensive analysis of serum peptidase activities in patients with different stages of chronic kidney disease (CKD) is presented, with special emphasis given to RAS peptidases • The serum activities of the peptidases angiotensin I-converting enzyme 2 and dipeptidyl peptidase 4 were identified as closely associated with kidney function, specifically with the estimated glomerular filtration rate. The findings are discussed in the context of available data suggesting protective roles for both enzymes in reno-cardiac diseases. • The data add to our understanding of pathomechanisms underlying development and progression of CKD and indicate that both enzymes might represent potential pharmacological targets for the preservation of renal function. PMID:28038565

Serum protease activity in chronic kidney disease patients: The GANI_MED renal cohort.

PubMed

Wolke, Carmen; Teumer, Alexander; Endlich, Karlhans; Endlich, Nicole; Rettig, Rainer; Stracke, Sylvia; Fiene, Beate; Aymanns, Simone; Felix, Stephan B; Hannemann, Anke; Lendeckel, Uwe

2017-03-01

Serum or plasma proteases have been associated with various diseases including cancer, inflammation, or reno-cardiovascular diseases. We aimed to investigate whether the enzymatic activities of serum proteases are associated with the estimated glomerular filtration rate (eGFR) in patients with different stages of chronic kidney disease (CKD). Our study population comprised 268 participants of the "Greifswald Approach to Individualized Medicine" (GANI_MED) cohort. Enzymatic activity of aminopeptidase A, aminopeptidase B, alanyl (membrane) aminopeptidase, insulin-regulated aminopeptidase, puromycin-sensitive aminopeptidase, leucine aminopeptidase 3, prolyl-endopeptidase (PEP), dipeptidyl peptidase 4 (DPP4), angiotensin I-converting enzyme, and angiotensin I-converting enzyme 2 (ACE2) proteases was measured in serum. Linear regression of the respective protease was performed on kidney function adjusted for age and sex. Kidney function was modeled either by the continuous Modification of Diet in Renal Disease (MDRD)-based eGFR or dichotomized by eGFR < 15 mL/min/1.73 m 2 or <45 mL/min/1.73 m 2 , respectively. Results with a false discovery rate below 0.05 were deemed statistically significant. Among the 10 proteases investigated, only the activities of ACE2 and DPP4 were correlated with eGFR. Patients with lowest eGFR exhibited highest DPP4 and ACE2 activities. DPP4 and PEP were correlated with age, but all other serum protease activities showed no associations with age or sex. Our data indicate that ACE2 and DPP4 enzymatic activity are associated with the eGFR in patients with CKD. This finding distinguishes ACE2 and DPP4 from other serum peptidases analyzed and clearly indicates that further analyses are warranted to identify the precise role of these serum ectopeptidases in the pathogenesis of CKD and to fully elucidate underlying molecular mechanisms. Impact statement • Renal and cardiac diseases are very common and often occur concomitantly, resulting in increased morbidity and mortality. Understanding of molecular mechanisms linking both diseases is limited, available fragmentary data point to a role of the renin-angiotensin system (RAS) and, in particular, Ras-related peptidases. • Here, a comprehensive analysis of serum peptidase activities in patients with different stages of chronic kidney disease (CKD) is presented, with special emphasis given to RAS peptidases • The serum activities of the peptidases angiotensin I-converting enzyme 2 and dipeptidyl peptidase 4 were identified as closely associated with kidney function, specifically with the estimated glomerular filtration rate. The findings are discussed in the context of available data suggesting protective roles for both enzymes in reno-cardiac diseases. • The data add to our understanding of pathomechanisms underlying development and progression of CKD and indicate that both enzymes might represent potential pharmacological targets for the preservation of renal function.

Functional role of a distal (3'-phosphate) group of CoA in the recombinant human liver medium-chain acyl-CoA dehydrogenase-catalysed reaction.

PubMed Central

Peterson, K L; Srivastava, D K

1997-01-01

The X-ray crystallographic structure of medium-chain acyl-CoA dehydrogenase (MCAD)-octenoyl-CoA complex reveals that the 3'-phosphate group of CoA is confined to the exterior of the protein structure [approx. 15 A (1.5 nm) away from the enzyme active site], and is fully exposed to the outside solvent environment. To ascertain whether such a distal (3'-phosphate) fragment of CoA plays any significant role in the enzyme catalysis, we investigated the recombinant human liver MCAD (HMCAD)-catalysed reaction by using normal (phospho) and 3'-phosphate-truncated (dephospho) forms of octanoyl-CoA and butyryl-CoA substrates. The steady-state kinetic data revealed that deletion of the 3'-phosphate group from octanoyl-CoA substrate increased the turnover rate of the enzyme to about one-quarter, whereas that from butyryl-CoA substrate decreased the turnover rate of the enzyme to about one-fifth; the Km values of both these substrates were increased by 5-10-fold on deletion of the 3'-phosphate group from the corresponding acyl-CoA substrates. The transient kinetics for the reductive half-reaction, oxidative half-reaction and the dissociation 'off-rate' (of the reaction product from the oxidized enzyme site) were all found to be affected by deletions of the 3'-phosphate group from octanoyl-CoA and butyryl-CoA substrates. A cumulative account of these results reveals that, although the 3'-phosphate group of acyl-CoA substrates might seem 'useless' on the basis of the structural data, it has an essential functional role during HMCAD catalysis. PMID:9271097

Probing the Intermediacy of Covalent RNA Enzyme Complexes in RNA Modification Enzymes

PubMed Central

Chervin, Stephanie M.; Kittendorf, Jeffrey D.; Garcia, George A.

2009-01-01

Within the large and diverse group of RNA-modifying enzymes, a number of enzymes seem to form stable covalent linkages to their respective RNA substrates. A complete understanding of the chemical and kinetic mechanisms of these enzymes, some of which have identified pathological roles, is lacking. As part of our ongoing work studying the posttranscriptional modification of tRNA with queuine, we wish to understand fully the chemical and kinetic mechanisms involved in this key transglycosylation reaction. In our previous investigations, we have used a gel mobility-shift assay to characterize an apparent covalent enzyme-RNA intermediate believed to be operative in the catalytic pathway. However, the simple observation of a covalent complex is not sufficient to prove intermediacy. To be a true intermediate, the complex must be both chemically and kinetically competent. As a case study for the proof of intermediacy, we report the use of this gel-shift assay under mildly denaturing conditions to probe the kinetic competency of the covalent association between RNA and the tRNA modifying enzyme tRNA-guanine transglycosylase (TGT). PMID:17673081

Excretory nitrogen metabolism in the juvenile axolotl Ambystoma mexicanum: differences in aquatic and terrestrial environments.

PubMed

Loong, Ai M; Chew, Shit F; Ip, Yuen K

2002-01-01

The fully grown but nonmetamorphosed (juvenile) axolotl Ambystoma mexicanum was ureogenic and primarily ureotelic in water. A complete ornithine-urea cycle (OUC) was present in the liver. Aerial exposure impeded urea (but not ammonia) excretion, leading to a decrease in the percentage of nitrogen excreted as urea in the first 24 h. However, urea and not ammonia accumulated in the muscle, liver, and plasma during aerial exposure. By 48 h, the rate of urea excretion recovered fully, probably due to the greater urea concentration gradient in the kidney. It is generally accepted that an increase in carbamoyl phosphate synthetase activity is especially critical in the developmental transition from ammonotelism to ureotelism in the amphibian. Results from this study indicate that such a transition in A. mexicanum would have occurred before migration to land. Aerial exposure for 72 h exhibited no significant effect on carbamoyl phosphate synthetase-I activity or that of other OUC enzymes (with the exception of ornithine transcarbamoylase) from the liver of the juvenile A. mexicanum. This supports our hypothesis that the capacities of OUC enzymes present in the liver of the aquatic juvenile axolotl were adequate to prepare it for its invasion of the terrestrial environment. The high OUC capacity was further supported by the capability of the juvenile A. mexicanum to survive in 10 mM NH(4)Cl without accumulating amino acids in its body. The majority of the accumulating endogenous and exogenous ammonia was detoxified to urea, which led to a greater than twofold increase in urea levels in the muscle, liver, and plasma and a significant increase in urea excretion by hour 96. Hence, it can be concluded that the juvenile axolotl acquired ureotelism while submerged in water, and its hepatic capacity of urea synthesis was more than adequate to handle the toxicity of endogenous ammonia during migration to land.

HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II.

PubMed

Boulon, Séverine; Pradet-Balade, Bérengère; Verheggen, Céline; Molle, Dorothée; Boireau, Stéphanie; Georgieva, Marya; Azzag, Karim; Robert, Marie-Cécile; Ahmad, Yasmeen; Neel, Henry; Lamond, Angus I; Bertrand, Edouard

2010-09-24

RNA polymerases are key multisubunit cellular enzymes. Microscopy studies indicated that RNA polymerase I assembles near its promoter. However, the mechanism by which RNA polymerase II is assembled from its 12 subunits remains unclear. We show here that RNA polymerase II subunits Rpb1 and Rpb3 accumulate in the cytoplasm when assembly is prevented and that nuclear import of Rpb1 requires the presence of all subunits. Using MS-based quantitative proteomics, we characterized assembly intermediates. These included a cytoplasmic complex containing subunits Rpb1 and Rpb8 associated with the HSP90 cochaperone hSpagh (RPAP3) and the R2TP/Prefoldin-like complex. Remarkably, HSP90 activity stabilized incompletely assembled Rpb1 in the cytoplasm. Our data indicate that RNA polymerase II is built in the cytoplasm and reveal quality-control mechanisms that link HSP90 to the nuclear import of fully assembled enzymes. hSpagh also bound the free RPA194 subunit of RNA polymerase I, suggesting a general role in assembling RNA polymerases. Copyright © 2010 Elsevier Inc. All rights reserved.

HSP90 and Its R2TP/Prefoldin-like Cochaperone Are Involved in the Cytoplasmic Assembly of RNA Polymerase II

PubMed Central

Boireau, Stéphanie; Georgieva, Marya; Azzag, Karim; Robert, Marie-Cécile; Ahmad, Yasmeen; Neel, Henry; Lamond, Angus I.; Bertrand, Edouard

2015-01-01

SUMMARY RNA polymerases are key multisubunit cellular enzymes. Microscopy studies indicated that RNA polymerase I assembles near its promoter. However, the mechanism by which RNA polymerase II is assembled from its 12 subunits remains unclear. We show here that RNA polymerase II subunits Rpb1 and Rpb3 accumulate in the cytoplasm when assembly is prevented and that nuclear import of Rpb1 requires the presence of all subunits. Using MS-based quantitative proteomics, we characterized assembly intermediates. These included a cytoplasmic complex containing subunits Rpb1 and Rpb8 associated with the HSP90 cochaperone hSpagh (RPAP3) and the R2TP/Prefoldin-like complex. Remarkably, HSP90 activity stabilized incompletely assembled Rpb1 in the cytoplasm. Our data indicate that RNA polymerase II is built in the cytoplasm and reveal quality-control mechanisms that link HSP90 to the nuclear import of fully assembled enzymes. hSpagh also bound the free RPA194 subunit of RNA polymerase I, suggesting a general role in assembling RNA polymerases. PMID:20864038

Extremely high intracellular concentration of glucose-6-phosphate and NAD(H) in Deinococcus radiodurans.

PubMed

Yamashiro, Takumi; Murata, Kousaku; Kawai, Shigeyuki

2017-03-01

Deinococcus radiodurans is highly resistant to ionizing radiation and UV radiation, and oxidative stress caused by such radiations. NADP(H) seems to be important for this resistance (Slade and Radman, Microbiol Mol Biol Rev 75:133-191; Slade, Radman, Microbiol Mol Biol Rev 75:133-191, 2011), but the mechanism underlying the generation of NADP(H) or NAD(H) in D. radiodurans has not fully been addressed. Intracellular concentrations of NAD + , NADH, NADP + , and NADPH in D. radiodurans are also not determined yet. We found that cell extracts of D. radiodurans catalyzed reduction of NAD(P) + in vitro, indicating that D. radiodurans cells contain both enzymes and a high concentration of substrates for this activity. The enzyme and the substrate were attributed to glucose-6-phosphate dehydrogenase and glucose-6-phosphate of which intracellular concentration was extremely high. Unexpectedly, the intracellular concentration of NAD(H) was also much greater than that of NADP(H), suggesting some significant roles of NADH. These unusual features of this bacterium would shed light on a new aspect of physiology of this bacterium.

Saccharomyces cerevisiae YOR071C encodes the high affinity nicotinamide riboside transporter Nrt1.

PubMed

Belenky, Peter A; Moga, Tiberiu G; Brenner, Charles

2008-03-28

NAD(+) is an essential coenzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+)-consuming enzymes. Nicotinamide riboside is a recently discovered eukaryotic NAD(+) precursor converted to NAD(+) via the nicotinamide riboside kinase pathway and by nucleosidase activity and nicotinamide salvage. Nicotinamide riboside supplementation of yeast extends replicative life span on high glucose medium. The molecular basis for nicotinamide riboside uptake was unknown in any eukaryote. Here, we show that deletion of a single gene, YOR071C, abrogates nicotinamide riboside uptake without altering nicotinic acid or nicotinamide import. The gene, which is negatively regulated by Sum1, Hst1, and Rfm1, fully restores nicotinamide riboside import and utilization when resupplied to mutant yeast cells. The encoded polypeptide, Nrt1, is a predicted deca-spanning membrane protein related to the thiamine transporter, which functions as a pH-dependent facilitator with a K(m) for nicotinamide riboside of 22 microm. Nrt1-related molecules are conserved in particular fungi, suggesting a similar basis for nicotinamide riboside uptake.

Mutants of Pseudomonas cepacia G4 defective in catabolism of aromatic compounds and trichloroethylene.

PubMed Central

Shields, M S; Montgomery, S O; Cuskey, S M; Chapman, P J; Pritchard, P H

1991-01-01

Pseudomonas cepacia G4 possesses a novel pathway of toluene catabolism that is shown to be responsible for the degradation of trichloroethylene (TCE). This pathway involves conversion of toluene via o-cresol to 3-methylcatechol. In order to determine the enzyme of toluene degradation that is responsible for TCE degradation, chemically induced mutants, blocked in the toluene ortho-monooxygenase (TOM) pathway of G4, were examined. Mutants of the phenotypic class designated TOM A- were all defective in their ability to oxidize toluene, o-cresol, m-cresol, and phenol, suggesting that a single enzyme is responsible for conversion of these compounds to their hydroxylated products (3-methylcatechol from toluene, o-cresol, and m-cresol and catechol from phenol) in the wild type. Mutants of this class did not degrade TCE. Two other mutant classes which were blocked in toluene catabolism, TOM B-, which lacked catechol-2,3-dioxygenase, and TOM C-, which lacked 2-hydroxy-6-oxoheptadienoic acid hydrolase activity, were fully capable of TCE degradation. Therefore, TCE degradation is directly associated with the monooxygenation capability responsible for toluene, cresol, and phenol hydroxylation. PMID:1892384

Cross-Genotypic Examination of Hepatitis C Virus Polymerase Inhibitors Reveals a Novel Mechanism of Action for Thumb Binders

PubMed Central

Eltahla, Auda A.; Tay, Enoch; Douglas, Mark W.

2014-01-01

Direct-acting antivirals (DAAs) targeting proteins encoded by the hepatitis C virus (HCV) genome have great potential for the treatment of HCV infections. However, the efficacy of DAAs designed to target genotype 1 (G1) HCV against non-G1 viruses has not been characterized fully. In this study, we investigated the inhibitory activities of nonnucleoside inhibitors (NNIs) against the HCV RNA-dependent RNA polymerase (RdRp). We examined the ability of six NNIs to inhibit G1b, G2a, and G3a subgenomic replicons in cell culture, as well as in vitro transcription by G1b and G3a recombinant RdRps. Of the six G1 NNIs, only the palm II binder nesbuvir demonstrated activity against G1, G2, and G3 HCV, in both replicon and recombinant enzyme models. The thumb I binder JTK-109 also inhibited G1b and G3a replicons and recombinant enzymes but was 41-fold less active against the G2a replicon. The four other NNIs, which included a palm I binder (setrobuvir), two thumb II binders (lomibuvir and filibuvir), and a palm β-hairpin binder (tegobuvir), all showed at least 40-fold decreases in potency against G2a and G3a replicons and the G3a enzyme. This antiviral resistance was largely conferred by naturally occurring amino acid residues in the G2a and G3a RdRps that are associated with G1 resistance. Lomibuvir and filibuvir (thumb II binders) inhibited primer-dependent but not de novo activity of the G1b polymerase. Surprisingly, these compounds instead specifically enhanced the de novo activity at concentrations of ≥100 nM. These findings highlight a potential differential mode of RdRp inhibition for HCV NNIs, depending on their prospective binding pockets, and also demonstrate a surprising enhancement of de novo activity for thumb RdRp binders. These results also provide a better understanding of the antiviral coverage for these polymerase inhibitors, which will likely be used in future combinational interferon-free therapies. PMID:25246395

Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

PubMed

Bi, Xiaodong; Liu, Zhen

2014-12-16

Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

Engineering Escherichia coli Nicotinic Acid Mononucleotide Adenylyltransferase for Fully Active Amidated NAD Biosynthesis.

PubMed

Wang, Xueying; Zhou, Yongjin J; Wang, Lei; Liu, Wujun; Liu, Yuxue; Peng, Chang; Zhao, Zongbao K

2017-07-01

NAD and its reduced form NADH function as essential redox cofactors and have major roles in determining cellular metabolic features. NAD can be synthesized through the deamidated and amidated pathways, for which the key reaction involves adenylylation of nicotinic acid mononucleotide (NaMN) and nicotinamide mononucleotide (NMN), respectively. In Escherichia coli , NAD de novo biosynthesis depends on the protein NadD-catalyzed adenylylation of NaMN to nicotinic acid adenine dinucleotide (NaAD), followed by NAD synthase-catalyzed amidation. In this study, we engineered NadD to favor NMN for improved amidated pathway activity. We designed NadD mutant libraries, screened by a malic enzyme-coupled colorimetric assay, and identified two variants, 11B4 (Y84V/Y118D) and 16D8 (A86W/Y118N), with a high preference for NMN. Whereas in the presence of NMN both variants were capable of enabling the viability of cells of E. coli BW25113-derived NAD-auxotrophic strain YJE003, for which the last step of the deamidated pathway is blocked, the 16D8 expression strain could grow without exogenous NMN and accumulated a higher cellular NAD(H) level than BW25113 in the stationary phase. These mutants established fully active amidated NAD biosynthesis and offered a new opportunity to manipulate NAD metabolism for biocatalysis and metabolic engineering. IMPORTANCE Adenylylation of nicotinic acid mononucleotide (NaMN) and adenylylation of nicotinamide mononucleotide (NMN), respectively, are the key steps in the deamidated and amidated pathways for NAD biosynthesis. In most organisms, canonical NAD biosynthesis follows the deamidated pathway. Here we engineered Escherichia coli NaMN adenylyltransferase to favor NMN and expressed the mutant enzyme in an NAD-auxotrophic E. coli strain that has the last step of the deamidated pathway blocked. The engineered strain survived in M9 medium, which indicated the implementation of a functional amidated pathway for NAD biosynthesis. These results enrich our understanding of NAD biosynthesis and are valuable for manipulation of NAD homeostasis for metabolic engineering. Copyright © 2017 American Society for Microbiology.

Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant.

PubMed

Kamal, Md Zahid; Mohammad, Tabrez Anwar Shamim; Krishnamoorthy, G; Rao, Nalam Madhusudhana

2012-01-01

Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site.

How ABA block polymers activate cytochrome c in toluene: molecular dynamics simulation and experimental observation.

PubMed

Chen, Gong; Kong, Xian; Zhu, Jingying; Lu, Diannan; Liu, Zheng

2015-04-28

While the conjugation of enzymes with ABA copolymers has resulted in increased enzymatic activities in organic solvents, by several orders of magnitude, the underpinning mechanism has not been fully uncovered, particularly at the molecular level. In the present work, a coarse-grained molecular dynamics simulation of cytochrome c (Cyt c) conjugated with a PEO-PPO-PEO block copolymer (ABA) in toluene was simulated with Cyt c as a control. It is shown that the hydrophilic segments (PEO) of the conjugated block copolymer molecules tend to entangle around the hydrophilic patch of Cyt c, while the hydrophobic segments (PPO) extend into the toluene. At a lower temperature, the PEO tails tend to form a hairpin structure outside the conjugated protein, whereas the Cyt c-ABA conjugates tend to form larger aggregates. At a higher temperature, however, the PEO tails tend to adsorb onto the hydrophilic protein surface, thus improving the suspension of the Cyt c-ABA conjugates and, consequently, the contact with the substrate. Moreover, the temperature increase drives the conformational transition of the active site of Cyt c-ABA from an "inactive state" to an "activated state" and thus results in an enhanced activity. To validate the above simulations, Cyt c was conjugated to F127, an extensively used ABA copolymer. By elevating the temperature, a decrease in the average size of the Cyt c-F127 conjugates along with a great increase in the apparent activity in toluene was observed, as can be predicted from the molecular dynamics simulation. The above mentioned molecular simulations offer a molecular insight into the temperature-responsive behaviour of protein-ABA copolymers, which is helpful for the design and application of enzyme-polymer conjugates for industrial biocatalysis.

13C ENDOR Spectroscopy of Lipoxygenase-Substrate Complexes Reveals the Structural Basis for C-H Activation by Tunneling.

PubMed

Horitani, Masaki; Offenbacher, Adam R; Carr, Cody A Marcus; Yu, Tao; Hoeke, Veronika; Cutsail, George E; Hammes-Schiffer, Sharon; Klinman, Judith P; Hoffman, Brian M

2017-02-08

In enzymatic C-H activation by hydrogen tunneling, reduced barrier width is important for efficient hydrogen wave function overlap during catalysis. For native enzymes displaying nonadiabatic tunneling, the dominant reactive hydrogen donor-acceptor distance (DAD) is typically ca. 2.7 Å, considerably shorter than normal van der Waals distances. Without a ground state substrate-bound structure for the prototypical nonadiabatic tunneling system, soybean lipoxygenase (SLO), it has remained unclear whether the requisite close tunneling distance occurs through an unusual ground state active site arrangement or by thermally sampling conformational substates. Herein, we introduce Mn 2+ as a spin-probe surrogate for the SLO Fe ion; X-ray diffraction shows Mn-SLO is structurally faithful to the native enzyme. 13 C ENDOR then reveals the locations of 13 C10 and reactive 13 C11 of linoleic acid relative to the metal; 1 H ENDOR and molecular dynamics simulations of the fully solvated SLO model using ENDOR-derived restraints give additional metrical information. The resulting three-dimensional representation of the SLO active site ground state contains a reactive (a) conformer with hydrogen DAD of ∼3.1 Å, approximately van der Waals contact, plus an inactive (b) conformer with even longer DAD, establishing that stochastic conformational sampling is required to achieve reactive tunneling geometries. Tunneling-impaired SLO variants show increased DADs and variations in substrate positioning and rigidity, confirming previous kinetic and theoretical predictions of such behavior. Overall, this investigation highlights the (i) predictive power of nonadiabatic quantum treatments of proton-coupled electron transfer in SLO and (ii) sensitivity of ENDOR probes to test, detect, and corroborate kinetically predicted trends in active site reactivity and to reveal unexpected features of active site architecture.

Hinokitiol Inhibits Migration of A549 Lung Cancer Cells via Suppression of MMPs and Induction of Antioxidant Enzymes and Apoptosis.

PubMed

Jayakumar, Thanasekaran; Liu, Chao-Hong; Wu, Guan-Yi; Lee, Tzu-Yin; Manubolu, Manjunath; Hsieh, Cheng-Ying; Yang, Chih-Hao; Sheu, Joen-Rong

2018-03-22

Hinokitiol, a natural monoterpenoid from the heartwood of Calocedrus formosana , has been reported to have anticancer effects against various cancer cell lines. However, the detailed molecular mechanisms and the inhibiting roles of hinokitiol on adenocarcinoma A549 cells remain to be fully elucidated. Thus, the current study was designed to evaluate the effect of hinokitiol on the migration of human lung adenocarcinoma A549 cells in vitro. The data demonstrates that hinokitiol does not effectively inhibit the viability of A549 cells at up to a 10 µM concentration. When treated with non-toxic doses (1-5 µM) of hinokitiol, the cell migration is markedly suppressed at 5 µM. Hinokitiol significantly reduced p53 expression, followed by attenuation of Bax in A549 cells. A dose-dependent inhibition of activated caspase-9 and -3 was observed in the presence of hinokitiol. An observed increase in protein expression of matrix metalloproteinases (MMPs) -2/-9 in A549 cells was significantly inhibited by hinokitiol. Remarkably, when A549 cells were subjected to hinokitiol (1-5 µM), there was an increase in the activities of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) from the reduction in cells. In addition, the incubation of A549 cells with hinokitiol significantly activated the cytochrome c expression, which may be triggered by activation of caspase-9 followed by caspase-3. These observations indicate that hinokitiol inhibited the migration of lung cancer A549 cells through several mechanisms, including the activation of caspases-9 and -3, induction of p53/Bax and antioxidant CAT and SOD, and reduction of MMP-2 and -9 activities. It also induces cytochrome c expression. These findings demonstrate a new therapeutic potential for hinokitiol in lung cancer chemoprevention.

Enzyme/non-enzyme discrimination and prediction of enzyme active site location using charge-based methods.

PubMed

Bate, Paul; Warwicker, Jim

2004-07-02

Calculations of charge interactions complement analysis of a characterised active site, rationalising pH-dependence of activity and transition state stabilisation. Prediction of active site location through large DeltapK(a)s or electrostatic strain is relevant for structural genomics. We report a study of ionisable groups in a set of 20 enzymes, finding that false positives obscure predictive potential. In a larger set of 156 enzymes, peaks in solvent-space electrostatic properties are calculated. Both electric field and potential match well to active site location. The best correlation is found with electrostatic potential calculated from uniform charge density over enzyme volume, rather than from assignment of a standard atom-specific charge set. Studying a shell around each molecule, for 77% of enzymes the potential peak is within that 5% of the shell closest to the active site centre, and 86% within 10%. Active site identification by largest cleft, also with projection onto a shell, gives 58% of enzymes for which the centre of the largest cleft lies within 5% of the active site, and 70% within 10%. Dielectric boundary conditions emphasise clefts in the uniform charge density method, which is suited to recognition of binding pockets embedded within larger clefts. The variation of peak potential with distance from active site, and comparison between enzyme and non-enzyme sets, gives an optimal threshold distinguishing enzyme from non-enzyme. We find that 87% of the enzyme set exceeds the threshold as compared to 29% of the non-enzyme set. Enzyme/non-enzyme homologues, "structural genomics" annotated proteins and catalytic/non-catalytic RNAs are studied in this context.

Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP).

PubMed

Ma, Hongyan; Delafield, Daniel G; Wang, Zhe; You, Jianlan; Wu, Si

2017-04-01

The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion. Graphical Abstract ᅟ.

Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP)

NASA Astrophysics Data System (ADS)

Ma, Hongyan; Delafield, Daniel G.; Wang, Zhe; You, Jianlan; Wu, Si

2017-04-01

The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion.

Co-immobilization of multiple enzymes by metal coordinated nucleotide hydrogel nanofibers: improved stability and an enzyme cascade for glucose detection

NASA Astrophysics Data System (ADS)

Liang, Hao; Jiang, Shuhui; Yuan, Qipeng; Li, Guofeng; Wang, Feng; Zhang, Zijie; Liu, Juewen

2016-03-01

Preserving enzyme activity and promoting synergistic activity via co-localization of multiple enzymes are key topics in bionanotechnology, materials science, and analytical chemistry. This study reports a facile method for co-immobilizing multiple enzymes in metal coordinated hydrogel nanofibers. Specifically, four types of protein enzymes, including glucose oxidase, Candida rugosa lipase, α-amylase, and horseradish peroxidase, were respectively encapsulated in a gel nanofiber made of Zn2+ and adenosine monophosphate (AMP) with a simple mixing step. Most enzymes achieved quantitative loading and retained full activity. At the same time, the entrapped enzymes were more stable against temperature variation (by 7.5 °C), protease attack, extreme pH (by 2-fold), and organic solvents. After storing for 15 days, the entrapped enzyme still retained 70% activity while the free enzyme nearly completely lost its activity. Compared to nanoparticles formed with AMP and lanthanide ions, the nanofiber gels allowed much higher enzyme activity. Finally, a highly sensitive and selective biosensor for glucose was prepared using the gel nanofiber to co-immobilize glucose oxidase and horseradish peroxidase for an enzyme cascade system. A detection limit of 0.3 μM glucose with excellent selectivity was achieved. This work indicates that metal coordinated materials using nucleotides are highly useful for interfacing with biomolecules.Preserving enzyme activity and promoting synergistic activity via co-localization of multiple enzymes are key topics in bionanotechnology, materials science, and analytical chemistry. This study reports a facile method for co-immobilizing multiple enzymes in metal coordinated hydrogel nanofibers. Specifically, four types of protein enzymes, including glucose oxidase, Candida rugosa lipase, α-amylase, and horseradish peroxidase, were respectively encapsulated in a gel nanofiber made of Zn2+ and adenosine monophosphate (AMP) with a simple mixing step. Most enzymes achieved quantitative loading and retained full activity. At the same time, the entrapped enzymes were more stable against temperature variation (by 7.5 °C), protease attack, extreme pH (by 2-fold), and organic solvents. After storing for 15 days, the entrapped enzyme still retained 70% activity while the free enzyme nearly completely lost its activity. Compared to nanoparticles formed with AMP and lanthanide ions, the nanofiber gels allowed much higher enzyme activity. Finally, a highly sensitive and selective biosensor for glucose was prepared using the gel nanofiber to co-immobilize glucose oxidase and horseradish peroxidase for an enzyme cascade system. A detection limit of 0.3 μM glucose with excellent selectivity was achieved. This work indicates that metal coordinated materials using nucleotides are highly useful for interfacing with biomolecules. Electronic supplementary information (ESI) available: Additional methods, IR and XRD spectroscopy, enzyme loading capacity, enzyme kinetic parameters, and enzyme stability data. See DOI: 10.1039/c5nr08734a

Living bacteria in silica gels

NASA Astrophysics Data System (ADS)

Nassif, Nadine; Bouvet, Odile; Noelle Rager, Marie; Roux, Cécile; Coradin, Thibaud; Livage, Jacques

2002-09-01

The encapsulation of enzymes within silica gels has been extensively studied during the past decade for the design of biosensors and bioreactors. Yeast spores and bacteria have also been recently immobilized within silica gels where they retain their enzymatic activity, but the problem of the long-term viability of whole cells in an inorganic matrix has never been fully addressed. It is a real challenge for the development of sol-gel processes. Generic tests have been performed to check the viability of Escherichia coli bacteria in silica gels. Surprisingly, more bacteria remain culturable in the gel than in an aqueous suspension. The metabolic activity of the bacteria towards glycolysis decreases slowly, but half of the bacteria are still viable after one month. When confined within a mineral environment, bacteria do not form colonies. The exchange of chemical signals between isolated bacteria rather than aggregates can then be studied, a point that could be very important for 'quorum sensing'.

Regulated Extracellular Choline Acetyltransferase Activity— The Plausible Missing Link of the Distant Action of Acetylcholine in the Cholinergic Anti-Inflammatory Pathway

PubMed Central

Aeinehband, Shahin; Behbahani, Homira; Grandien, Alf; Nilsson, Bo; Ekdahl, Kristina N.; Lindblom, Rickard P. F.; Piehl, Fredrik; Darreh-Shori, Taher

2013-01-01

Acetylcholine (ACh), the classical neurotransmitter, also affects a variety of nonexcitable cells, such as endothelia, microglia, astrocytes and lymphocytes in both the nervous system and secondary lymphoid organs. Most of these cells are very distant from cholinergic synapses. The action of ACh on these distant cells is unlikely to occur through diffusion, given that ACh is very short-lived in the presence of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), two extremely efficient ACh-degrading enzymes abundantly present in extracellular fluids. In this study, we show compelling evidence for presence of a high concentration and activity of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT) in human cerebrospinal fluid (CSF) and plasma. We show that ChAT levels are physiologically balanced to the levels of its counteracting enzymes, AChE and BuChE in the human plasma and CSF. Equilibrium analyses show that soluble ChAT maintains a steady-state ACh level in the presence of physiological levels of fully active ACh-degrading enzymes. We show that ChAT is secreted by cultured human-brain astrocytes, and that activated spleen lymphocytes release ChAT itself rather than ACh. We further report differential CSF levels of ChAT in relation to Alzheimer’s disease risk genotypes, as well as in patients with multiple sclerosis, a chronic neuroinflammatory disease, compared to controls. Interestingly, soluble CSF ChAT levels show strong correlation with soluble complement factor levels, supporting a role in inflammatory regulation. This study provides a plausible explanation for the long-distance action of ACh through continuous renewal of ACh in extracellular fluids by the soluble ChAT and thereby maintenance of steady-state equilibrium between hydrolysis and synthesis of this ubiquitous cholinergic signal substance in the brain and peripheral compartments. These findings may have important implications for the role of cholinergic signaling in states of inflammation in general and in neurodegenerative disease, such as Alzheimer’s disease and multiple sclerosis in particular. PMID:23840379

Single or Multiple Access Channels to the CYP450s Active Site? An Answer from Free Energy Simulations of the Human Aromatase Enzyme.

PubMed

Magistrato, Alessandra; Sgrignani, Jacopo; Krause, Rolf; Cavalli, Andrea

2017-05-04

Cytochromes P450 (CYP450s), in particular, CYP19A1 and CYP17A1, are key clinical targets of breast and prostate anticancer therapies, critical players in drug metabolism, and their overexpression in tumors is associated with drug resistance. In these enzymes, ligand (substrates, drugs) metabolism occurs in deeply buried active sites accessible only via several grueling channels, whose exact biological role remains unclear. Gaining direct insights on the mechanism by which ligands travel in and out is becoming increasingly important given that channels are involved in the modulation of binding/dissociation kinetics and the specificity of ligands toward a CYP450. This has profound implications for enzymatic efficiency and drug efficacy/toxicity. Here, by applying free energy methods, for a cumulative simulation time of 20 μs, we provide detailed atomistic characterization and free energy profiles of the entry/exit routes preferentially followed by a substrate (androstenedione) and a last-generation inhibitor (letrozole) to/from the catalytic site of CYP19A1 (the human aromatase (HA) enzyme), a key clinical target against breast cancer, studied here as prototypical CYP450. Despite the remarkably different size/shape/hydrophobicity of the ligands, two channels appear accessible to their entrance, while only one exit route appears to be preferential. Our study shows that the preferential paths may be conserved among different CYP450s. Moreover, our results highlight that, at least in the case of HA, ligand channeling is associated with large enzyme structural rearrangements. A wise choice of the computational method and very long simulations are, thus, required to obtain fully converged quantitative free energy profiles, which might be used to design novel biocatalysts or next-generation cytochrome inhibitors with an in silico tuned K m .

A role for the peroxisomal 3-ketoacyl-CoA thiolase B enzyme in the control of PPARα-mediated upregulation of SREBP-2 target genes in the liver.

PubMed

Fidaleo, Marco; Arnauld, Ségolène; Clémencet, Marie-Claude; Chevillard, Grégory; Royer, Marie-Charlotte; De Bruycker, Melina; Wanders, Ronald J A; Athias, Anne; Gresti, Joseph; Clouet, Pierre; Degrace, Pascal; Kersten, Sander; Espeel, Marc; Latruffe, Norbert; Nicolas-Francès, Valérie; Mandard, Stéphane

2011-05-01

Peroxisomal 3-ketoacyl-CoA thiolase B (Thb) catalyzes the final step in the peroxisomal β-oxidation of straight-chain acyl-CoAs and is under the transcription control of the nuclear hormone receptor PPARα. PPARα binds to and is activated by the synthetic compound Wy14,643 (Wy). Here, we show that the magnitude of Wy-mediated induction of peroxisomal β-oxidation of radiolabeled (1-(14)C) palmitate was significantly reduced in mice deficient for Thb. In contrast, mitochondrial β-oxidation was unaltered in Thb(-/-) mice. Given that Wy-treatment induced Acox1 and MFP-1/-2 activity at a similar level in both genotypes, we concluded that the thiolase step alone was responsible for the reduced peroxisomal β-oxidation of fatty acids. Electron microscopic analysis and cytochemical localization of catalase indicated that peroxisome proliferation in the liver after Wy-treatment was normal in Thb(-/-) mice. Intriguingly, micro-array analysis revealed that mRNA levels of genes encoding cholesterol biosynthesis enzymes were upregulated by Wy in Wild-Type (WT) mice but not in Thb(-/-) mice, which was confirmed at the protein level for the selected genes. The non-induction of genes encoding cholesterol biosynthesis enzymes by Wy in Thb(-/-) mice appeared to be unrelated to defective SREBP-2 or PPARα signaling. No difference was observed in the plasma lathosterol/cholesterol ratio (a marker for de novo cholesterol biosynthesis) between Wy-treated WT and Thb(-/-) mice, suggesting functional compensation. Overall, we conclude that ThA and SCPx/SCP2 thiolases cannot fully compensate for the absence of ThB. In addition, our data indicate that ThB is involved in the regulation of genes encoding cholesterol biosynthesis enzymes in the liver, suggesting that the peroxisome could be a promising candidate for the correction of cholesterol imbalance in dyslipidemia. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Structural and functional insights into RHA-P, a bacterial GH106 α-L-rhamnosidase from Novosphingobium sp. PP1Y.

PubMed

Mensitieri, Francesca; De Lise, Federica; Strazzulli, Andrea; Moracci, Marco; Notomista, Eugenio; Cafaro, Valeria; Bedini, Emiliano; Sazinsky, Matthew Howard; Trifuoggi, Marco; Di Donato, Alberto; Izzo, Viviana

2018-04-18

α-L-Rhamnosidases (α-RHAs, EC 3.2.1.40) are glycosyl hydrolases (GHs) hydrolyzing terminal α-l-rhamnose residues from different substrates such as heteropolysaccharides, glycosylated proteins and natural flavonoids. Although the possibility to hydrolyze rhamnose from natural flavonoids has boosted the use of these enzymes in several biotechnological applications over the past decades, to date only few bacterial rhamnosidases have been fully characterized and only one crystal structure of a rhamnosidase of the GH106 family has been described. In our previous work, an α-l-rhamnosidase belonging to this family, named RHA-P, was isolated from the marine microorganism Novosphingobium sp. PP1Y. The initial biochemical characterization highlighted the biotechnological potential of RHA-P for bioconversion applications. In this work, further functional and structural characterization of the enzyme is provided. The recombinant protein was obtained fused to a C-terminal His-tag and, starting from the periplasmic fractions of induced recombinant cells of E. coli strain BL21(DE3), was purified through a single step purification protocol. Homology modeling of RHA-P in combination with a site directed mutagenesis analysis confirmed the function of residues D503, E506, E644, likely located at the catalytic site of RHA-P. In addition, a kinetic characterization of the enzyme on natural flavonoids such as naringin, rutin, hesperidin and quercitrin was performed. RHA-P showed activity on all flavonoids tested, with a catalytic efficiency comparable or even higher than other bacterial α-RHAs described in literature. The results confirm that RHA-P is able to hydrolyze both α-1,2 and α-1,6 glycosidic linkages, and suggest that the enzyme may locate different polyphenolic aromatic moities in the active site. Copyright © 2018 Elsevier Inc. All rights reserved.

County-scale spatial distribution of soil enzyme activities and enzyme activity indices in agricultural land: implications for soil quality assessment.

PubMed

Tan, Xiangping; Xie, Baoni; Wang, Junxing; He, Wenxiang; Wang, Xudong; Wei, Gehong

2014-01-01

Here the spatial distribution of soil enzymatic properties in agricultural land was evaluated on a county-wide (567 km(2)) scale in Changwu, Shaanxi Province, China. The spatial variations in activities of five hydrolytic enzymes were examined using geostatistical methods. The relationships between soil enzyme activities and other soil properties were evaluated using both an integrated total enzyme activity index (TEI) and the geometric mean of enzyme activities (GME). At the county scale, soil invertase, phosphatase, and catalase activities were moderately spatially correlated, whereas urease and dehydrogenase activities were weakly spatially correlated. Correlation analysis showed that both TEI and GME were better correlated with selected soil physicochemical properties than single enzyme activities. Multivariate regression analysis showed that soil OM content had the strongest positive effect while soil pH had a negative effect on the two enzyme activity indices. In addition, total phosphorous content had a positive effect on TEI and GME in orchard soils, whereas alkali-hydrolyzable nitrogen and available potassium contents, respectively, had negative and positive effects on these two enzyme indices in cropland soils. The results indicate that land use changes strongly affect soil enzyme activities in agricultural land, where TEI provides a sensitive biological indicator for soil quality.

Glycyl radical activating enzymes: Structure, mechanism, and substrate interactions☆

PubMed Central

Shisler, Krista A.; Broderick, Joan B.

2014-01-01

The glycyl radical enzyme activating enzymes (GRE–AEs) are a group of enzymes that belong to the radical S-adenosylmethionine (SAM) superfamily and utilize a [4Fe–4S] cluster and SAM to catalyze H-atom abstraction from their substrate proteins. GRE–AEs activate homodimeric proteins known as glycyl radical enzymes (GREs) through the production of a glycyl radical. After activation, these GREs catalyze diverse reactions through the production of their own substrate radicals. The GRE–AE pyruvate formate lyase activating enzyme (PFL-AE) is extensively characterized and has provided insights into the active site structure of radical SAM enzymes including GRE–AEs, illustrating the nature of the interactions with their corresponding substrate GREs and external electron donors. This review will highlight research on PFL-AE and will also discuss a few GREs and their respective activating enzymes. PMID:24486374

Nitric oxide mitigates arsenic-induced oxidative stress and genotoxicity in Vicia faba L.

PubMed

Shukla, Pratiksha; Singh, A K

2015-09-01

The protective effects of nitric oxide (NO) against arsenic (As)-induced structural disturbances in Vicia faba have been investigated. As treatment (0.25, 0.50, and 1 mM) resulted in a declined growth of V. faba seedlings. Arsenic treatment stimulates the activity of SOD and CAT while the activities of APX and GST content were decreased. The oxidative stress markers such as superoxide radical, hydrogen peroxide and malondialdehyde (lipid peroxidation) contents were enhanced by As. Overall results revealed that significant accumulation of As suppressed growth, photosynthesis, antioxidant enzymes (SOD, CAT, APX, and GST activity), mitotic index, and induction of different chromosomal abnormalities, hence led to oxidative stress. The concentration of SNP (0.02 mM) was very effective in counteracting the adverse effect of As toxicity. These abnormalities use partially or fully reversed by a simultaneous application of As and NO donor and sodium nitroprusside and has an ameliorating effect against As-induced oxidative stress and genotoxicity in V. faba roots.

[Interaction between CYP450 enzymes and metabolism of traditional Chinese medicine as well as enzyme activity assay].

PubMed

Lu, Tu-lin; Su, Lian-lin; Ji, De; Gu, Wei; Mao, Chun-qin

2015-09-01

Drugs are exogenous compounds for human bodies, and will be metabolized by many enzymes after administration. CYP450 enzyme, as a major metabolic enzyme, is an important phase I drug metabolizing enzyme. In human bodies, about 75% of drug metabolism is conducted by CYP450 enzymes, and CYP450 enzymes is the key factor for drug interactions between traditional Chinese medicine( TCM) -TCM, TCM-medicine and other drug combination. In order to make clear the interaction between metabolic enzymes and TCM metabolism, we generally chose the enzymatic activity as an evaluation index. That is to say, the enhancement or reduction of CYP450 enzyme activity was used to infer the inducing or inhibitory effect of active ingredients and extracts of traditional Chinese medicine on enzymes. At present, the common method for measuring metabolic enzyme activity is Cocktail probe drugs, and it is the key to select the suitable probe substrates. This is of great significance for study drug's absorption, distribution, metabolism and excretion (ADME) process in organisms. The study focuses on the interaction between TCMs, active ingredients, herbal extracts, cocktail probe substrates as well as CYP450 enzymes, in order to guide future studies.

Omics Analyses of Trichoderma reesei CBS999.97 and QM6a Indicate the Relevance of Female Fertility to Carbohydrate-Active Enzyme and Transporter Levels.

PubMed

Tisch, Doris; Pomraning, Kyle R; Collett, James R; Freitag, Michael; Baker, Scott E; Chen, Chia-Ling; Hsu, Paul Wei-Che; Chuang, Yu Chien; Schuster, Andre; Dattenböck, Christoph; Stappler, Eva; Sulyok, Michael; Böhmdorfer, Stefan; Oberlerchner, Josua; Wang, Ting-Fang; Schmoll, Monika

2017-11-15

The filamentous fungus Trichoderma reesei is found predominantly in the tropics but also in more temperate regions, such as Europe, and is widely known as a producer of large amounts of plant cell wall-degrading enzymes. We sequenced the genome of the sexually competent isolate CBS999.97, which is phenotypically different from the female sterile strain QM6a but can cross sexually with QM6a. Transcriptome data for growth on cellulose showed that entire carbohydrate-active enzyme (CAZyme) families are consistently differentially regulated between these strains. We evaluated backcrossed strains of both mating types, which acquired female fertility from CBS999.97 but maintained a mostly QM6a genetic background, and we could thereby distinguish between the effects of strain background and female fertility or mating type. We found clear regulatory differences associated with female fertility and female sterility, including regulation of CAZyme and transporter genes. Analysis of carbon source utilization, transcriptomes, and secondary metabolites in these strains revealed that only a few changes in gene regulation are consistently correlated with different mating types. Different strain backgrounds (QM6a versus CBS999.97) resulted in the most significant alterations in the transcriptomes and in carbon source utilization, with decreased growth of CBS999.97 on several amino acids (for example proline or alanine), which further correlated with the downregulation of genes involved in the respective pathways. In combination, our findings support a role of fertility-associated processes in physiology and gene regulation and are of high relevance for the use of sexual crossing in combining the characteristics of two compatible strains or quantitative trait locus (QTL) analysis. IMPORTANCE Trichoderma reesei is a filamentous fungus with a high potential for secretion of plant cell wall-degrading enzymes. We sequenced the genome of the fully fertile field isolate CBS999.97 and analyzed its gene regulation characteristics in comparison with the commonly used laboratory wild-type strain QM6a, which is not female fertile. Additionally, we also evaluated fully fertile strains with genotypes very close to that of QM6a in order to distinguish between strain-specific and fertility-specific characteristics. We found that QM6a and CBS999.97 clearly differ in their growth patterns on different carbon sources, CAZyme gene regulation, and secondary metabolism. Importantly, we found altered regulation of 90 genes associated with female fertility, including CAZyme genes and transporter genes, but only minor mating type-dependent differences. Hence, when using sexual crossing in research and for strain improvement, it is important to consider female fertile and female sterile strains for comparison with QM6a and to achieve optimal performance. Copyright © 2017 Tisch et al.

Stabilization of enzymes in ionic liquids via modification of enzyme charge.

PubMed

Nordwald, Erik M; Kaar, Joel L

2013-09-01

Due to the propensity of ionic liquids (ILs) to inactivate enzymes, the development of strategies to improve enzyme utility in these solvents is critical to fully exploit ILs for biocatalysis. We have developed a strategy to broadly improve enzyme utility in ILs based on elucidating the effect of charge modifications on the function of enzymes in IL environments. Results of stability studies in aqueous-IL mixtures indicated a clear connection between the ratio of enzyme-containing positive-to-negative sites and enzyme stability in ILs. Stability studies of the effect of [BMIM][Cl] and [EMIM][EtSO4 ] on chymotrypsin specifically found an optimum ratio of positively-charged amine-to-negatively-charged acid groups (0.39). At this ratio, the half-life of chymotrypsin was increased 1.6- and 4.3-fold relative to wild-type chymotrypsin in [BMIM][Cl] and [EMIM][EtSO4 ], respectively. The half-lives of lipase and papain were similarly increased as much as 4.0 and 2.4-fold, respectively, in [BMIM][Cl] by modifying the ratio of positive-to-negative sites of each enzyme. More generally, the results of stability studies found that modifications that reduce the ratio of enzyme-containing positive-to-negative sites improve enzyme stability in ILs. Understanding the impact of charge modification on enzyme stability in ILs may ultimately be exploited to rationally engineer enzymes for improved function in IL environments. Copyright © 2013 Wiley Periodicals, Inc.

Distribution of enzyme activity hotspots induced by earthworms in top- and subsoil

NASA Astrophysics Data System (ADS)

Hoang, D. T. T.

2016-12-01

Earthworms (Lumbricus terrestris L.) not only affect soil physics, but they also boost microbial activities and consequently create important hotspots of microbial mediated carbon and nutrient turnover through their burrowing activity. However, it is still unknown to which extend earthworms change the enzyme distribution and activity inside their burrows in top- and subsoil horizons. We hypothesized that earthworm burrows, which are enriched in available substrates, have higher percentage of enzyme activity hotspots than soil without earthworms, and that enzyme activities decreased with increasing depth because of the increasing recalcitrance of organic matter in subsoil. We visualized enzyme distribution inside and outside of worm burrows (biopores) by in situ soil zymography and measured enzyme kinetics of 6 enzymes - β-glucosidase (GLU), cellobiohydrolase (CBH), xylanase (XYL), chitinase (NAG), leucine aminopeptidase (LAP) and acid phosphatase (APT) - in pore and bulk soil material up to 105 cm. Zymography showed a heterogeneous distribution of hotspots in worm burrows. The hotspot areas was 2.4 to 14 times larger in the burrows than in soil without earthworms. However, the dispersion index of hotspot distribution showed more aggregated hotspots in soil without earthworms than in soil with earthworms and burrow wall. Enzyme activities decreased with depth, by a factor of 2 to 8 due to fresh C input from the soil surface. Compared to bulk soil, enzyme activities in topsoil biopores were up to 11 times higher for all enzymes, but in the subsoil activities of XYL, NAG and APT were lower in earthworm biopores than bulk soil. In conclusion, hotspots were twice as concentrated close to earthworm burrows as in surrounding soil. Earthworms exerted stronger effects on enzyme activities in biopores in the topsoil than in subsoil. Keywords: Earthworms, hotspots, enzyme activities, enzyme distribution, subsoil

Current concepts on selected plant secondary metabolites with promising inhibitory effects against enzymes linked to Alzheimer's disease.

PubMed

Orhan, I Erdogan

2012-01-01

Alzheimer's disease (AD) has become one of the deadliest diseases for human beings with special incidence in elderly population. It is a progressive neurodegenerative disease and the most prevalent cause of dementia. The neuropathology of AD has not been fully elucidated yet, however, cholinergic hypothesis is the most accepted theory nowadays, resulting from the cholinergic deficit emerging in the brains of AD patients. Shortage of the neurotransmitters, acetylcholine and butyrylcholine has been demonstrated, and therefore, inhibition of the enzymes; acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) that break down acetylcholine and butyrylcholine has become a standard approach for AD treatment. However, cholinesterase inhibitors are only effective in symptomatic treatment and have no ability to impede the disease. The pathogenesis of AD is highly complex and another hypothesis is the formation of amyloid plaques containing beta-amyloid peptide, which causes neurolesions in the brains of AD patients. Beta-amyloid peptide is generated after the sequential cleavage of amyloid precursor protein, especially by the beta- and gamma-secretase in the amyloidogenic pathway. The secretases involved in the processing of amyloid precursor protein are of particular interest and, consequently, the inhibition of secretase enzyme family of protease type has become another desired treatment strategy for AD. On the other hand, medicinal plants are attractive sources for drug research and development as they produce chemically-varying molecules with preferred biological activities. The aim of this article is to review the available data on selected inhibitors from plant secondary metabolites with emphasis on cholinesterase, prolyl endopeptidase, and secretase enzyme families as being the current treatments of AD.

A biochemical and pharmacological comparison of enzyme replacement therapies for the glycolipid storage disorder Fabry disease.

PubMed

Lee, Karen; Jin, Xiaoying; Zhang, Kate; Copertino, Lorraine; Andrews, Laura; Baker-Malcolm, Jennifer; Geagan, Laura; Qiu, Huawei; Seiger, Keirsten; Barngrover, Debra; McPherson, John M; Edmunds, Tim

2003-04-01

Fabry disease is a lysosomal storage disease arising from deficiency of the enzyme alpha-galactosidase A. Two recombinant protein therapeutics, Fabrazyme (agalsidase beta) and Replagal (agalsidase alfa), have been approved in Europe as enzyme replacement therapies for Fabry disease. Both contain the same human enzyme, alpha-galactosidase A, but they are produced using different protein expression systems and have been approved for administration at different doses. To determine if there is recognizable biochemical basis for the different doses, we performed a comparison of the two drugs, focusing on factors that are likely to influence biological activity and availability. The two drugs have similar glycosylation, both in the type and location of the oligosaccharide structures present. Differences in glycosylation were mainly limited to the levels of sialic acid and mannose-6-phosphate present, with Fabrazyme having a higher percentage of fully sialylated oligosaccharides and a higher level of phosphorylation. The higher levels of phosphorylated oligomannose residues correlated with increased binding to mannose-6-phosphate receptors and uptake into Fabry fibroblasts in vitro. Biodistribution studies in a mouse model of Fabry disease showed similar organ uptake. Likewise, antigenicity studies using antisera from Fabry patients demonstrated that both drugs were indistinguishable in terms of antibody cross-reactivity. Based on these studies and present knowledge regarding the influence of glycosylation on protein biodistribution and cellular uptake, the two protein preparations appear to be functionally indistinguishable. Therefore, the data from these studies provide no rationale for the use of these proteins at different therapeutic doses.

Occurrence of trans monounsaturated and polyunsaturated fatty acids in Colwellia psychrerythraea strain 34H.

PubMed

Hashimoto, Mikako; Orikasa, Yoshitake; Hayashi, Hidenori; Watanabe, Kentaro; Yoshida, Kiyohito; Okuyama, Hidetoshi

2015-07-01

Colwellia psychrerythraea strain 34H is an obligately psychrophilic bacterium that has been used as a model cold-adapted microorganism because of its psychrophilic growth profile, significant production of cold-active enzymes, and cryoprotectant extracellular polysaccharide substances. However, its fatty acid components, particularly trans unsaturated fatty acids and long-chain polyunsaturated fatty acids (LC-PUFAs), have not been fully investigated. In this study, we biochemically identified Δ9-trans hexadecenoic acid [16:1(9t)] and LC-PUFAs such as docosahexaenoic acid. These results are comparable with the fact that the strain 34H genome sequence includes pfa and cti genes that are responsible for the biosynthesis of LC-PUFAs and trans unsaturated fatty acids, respectively. Strain 34H cells grown under static conditions at 5 °C had higher levels of 16:1(9t) than those grown under shaken conditions, and this change was accompanied by an antiparallel decrease in the levels of Δ9-cis hexadecenoic acid [16:1(9c)], suggesting that the cis-to-trans isomerization reaction of 16:1(9c) is activated under static (microanaerobic) culture conditions, that is, the enzyme could be activated by the decreased dissolved oxygen concentration of cultures. On the other hand, the levels of LC-PUFAs were too low (less than 3% of the total), even for cells grown at 5 °C, to evaluate their cold-adaptive function in this bacterium. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Emerging therapeutic delivery capabilities and challenges utilizing enzyme/protein packaged bacterial vesicles.

PubMed

Alves, Nathan J; Turner, Kendrick B; Medintz, Igor L; Walper, Scott A

2015-07-01

Nanoparticle-based therapeutics are poised to play a critical role in treating disease. These complex multifunctional drug delivery vehicles provide for the passive and active targeted delivery of numerous small molecule, peptide and protein-derived pharmaceuticals. This article will first discuss some of the current state of the art nanoparticle classes (dendrimers, lipid-based, polymeric and inorganic), highlighting benefits/drawbacks associated with their implementation. We will then discuss an emerging class of nanoparticle therapeutics, bacterial outer membrane vesicles, that can provide many of the nanoparticle benefits while simplifying assembly. Through molecular biology techniques; outer membrane vesicle hijacking potentially allows for stringent control over nanoparticle production allowing for targeted protein packaged nanoparticles to be fully synthesized by bacteria.

Automated homogeneous liposome immunoassay systems for anticonvulsant drugs.

PubMed

Kubotsu, K; Goto, S; Fujita, M; Tuchiya, H; Kida, M; Takano, S; Matsuura, S; Sakurabayashi, I

1992-06-01

We developed automated homogeneous immunoassays, based on immunolysis of liposomes, for measuring phenytoin, phenobarbital, and carbamazepine from serum. Liposome lysis was detected spectrophotometrically from entrapped glucose-6-phosphate dehydrogenase activity. The procedure was fully automated on a routine automated clinical analyzer. Within-run, between-run, dilution, and recovery tests showed good accuracies and reproducibilities. Bilirubin, hemoglobin, triglycerides, and Intrafat did not affect assay results. The results obtained by liposome immunoassays for phenytoin, phenobarbital, and carbamazepine correlated well with those obtained by enzyme-multiplied immunoassay (Syva EMIT) kits (r = 0.995, 0.986, and 0.988, respectively) and fluorescence polarization immunoassay (Abbott TDx) kits (r = 0.990, 0.991, and 0.975, respectively). The proposed method should be useful for monitoring anticonvulsant drug concentrations in blood.

Antioxidant Activity and Inhibitory Potential of Cistus salviifolius (L.) and Cistus monspeliensis (L.) Aerial Parts Extracts against Key Enzymes Linked to Hyperglycemia

PubMed Central

Marmouzi, Ilias; Cherrah, Yahia; Faouzi, My El Abbes

2017-01-01

Cistus genus (Cistaceae) comprises several medicinal plants used in traditional medicines to treat several pathological conditions including hyperglycemia. These include Cistus salviifolius L. (CS) and Cistus monspeliensis L. (CM), still not fully explored as a source of metabolites with therapeutic potential for human diseases. In this study, the antioxidant α-amylase and α-glucosidase enzyme inhibitory effects of aqueous and hydromethanolic extracts from the aerial parts of Moroccan CS and CM were investigated. Antioxidant activity has been assessed using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radicals and ferric reducing/antioxidant power (FRAP) methods. The α-amylase and α-glucosidase inhibitory activity has been assessed using an in vitro model. Moreover, mineral and phenolic contents of CS and CM were analyzed. The extracts of both species exhibited potent antioxidant activity in all used systems and possess strong inhibitory effect towards α-glucosidase (IC50: 0.95 ± 0.14 to 14.58 ± 1.26 μg/mL) and significant inhibitory potential against α-amylase (IC50: 217.10 ± 0.15 to 886.10 ± 0.10 μg/mL). Furthermore, the result showed high levels of phenolic content and unexpectedly some higher levels of mineral content in CS. The results suggest that the phenolic rich extracts of CS and CM may have a therapeutic potential against diseases associated with oxidative stress and may be useful in the management of hyperglycemia in diabetic patients. PMID:28116307

Human neuronal coenzyme Q10 deficiency results in global loss of mitochondrial respiratory chain activity, increased mitochondrial oxidative stress and reversal of ATP synthase activity: implications for pathogenesis and treatment.

PubMed

Duberley, Kate E C; Abramov, Andrey Y; Chalasani, Annapurna; Heales, Simon J; Rahman, Shamima; Hargreaves, Iain P

2013-01-01

Disorders of coenzyme Q(10) (CoQ(10)) biosynthesis represent the most treatable subgroup of mitochondrial diseases. Neurological involvement is frequently observed in CoQ(10) deficiency, typically presenting as cerebellar ataxia and/or seizures. The aetiology of the neurological presentation of CoQ(10) deficiency has yet to be fully elucidated and therefore in order to investigate these phenomena we have established a neuronal cell model of CoQ(10) deficiency by treatment of neuronal SH-SY5Y cell line with para-aminobenzoic acid (PABA). PABA is a competitive inhibitor of the CoQ(10) biosynthetic pathway enzyme, COQ2. PABA treatment (1 mM) resulted in a 54 % decrease (46 % residual CoQ(10)) decrease in neuronal CoQ(10) status (p < 0.01). Reduction of neuronal CoQ(10) status was accompanied by a progressive decrease in mitochondrial respiratory chain enzyme activities, with a 67.5 % decrease in cellular ATP production at 46 % residual CoQ(10). Mitochondrial oxidative stress increased four-fold at 77 % and 46 % residual CoQ(10). A 40 % increase in mitochondrial membrane potential was detected at 46 % residual CoQ(10) with depolarisation following oligomycin treatment suggesting a reversal of complex V activity. This neuronal cell model provides insights into the effects of CoQ(10) deficiency on neuronal mitochondrial function and oxidative stress, and will be an important tool to evaluate candidate therapies for neurological conditions associated with CoQ(10) deficiency.

Activation of Gq/11 in the Mouse Corpus Luteum Is Required for Parturition

PubMed Central

Mejia, Rachel; Waite, Courtney

2015-01-01

Mice with a deletion of Gαq/11 in granulosa cells were previously shown to be subfertile. They also have a reduced ovulatory response due to a deficiency in the ability of the activated LH receptor to fully induce the granulosa cell progesterone receptor. Because this conditional deletion of Gαq/11 will interfere with the actions of any G protein-coupled receptor that activates Gq/11 in granulosa or luteal cells, we sought to determine whether the actions of other hormones that contribute to fertility were also impaired. We focused our attention on prostaglandin F2 (PGF2)α, because this hormone is known to activate phospholipase C (a prominent Gαq/11 effector) in luteal cells and because the action of PGF2α on luteal cells is the first step in the murine parturition pathway. Our data show that the conditional deletion of Gαq/11 from granulosa cells prevents the ability of PGF2α to induce Akr1c18 in luteal cells. Akr1c18 codes for 20α-hydroxysteroid dehydrogenase, an enzyme that inactivates progesterone. The PGF2α-mediated induction of this enzyme towards the end of pregnancy increases the inactivation of progesterone and precipitates parturition in mice. Thus, the conditional deletion of Gαq/11 from granulosa/luteal cells prevents the progesterone withdrawal that occurs at the end of pregnancy and impairs parturition. This novel molecular defect contributes to the subfertile phenotype of the mice with a deletion of Gαq/11 from granulosa cells. PMID:25495873

Activation of Gq/11 in the mouse corpus luteum is required for parturition.

PubMed

Mejia, Rachel; Waite, Courtney; Ascoli, Mario

2015-02-01

Mice with a deletion of Gα(q/11) in granulosa cells were previously shown to be subfertile. They also have a reduced ovulatory response due to a deficiency in the ability of the activated LH receptor to fully induce the granulosa cell progesterone receptor. Because this conditional deletion of Gα(q/11) will interfere with the actions of any G protein-coupled receptor that activates G(q/11) in granulosa or luteal cells, we sought to determine whether the actions of other hormones that contribute to fertility were also impaired. We focused our attention on prostaglandin F2 (PGF2)α, because this hormone is known to activate phospholipase C (a prominent Gα(q/11) effector) in luteal cells and because the action of PGF2α on luteal cells is the first step in the murine parturition pathway. Our data show that the conditional deletion of Gα(q/11) from granulosa cells prevents the ability of PGF2α to induce Akr1c18 in luteal cells. Akr1c18 codes for 20α-hydroxysteroid dehydrogenase, an enzyme that inactivates progesterone. The PGF2α-mediated induction of this enzyme towards the end of pregnancy increases the inactivation of progesterone and precipitates parturition in mice. Thus, the conditional deletion of Gαq/11 from granulosa/luteal cells prevents the progesterone withdrawal that occurs at the end of pregnancy and impairs parturition. This novel molecular defect contributes to the subfertile phenotype of the mice with a deletion of Gα(q/11) from granulosa cells.

Glycyl radical activating enzymes: structure, mechanism, and substrate interactions.

PubMed

Shisler, Krista A; Broderick, Joan B

2014-03-15

The glycyl radical enzyme activating enzymes (GRE-AEs) are a group of enzymes that belong to the radical S-adenosylmethionine (SAM) superfamily and utilize a [4Fe-4S] cluster and SAM to catalyze H-atom abstraction from their substrate proteins. GRE-AEs activate homodimeric proteins known as glycyl radical enzymes (GREs) through the production of a glycyl radical. After activation, these GREs catalyze diverse reactions through the production of their own substrate radicals. The GRE-AE pyruvate formate lyase activating enzyme (PFL-AE) is extensively characterized and has provided insights into the active site structure of radical SAM enzymes including GRE-AEs, illustrating the nature of the interactions with their corresponding substrate GREs and external electron donors. This review will highlight research on PFL-AE and will also discuss a few GREs and their respective activating enzymes. Copyright © 2014. Published by Elsevier Inc.

Anion channels and the stimulation of anthocyanin accumulation by blue light in Arabidopsis seedlings

NASA Technical Reports Server (NTRS)

Noh, B.; Spalding, E. P.; Evans, M. H. (Principal Investigator)

1998-01-01

Activation of anion channels by blue light begins within seconds of irradiation in seedlings and is related to the ensuing growth inhibition. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) is a potent, selective, and reversible blocker of these anion channels in Arabidopsis thaliana. Here we show that 20 microM NPPB blocked 72% of the blue-light-induced accumulation of anthocyanin pigments in seedlings. Feeding biosynthetic intermediates to wild-type and tt5 seedlings provided evidence that NPPB prevented blue light from up-regulating one or more steps between and including phenylalanine ammonia lyase and chalcone isomerase. NPPB was found to have no significant effect on the blue-light-induced increase in transcript levels of PAL1, CHS, CHI, or DFR, which are genes that encode anthocyanin-biosynthetic enzymes. Immunoblots revealed that NPPB also did not inhibit the accumulation of the chalcone synthase, chalcone isomerase, or flavanone-3-hydroxylase proteins. This is in contrast to the reduced anthocyanin accumulation displayed by a mutant lacking the HY4 blue-light receptor, as hy4 displayed reduced expression of the above enzymes. Taken together, the data indicate that blue light acting through HY4 leads to an increase in the amount of biosynthetic enzymes but blue light must also act through a separate, anion-channel-dependent system to create a fully functional biosynthetic pathway.

Lotus japonicus flowers are defended by a cyanogenic β-glucosidase with highly restricted expression to essential reproductive organs.

PubMed

Lai, Daniela; Pičmanová, Martina; Abou Hachem, Maher; Motawia, Mohammed Saddik; Olsen, Carl Erik; Møller, Birger Lindberg; Rook, Fred; Takos, Adam M

2015-09-01

Flowers and leaves of Lotus japonicus contain α-, β-, and γ-hydroxynitrile glucoside (HNG) defense compounds, which are bioactivated by β-glucosidase enzymes (BGDs). The α-HNGs are referred to as cyanogenic glucosides because their hydrolysis upon tissue disruption leads to release of toxic hydrogen cyanide gas, which can deter herbivore feeding. BGD2 and BGD4 are HNG metabolizing BGD enzymes expressed in leaves. Only BGD2 is able to hydrolyse the α-HNGs. Loss of function mutants of BGD2 are acyanogenic in leaves but fully retain cyanogenesis in flowers pointing to the existence of an alternative cyanogenic BGD in flowers. This enzyme, named BGD3, is identified and characterized in this study. Whereas all floral tissues contain α-HNGs, only those tissues in which BGD3 is expressed, the keel and the enclosed reproductive organs, are cyanogenic. Biochemical analysis, active site architecture molecular modelling, and the observation that L. japonicus accessions lacking cyanogenic flowers contain a non-functional BGD3 gene, all support the key role of BGD3 in floral cyanogenesis. The nectar of L. japonicus flowers was also found to contain HNGs and additionally their diglycosides. The observed specialisation in HNG based defence in L. japonicus flowers is discussed in the context of balancing the attraction of pollinators with the protection of reproductive structures against herbivores.

Dichlone-induced oxidative stress in a model insect species, Spodoptera eridania.

PubMed

Ahmad, S; Zaman, K; MacGill, R S; Batcabe, J P; Pardini, R S

1995-11-01

Southern armyworm, Spodoptera eridania, larvae were provided ad libitum 0.002-0.25% w/w dichlone, 2,3-dichloro-1,4-naphthoquinone (CNQ). Larval mortality occurred in a time-and-dose dependent manner, with an LC17 of 0.01% and an LC50 of 0.26% CNQ at day-5. Extracts of larvae fed control, 0.01, and 0.25% CNQ diets for 5 days were assayed for antioxidant enzymes. While 0.01% CNQ had a mild effect, 0.25% CNQ profoundly increased levels of all antioxidant enzymes that were examined. The increases as compared to control were: 5.3-, 1.9-, 3.2-, 2.6-, 2.8-, and 3.5-fold higher for superoxide dismutase, catalase, glutathione transferase and its peroxidase activity, glutathione reductase and DT-diaphorase, respectively. At 0.01% CNQ, the thiobarbituric acid reactive substances (TBARS) were similar to the control group. However, despite the induction from 0.25% CNQ of all enzymes examined, the lipid peroxidation was not attenuated; the TBARS were 29.7% over the control value. High mortalities and CNQ-induced pathologies reflected in retarded growth, wasting syndrome, and diuresis clearly indicated that the insect sustained severe oxidant-induced injuries before appropriate defenses were fully mobilized. Thus, this quinone causes an oxidative stress in a model insect species analogous to that observed in mammalian species.

Divergent hypoxia tolerance in adult males and females of the plainfin midshipman (Porichthys notatus).

PubMed

LeMoine, Christophe M R; Bucking, Carol; Craig, Paul M; Walsh, Patrick J

2014-01-01

In the summer, the plainfin midshipman (Poricththys notatus) migrates to reproduce in the nearshore environment, where oxygen levels are influenced by the tidal cycles. Parental males establish nests under rocks in the intertidal zone, where they reside until the eggs they guard are fully developed. In contrast, females and sneaker males leave the nests shortly after spawning. We examined the physiological resistance and metabolic response of parental male and female adult midshipman to hypoxia to test whether they exhibited sex-specific differences reflecting their reproductive strategies. Further, we assessed whether metabolic enzymes and metabolites were differentially enriched in tissues of parental males and females to explain the differences observed in their hypoxia tolerance. While parental males and females exhibited similar depression of their oxygen consumption in response to graded hypoxia, parental males could withstand significantly longer exposures to severe hypoxic stress. At the biochemical level, parental males showed higher hepatic glycogen reserves and higher glycolytic enzyme capacities in gills and skeletal muscles than females. Although some of these enzymatic variations could be explained by differences in body size, we also observed a significant effect of sex on some of these factors. These results suggest that parental male midshipman may benefit from sexual dimorphism at the whole-organismal (larger body size) and biochemical (enzyme activities) levels, conferring on them a higher glycolytic potential to sustain the extensive hypoxia bouts they experience in nature.

Spatial distribution of enzyme activities along the root and in the rhizosphere of different plants

NASA Astrophysics Data System (ADS)

Razavi, Bahar S.; Zarebanadkouki, Mohsen; Blagodatskaya, Evgenia; Kuzyakov, Yakov

2015-04-01

Extracellular enzymes are important for decomposition of many biological macromolecules abundant in soil such as cellulose, hemicelluloses and proteins. Activities of enzymes produced by both plant roots and microbes are the primary biological drivers of organic matter decomposition and nutrient cycling. So far acquisition of in situ data about local activity of different enzymes in soil has been challenged. That is why there is an urgent need in spatially explicit methods such as 2-D zymography to determine the variation of enzymes along the roots in different plants. Here, we developed further the zymography technique in order to quantitatively visualize the enzyme activities (Spohn and Kuzyakov, 2013), with a better spatial resolution We grew Maize (Zea mays L.) and Lentil (Lens culinaris) in rhizoboxes under optimum conditions for 21 days to study spatial distribution of enzyme activity in soil and along roots. We visualized the 2D distribution of the activity of three enzymes:β-glucosidase, leucine amino peptidase and phosphatase, using fluorogenically labelled substrates. Spatial resolution of fluorescent images was improved by direct application of a substrate saturated membrane to the soil-root system. The newly-developed direct zymography shows different pattern of spatial distribution of enzyme activity along roots and soil of different plants. We observed a uniform distribution of enzyme activities along the root system of Lentil. However, root system of Maize demonstrated inhomogeneity of enzyme activities. The apical part of an individual root (root tip) in maize showed the highest activity. The activity of all enzymes was the highest at vicinity of the roots and it decreased towards the bulk soil. Spatial patterns of enzyme activities as a function of distance from the root surface were enzyme specific, with highest extension for phosphatase. We conclude that improved zymography is promising in situ technique to analyze, visualize and quantify spatial distribution of enzyme activities in the rhizosphere hotspots. References Spohn, M., Kuzyakov, Y., 2013. Phosphorus mineralization can be driven by microbial need for carbon. Soil Biology & Biochemistry 61: 69-75

Erythrocyte enzymes in sheep: comparison of activity in fetal, newborn, maternal and nonpregnant ewe erythrocytes.

PubMed

Noble, N A; Cabalum, T C; Nathanielsz, P W; Tanaka, K R

1982-01-01

Hematological data and the activities of 21 red cell enzymes were measured in 8 nonpregnant ewes, 13 chronically catheterized fetuses at 125-135 days of gestation, and 8 of their mothers. In addition, 7 lambs were followed from birth to 17 days of age. Fetal sheep red cells have dramatically increased activities for 17 of 21 enzymes measured compared with adult nonpregnant ewes. The pattern of decline of enzyme activities with development varies considerably among enzymes. The activity of seven enzymes showed an orderly decline from fetal to adult life. For seven enzymes very little or no decline in activity was observed between 125 and 135 days of gestation and birth. Pyruvate kinase activity declined to adult levels by birth. Phosphoglucose isomerase and nucleoside phosphorylase activity increased, and glutathione peroxidase activity decreased in newborn lamb red cells compared to fetal cells. Differences in blood cell variables were also found among these groups.

Progress and obstacles in the production and application of recombinant lignin-degrading peroxidases

PubMed Central

Lambertz, Camilla; Ece, Selin; Fischer, Rainer; Commandeur, Ulrich

2016-01-01

ABSTRACT Lignin is 1 of the 3 major components of lignocellulose. Its polymeric structure includes aromatic subunits that can be converted into high-value-added products, but this potential cannot yet been fully exploited because lignin is highly recalcitrant to degradation. Different approaches for the depolymerization of lignin have been tested, including pyrolysis, chemical oxidation, and hydrolysis under supercritical conditions. An additional strategy is the use of lignin-degrading enzymes, which imitates the natural degradation process. A versatile set of enzymes for lignin degradation has been identified, and research has focused on the production of recombinant enzymes in sufficient amounts to characterize their structure and reaction mechanisms. Enzymes have been analyzed individually and in combinations using artificial substrates, lignin model compounds, lignin and lignocellulose. Here we consider progress in the production of recombinant lignin-degrading peroxidases, the advantages and disadvantages of different expression hosts, and obstacles that must be overcome before such enzymes can be characterized and used for the industrial processing of lignin. PMID:27295524

Helicobacter pylori purine nucleoside phosphorylase shows new distribution patterns of open and closed active site conformations and unusual biochemical features.

PubMed

Narczyk, Marta; Bertoša, Branimir; Papa, Lucija; Vuković, Vedran; Leščić Ašler, Ivana; Wielgus-Kutrowska, Beata; Bzowska, Agnieszka; Luić, Marija; Štefanić, Zoran

2018-04-01

Even with decades of research, purine nucleoside phosphorylases (PNPs) are enzymes whose mechanism is yet to be fully understood. This is especially true in the case of hexameric PNPs, and is probably, in part, due to their complex oligomeric nature and a whole spectrum of active site conformations related to interactions with different ligands. Here we report an extensive structural characterization of the apo forms of hexameric PNP from Helicobacter pylori (HpPNP), as well as its complexes with phosphate (P i ) and an inhibitor, formycin A (FA), together with kinetic, binding, docking and molecular dynamics studies. X-ray structures show previously unseen distributions of open and closed active sites. Microscale thermophoresis results indicate that a two-site model describes P i binding, while a three-site model is needed to characterize FA binding, irrespective of P i presence. The latter may be related to the newly found nonstandard mode of FA binding. The ternary complex of the enzyme with P i and FA shows, however, that P i binding stabilizes the standard mode of FA binding. Surprisingly, HpPNP has low affinity towards the natural substrate adenosine. Molecular dynamics simulations show that P i moves out of most active sites, in accordance with its weak binding. Conformational changes between nonstandard and standard binding modes of nucleoside are observed during the simulations. Altogether, these findings show some unique features of HpPNP and provide new insights into the functioning of the active sites, with implications for understanding the complex mechanism of catalysis of this enzyme. The atomic coordinates and structure factors have been deposited in the Protein Data Bank: with accession codes 6F52 (HpPNPapo_1), 6F5A (HpPNPapo_2), 6F5I (HpPNPapo_3), 5LU0 (HpPNP_PO4), 6F4W (HpPNP_FA) and 6F4X (HpPNP_PO4_FA). Purine nucleoside orthophosphate ribosyl transferase, EC2.4.2.1, UniProtID: P56463. © 2018 Federation of European Biochemical Societies.

Grapevine Botryosphaeria dieback fungi have specific aggressiveness factor repertory involved in wood decay and stilbene metabolization

PubMed Central

Wilhelm, Kim; Tarnus, Céline; Bertsch, Christophe

2017-01-01

Grapevine trunk diseases: Eutypa dieback, esca and Botryosphaeria dieback, which incidence has increased recently, are associated with several symptoms finally leading to the plant death. In the absence of efficient treatments, these diseases are a major problem for the viticulture; however, the factors involved in disease progression are not still fully identified. In order to get a better understanding of Botryosphaeria dieback development in grapevine, we have investigated different factors involved in Botryosphaeriaceae fungi aggressiveness. We first evaluated the activity of the wood-degrading enzymes of different isolates of Neofusicoccum parvum and Diplodia seriata, two major fungi associated with Botryosphaeria dieback. We further examinated the ability of these fungi to metabolize major grapevine phytoalexins: resveratrol and δ-viniferin. Our results demonstrate that Botryosphaeriaceae were characterized by differential wood decay enzymatic activities and have the capacity to rapidly degrade stilbenes. N. parvum is able to degrade parietal polysaccharides, whereas D. seriata has a better capacity to degrade lignin. Growth of both fungi exhibited a low sensitivity to resveratrol, whereas δ-viniferin has a fungistatic effect, especially on N. parvum Bourgogne S-116. We further show that Botryosphaeriaceae are able to metabolize rapidly resveratrol and δ-viniferin. The best stilbene metabolizing activity was measured for D. seriata. In conclusion, the different Botryosphaeriaceae isolates are characterized by a specific aggressiveness repertory. Wood and phenolic compound decay enzymatic activities could enable Botryosphaeriaceae to bypass chemical and physical barriers of the grapevine plant. The specific signature of Botryosphaeriaceae aggressiveness factors could explain the importance of fungi complexes in synergistic activity in order to fully colonize the host. PMID:29261692

Detailed reaction mechanism of macrophomate synthase. Extraordinary enzyme catalyzing five-step transformation from 2-pyrones to benzoates.

PubMed

Watanabe, K; Mie, T; Ichihara, A; Oikawa, H; Honma, M

2000-12-08

Macrophomate synthase from the fungus Macrophoma commelinae IFO 9570 is a Mg(II)-dependent dimeric enzyme that catalyzes an extraordinary, complex five-step chemical transformation from 2-pyrone and oxalacetate to benzoate involving decarboxylation, C-C bond formation, and dehydration. The catalytic mechanism of the whole pathway was investigated in three separate chemical steps. In the first decarboxylation step, the enzyme loses oxalacetate decarboxylation activity upon incubation with EDTA. Activity is fully restored by addition of Mg(II) and is not restored with other divalent metal cations. The dissociation constant of 0.93 x 10(-)(7) for Mg(II) and atomic absorption analysis established a 1:1 stoichiometric complex. Inhibition of pyruvate formation with 2-pyrone revealed that the actual product in the first step is a pyruvate enolate, which undergoes C-C bond formation in the presence of 2-pyrone. Incubation of substrate analogs provided aberrant adducts that were produced via C-C bond formation and rearrangement. This strongly indicates that the second step is two C-C bond formations, affording a bicyclic intermediate. Based on the stereospecificity, involvement of a Diels-Alder reaction at the second step is proposed. Incubation of the stereospecifically deuterium-labeled malate with 2-pyrones in the presence of malate dehydrogenase provided information for the stereochemical course of the reaction catalyzed by macrophomate synthase, indicating that the first decarboxylation provides pyruvate (Z)-[3-(2)H]enolate and that dehydration at the final step occurs with anti-elimination accompanied by concomitant decarboxylation. Examination of kinetic parameters in the individual steps suggests that the third step is the rate-determining step of the overall transformation.

Reversible and strong immobilization of proteins by ionic exchange on supports coated with sulfate-dextran.

PubMed

Fuentes, Manuel; Pessela, Benevides C C; Maquiese, Jorgette V; Ortiz, Claudia; Segura, Rosa L; Palomo, Jose M; Abian, Olga; Torres, Rodrigo; Mateo, Cesar; Fernández-Lafuente, Roberto; Guisán, J M

2004-01-01

New and strong ionic exchange resins have been prepared by the simple and rapid ionic adsorption of anionic polymers (sulfate-dextran) on porous supports activated with the opposite ionic group (DEAE/MANAE). Ionic exchange properties of such composites were strongly dependent on the size of the ionic polymers as well as on the conditions of the ionic coating of the solids with the ionic polymers (optimal conditions were 400 mg of sulfate-dextran 5000 kDa per gram of support). Around 80% of the proteins contained in crude extracts from Escherichia coli and Acetobacter turbidans could be adsorbed on these porous composites even at pH 7. This interaction was stronger than that using conventional carboxymethyl cellulose (CMC) and even others such as supports coated with aspartic-dextran polymer. By means of the sequential use of the new supports and supports coated with polyethyleneimine (PEI), all proteins from crude extracts could be immobilized. In fact, a large percentage (over 50%) could be immobilized on both supports. Finally, some industrially relevant enzymes (beta-galactosidases from Aspergillus oryzae, Kluyveromyces lactis, and Thermussp. strain T2, lipases from Candida antarctica A and B, Candida rugosa, Rhizomucor miehei, and Rhyzopus oryzae and bovine pancreas trypsin and chymotrypsin) have been immobilized on these supports with very high activity recoveries and immobilization rates. After enzyme inactivation, the protein could be fully desorbed from the support, and then the support could be reused for several cycles. Moreover, in some instances the enzyme stability was significantly improved, mainly in the presence of organic solvents, perhaps as a consequence of the highly hydrophilic microenvironment of the support.

Genetically programmed chiral organoborane synthesis

NASA Astrophysics Data System (ADS)

Kan, S. B. Jennifer; Huang, Xiongyi; Gumulya, Yosephine; Chen, Kai; Arnold, Frances H.

2017-12-01

Recent advances in enzyme engineering and design have expanded nature’s catalytic repertoire to functions that are new to biology. However, only a subset of these engineered enzymes can function in living systems. Finding enzymatic pathways that form chemical bonds that are not found in biology is particularly difficult in the cellular environment, as this depends on the discovery not only of new enzyme activities, but also of reagents that are both sufficiently reactive for the desired transformation and stable in vivo. Here we report the discovery, evolution and generalization of a fully genetically encoded platform for producing chiral organoboranes in bacteria. Escherichia coli cells harbouring wild-type cytochrome c from Rhodothermus marinus (Rma cyt c) were found to form carbon-boron bonds in the presence of borane-Lewis base complexes, through carbene insertion into boron-hydrogen bonds. Directed evolution of Rma cyt c in the bacterial catalyst provided access to 16 novel chiral organoboranes. The catalyst is suitable for gram-scale biosynthesis, providing up to 15,300 turnovers, a turnover frequency of 6,100 h-1, a 99:1 enantiomeric ratio and 100% chemoselectivity. The enantiopreference of the biocatalyst could also be tuned to provide either enantiomer of the organoborane products. Evolved in the context of whole-cell catalysts, the proteins were more active in the whole-cell system than in purified forms. This study establishes a DNA-encoded and readily engineered bacterial platform for borylation; engineering can be accomplished at a pace that rivals the development of chemical synthetic methods, with the ability to achieve turnovers that are two orders of magnitude (over 400-fold) greater than those of known chiral catalysts for the same class of transformation. This tunable method for manipulating boron in cells could expand the scope of boron chemistry in living systems.

Mercury exposure induces proinflammatory enzymes in vascular fibroblasts.

PubMed

Millán Longo, Alberto; Montero Saiz, Óscar; Sarró Fuente, Claudia; Aguado Martínez, Andrea; Salaices Sánchez, Mercedes

Previous studies show that mercury exposure increases cardiovascular risk, although the underlying cellular mechanisms have still not been fully studied. The aim of this project is to study, in vascular fibroblasts (VF), the effect of HgCl 2 exposure on the expression of enzymes involved in the synthesis of prostanoids and reactive oxygen species (ROS). These molecules have been shown to participate in the inflammatory response associated with cardiovascular diseases. Adventitial VF cultures of Sprague-Dawley rat aortas, shown to be α-actin negative by immunofluorescence, were exposed to HgCl 2 (0.05-5μg/mL) for 48h. mRNA and protein levels of cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase 1 (mPGES-1), thromboxane A 2 synthase (TXAS), NADPH oxidase 1 (NOX-1), and 4 (NOX-4) where analyzed using qRT-PCR and western blot, respectively. NOX activity was determined by chemiluminescence. HgCl 2 exposure increased COX-2, mPGES-1, TXAS, and NOX-1 expression and NOX activity, and decreased NOX-4 expression. The increase in NOX-1 and COX-2 expression was abolished by the treatment with inhibitors of COX-2 (10μM celecoxib) and NOX (300μM apocynin, 0.5μM ML-171). 1) HgCl 2 increases the expression of pro-inflammatory enzymes involved in ROS and prostanoid synthesis in VF. 2) There is a reciprocal regulation between COX-2 and NOX-1 pathways. 3) These effects can contribute to explain the increase in cardiovascular risk associated to mercury. Copyright © 2017 Sociedad Española de Arteriosclerosis. Publicado por Elsevier España, S.L.U. All rights reserved.

Crystal structure of the 28 kDa glutathione S-transferase from Schistosoma haematobium.

PubMed

Johnson, Kenneth A; Angelucci, Francesco; Bellelli, Andrea; Hervé, Maxime; Fontaine, Josette; Tsernoglou, Demetrious; Capron, André; Trottein, François; Brunori, Maurizio

2003-09-02

Schistomiasis is a debilitating parasitic disease which affects 200 million people, causing life-threatening complications in 10% of the patients. This paper reports the crystal structure of the Schistosoma haematobium 28 kDa glutathione S-transferase, a multifunctional enzyme involved in host-parasite interactions and presently considered as a promising vaccine candidate against schistosomiasis. The structures of the GSH-free enzyme, as well as the partially (approximately 40%) and almost fully (approximately 80%) GSH-saturated enzyme, exhibit a unique feature, absent in previous GST structures, concerning the crucial and invariant Tyr10 side chain which occupies two alternative positions. The canonical conformer, which allows an H-bond to be formed between the side chain hydroxyl group and the activated thiolate of GSH, is somewhat less than 50% occupied. The new conformer, with the phenoxyl ring on the opposite side of the mobile loop connecting strand 1 and helix 1, is stabilized by a polar interaction with the guanidinium group of the conserved Arg21 side chain. The presence of two conformers of Tyr10 may provide a clue about clarifying the multiple catalytic functions of Sh28GST and might prove to be relevant for the design of specific antischistosomal drugs. The K(d) for GSH binding was determined by equilibrium fluorescence titrations to be approximately 3 microM and by stopped-flow rapid mixing experiments to be approximately 9 microM. The relatively tight binding of GSH by Sh28GST explains the residually bound GSH in the crystal and supports a possible role of GSH as a tightly bound cofactor involved in the catalytic mechanism for prostaglandin D(2) synthase activity.

Harman induces CYP1A1 enzyme through an aryl hydrocarbon receptor mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

El Gendy, Mohamed A.M.; El-Kadi, Ayman O.S., E-mail: aelkadi@pharmacy.ualberta.c

Harman is a common compound in several foods, plants and beverages. Numerous studies have demonstrated its mutagenic, co-mutagenic and carcinogenic effects; however, the exact mechanism has not been fully identified. Aryl hydrocarbon receptor (AhR) is a transcription factor regulating the expression of the carcinogen-activating enzyme; cytochrome P450 1A1 (CYP1A1). In the present study, we examined the ability of harman to induce AhR-mediated signal transduction in human and rat hepatoma cells; HepG2 and H4IIE cells. Our results showed that harman significantly induced CYP1A1 mRNA in a time- and concentration-dependent manner. Similarly, harman significantly induced CYP1A1 at protein and activity levels inmore » a concentration-dependent manner. Moreover, the AhR antagonist, resveratrol, inhibited the increase in CYP1A1 activity by harman. The RNA polymerase inhibitor, actinomycin D, completely abolished the CYP1A1 mRNA induction by harman, indicating a transcriptional activation. The role of AhR in CYP1A1 induction by harman was confirmed by using siRNA specific for human AhR. The ability of harman to induce CYP1A1 was strongly correlated with its ability to stimulate AhR-dependent luciferase activity and electrophoretic mobility shift assay. At post-transcriptional and post-translational levels, harman did not affect the stability of CYP1A1 at the mRNA and the protein levels, excluding other mechanisms participating in the obtained effects. We concluded that harman can directly induce CYP1A1 gene expression in an AhR-dependent manner and may represent a novel mechanism by which harman promotes mutagenicity, co-mutagenicity and carcinogenicity.« less

Production, characterization, and identification using proteomic tools of a polygalacturonase from Fusarium graminearum.

PubMed

Ortega, Leonel M; Kikot, Gisele E; Rojas, Natalia L; López, Laura M I; Astoreca, Andrea L; Alconada, Teresa M

2014-07-01

Since enzymatic degradation is a mechanism or component of the aggressiveness of a pathogen, enzymatic activities from a Fusarium graminearum isolate obtained from infected wheat spikes of Argentina Pampa region were studied in order to understand the disease progression, tending to help disease control. In particular, the significance of the study of polygalacturonase activity is based on that such activity is produced in the early stages of infection on the host, suggesting a crucial role in the establishment of disease. In this sense, polygalacturonase activity produced by this microorganism has been purified 375 times from 2-day-old culture filtrates by gel filtration and ion-exchange chromatography successively. The purified sample showed two protein bands in sodium dodecyl sulfate-polyacrylamide gels, with a molecular mass of 40 and 55 kDa. The protein bands were identified as an endopolygalacturonase and as a serine carboxypeptidase of F. graminearum, respectively, by peptide mass fingerprinting (matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF/TOF) fragment ion analysis). The pattern of substrate degradation analyzed by thin layer chromatography confirmed the mode of action of the enzyme as an endopolygalacturonase. High activity of the polygalacturonase against polygalacturonic acid was observed between 4 and 6 of pH, and between 30 and 50 °C, being 5 and 50 °C the optimum pH and temperature, respectively. The enzyme was fully stable at pH 5 for 120 min and 30 °C and sensible to the presence of some metal ions. This information would contribute to understand the most favorable environmental conditions for establishment of the disease. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification and gene expression of anaerobically induced enolase in Echinochloa phyllopogon and Echinochloa crus-pavonis.

PubMed Central

Fox, T C; Mujer, C V; Andrews, D L; Williams, A S; Cobb, B G; Kennedy, R A; Rumpho, M E

1995-01-01

Enolase (2-phospho-D-glycerate hydrolase, EC 4.2.1.11) has been identified as an anaerobic stress protein in Echinochloa oryzoides based on the homology of its internal amino acid sequence with those of enolases from other organisms, by immunological reactivity, and induction of catalytic activity during anaerobic stress. Enolase activity was induced 5-fold in anoxically treated seedlings of three flood-tolerant species (E. oryzoides, Echinochloa phyllopogon, and rice [Oryza sativa L.]) but not in the flood-intolerant species (Echinochloa crus-pavonis). A 540-bp fragment of the enolase gene was amplified by polymerase chain reaction from cDNAs of E. phyllopogon and maize (Zea mays L.) and used to estimate the number of enolase genes and to study the expression of enolase transcripts in E. phyllopogon, E. crus-pavonis, and maize. Southern blot analysis indicated that only one enolase gene is present in either E. phyllopogon or E. crus-pavonis. Three patterns of enolase gene expression were observed in the three species studied. In E. phyllopogon, enolase induction at both the mRNA and enzyme activity levels was sustained at all times with a further induction after 48 h of anoxia. In contrast, enolase was induced in hypoxically treated maize root tips only at the mRNA level. In E. crus-pavonis, enolase mRNA and enzyme activity were induced during hypoxia, but activity was only transiently elevated. These results suggest that enolase expression in maize and E. crus-pavonis during anoxia are similarly regulated at the transcriptional level but differ in posttranslational regulation, whereas enolase is fully induced in E. phyllopogon during anaerobiosis. PMID:7480340

Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

PubMed Central

Lauretti, Elisabetta; Hulse, Michael; Siciliano, Micheal; Lupey-Green, Lena N.; Abraham, Aaron; Skorski, Tomasz; Tempera, Italo

2018-01-01

The enzyme Poly(ADP-ribose) polymerase 1 (PARP1) plays a very important role in the DNA damage response, but its role in numerous aspects is not fully understood. We recently showed that in the absence of DNA damage, PARP1 regulates the expression of the chromatin-modifying enzyme EZH2. Work from other groups has shown that EZH2 participates in the DNA damage response. These combined data suggest that EZH2 could be a target of PARP1 in both untreated and genotoxic agent-treated conditions. In this work we tested the hypothesis that, in response to DNA damage, PARP1 regulates EZH2 activity. Here we report that PARP1 regulates EZH2 activity after DNA damage. In particular, we find that EZH2 is a direct target of PARP1 upon induction of alkylating and UV-induced DNA damage in cells and in vitro. PARylation of EZH2 inhibits EZH2 histone methyltransferase (H3K27me) enzymatic activity. We observed in cells that the induction of PARP1 activity by DNA alkylating agents decreases the association of EZH2 with chromatin, and PARylation of histone H3 reduces EZH2 affinity for its target histone H3. Our findings establish that PARP1 and PARylation are important regulators of EZH2 function and link EZH2-mediated heterochromatin formation, DNA damage and PARylation. These findings may also have clinical implications, as they suggest that inhibitors of EZH2 can improve anti-tumor effects of PARP1 inhibitors in BRCA1/2-deficient cancers. PMID:29535829

Process for preparing multilayer enzyme coating on a fiber

DOEpatents

Kim, Jungbae [Richland, WA; Kwak, Ja Hun [Richland, WA; Grate, Jay W [West Richland, WA

2009-11-03

A process for preparing high stability, high activity biocatalytic materials is disclosed and processes for using the same. The process involves coating of a material or fiber with enzymes and enzyme aggregate providing a material or fiber with high biocatalytic activity and stability useful in heterogeneous environments. In one illustrative approach, enzyme "seeds" are covalently attached to polymer nanofibers followed by treatment with a reagent that crosslinks additional enzyme molecules to the seed enzymes forming enzyme aggregates thereby improving biocatalytic activity due to increased enzyme loading and enzyme stability. This approach creates a useful new biocatalytic immobilized enzyme system with potential applications in bioconversion, bioremediation, biosensors, and biofuel cells.

In Situ Enzyme Activity in the Dissolved and Particulate Fraction of the Fluid from Four Pitcher Plant Species of the Genus Nepenthes

PubMed Central

Takeuchi, Yayoi; Salcher, Michaela M.; Ushio, Masayuki; Shimizu-Inatsugi, Rie; Kobayashi, Masaki J.; Diway, Bibian; von Mering, Christian; Pernthaler, Jakob; Shimizu, Kentaro K.

2011-01-01

The genus Nepenthes, a carnivorous plant, has a pitcher to trap insects and digest them in the contained fluid to gain nutrient. A distinctive character of the pitcher fluid is the digestive enzyme activity that may be derived from plants and dwelling microbes. However, little is known about in situ digestive enzymes in the fluid. Here we examined the pitcher fluid from four species of Nepenthes. High bacterial density was observed within the fluids, ranging from 7×106 to 2.2×108 cells ml−1. We measured the activity of three common enzymes in the fluid: acid phosphatases, β-d-glucosidases, and β-d-glucosaminidases. All the tested enzymes detected in the liquid of all the pitcher species showed activity that considerably exceeded that observed in aquatic environments such as freshwater, seawater, and sediment. Our results indicate that high enzyme activity within a pitcher could assist in the rapid decomposition of prey to maximize efficient nutrient use. In addition, we filtered the fluid to distinguish between dissolved enzyme activity and particle-bound activity. As a result, filtration treatment significantly decreased the activity in all enzymes, while pH value and Nepenthes species did not affect the enzyme activity. It suggested that enzymes bound to bacteria and other organic particles also would significantly contribute to the total enzyme activity of the fluid. Since organic particles are themselves usually colonized by attached and highly active bacteria, it is possible that microbe-derived enzymes also play an important role in nutrient recycling within the fluid and affect the metabolism of the Nepenthes pitcher plant. PMID:21949872

In situ enzyme activity in the dissolved and particulate fraction of the fluid from four pitcher plant species of the genus Nepenthes.

PubMed

Takeuchi, Yayoi; Salcher, Michaela M; Ushio, Masayuki; Shimizu-Inatsugi, Rie; Kobayashi, Masaki J; Diway, Bibian; von Mering, Christian; Pernthaler, Jakob; Shimizu, Kentaro K

2011-01-01

The genus Nepenthes, a carnivorous plant, has a pitcher to trap insects and digest them in the contained fluid to gain nutrient. A distinctive character of the pitcher fluid is the digestive enzyme activity that may be derived from plants and dwelling microbes. However, little is known about in situ digestive enzymes in the fluid. Here we examined the pitcher fluid from four species of Nepenthes. High bacterial density was observed within the fluids, ranging from 7×10(6) to 2.2×10(8) cells ml(-1). We measured the activity of three common enzymes in the fluid: acid phosphatases, β-D-glucosidases, and β-D-glucosaminidases. All the tested enzymes detected in the liquid of all the pitcher species showed activity that considerably exceeded that observed in aquatic environments such as freshwater, seawater, and sediment. Our results indicate that high enzyme activity within a pitcher could assist in the rapid decomposition of prey to maximize efficient nutrient use. In addition, we filtered the fluid to distinguish between dissolved enzyme activity and particle-bound activity. As a result, filtration treatment significantly decreased the activity in all enzymes, while pH value and Nepenthes species did not affect the enzyme activity. It suggested that enzymes bound to bacteria and other organic particles also would significantly contribute to the total enzyme activity of the fluid. Since organic particles are themselves usually colonized by attached and highly active bacteria, it is possible that microbe-derived enzymes also play an important role in nutrient recycling within the fluid and affect the metabolism of the Nepenthes pitcher plant.

Enhanced enzyme stability through site-directed covalent immobilization.

PubMed

Wu, Jeffrey Chun Yu; Hutchings, Christopher Hayden; Lindsay, Mark Jeffrey; Werner, Christopher James; Bundy, Bradley Charles

2015-01-10

Breakthroughs in enzyme immobilization have enabled increased enzyme recovery and reusability, leading to significant decreases in the cost of enzyme use and fueling biocatalysis growth. However, current enzyme immobilization techniques suffer from leaching, enzyme stability, and recoverability and reusability issues. Moreover, these techniques lack the ability to control the orientation of the immobilized enzymes. To determine the impact of orientation on covalently immobilized enzyme activity and stability, we apply our PRECISE (Protein Residue-Explicit Covalent Immobilization for Stability Enhancement) system to a model enzyme, T4 lysozyme. The PRECISE system uses non-canonical amino acid incorporation and the Huisgen 1,3-dipolar cycloaddition "click" reaction to enable directed enzyme immobilization at rationally chosen residues throughout an enzyme. Unlike previous site-specific systems, the PRECISE system is a truly covalent immobilization method. Utilizing this system, enzymes immobilized at proximate and distant locations from the active site were tested for activity and stability under denaturing conditions. Our results demonstrate that orientation control of covalently immobilized enzymes can provide activity and stability benefits exceeding that of traditional random covalent immobilization techniques. PRECISE immobilized enzymes were 50 and 73% more active than randomly immobilized enzymes after harsh freeze-thaw and chemical denaturant treatments. Copyright © 2014 Elsevier B.V. All rights reserved.

Nanotechnology applied to treatment of mucopolysaccharidoses.

PubMed

Schuh, Roselena S; Baldo, Guilherme; Teixeira, Helder F

2016-12-01

Mucopolysaccharidoses (MPS) are genetic disorders caused by the accumulation of glycosaminoglycans due to deficiencies in the lysosomal enzymes responsible for their catabolism. Current treatments are not fully effective and are not available for all MPS types. Accordingly, researchers have tested novel therapies for MPS, including nanotechnology-based enzyme delivery systems and gene therapy. In this review, we aim to analyze some of the approaches involving nanotechnology as alternative treatments for MPS. Areas covered: We analyze nanotechnology-based systems, focusing on the biomaterials, such as polymers and lipids, that comprise these nanostructures, and we have highlighted studies that describe their use as enzyme and gene delivery systems for the treatment of MPS diseases. Expert opinion: Some protocols, such as the use of polymer-based systems or nanostructured carriers associated with enzymes and nanotechnology-based carriers for gene therapy, along with combined approaches, seem to be the future of MPS therapy.

Spatial characterization of proteolytic enzyme activity in the foregut region of the adult necrophagous fly, Protophormia terraenovae.

PubMed

Rivers, David B; Acca, Gillian; Fink, Marc; Brogan, Rebecca; Schoeffield, Andrew

2014-08-01

The spatial distribution of proteolytic enzymes in the adult foregut of Protophormia terraenovae was studied in the context of protein digestion and regurgitation. Based on substrate specificity, pH optima, and use of specific protease inhibitors, all adults tested displayed enzyme activity in the foregut consistent with pepsin, trypsin and chymotrypsin. Chymotrypsin-like and trypsin-like enzyme activity were detected in all gut fluids and tissues tested, with chymotrypsin displaying the highest activity in saliva and salivary gland tissue, whereas maximal trypsin activity was evident in the crop. Pepsin-like activity was only evident in crop fluids and tissues. The activity of all three enzymes was low or undetectable (pepsin) in the fluids and tissue homogenates derived from the esophagus and cardia of any of the adults assayed. Fed adult females displayed higher enzyme activities than fed males, and the activity of all three enzymes were much more prevalent in fed adults than starved. The pH optimum of the trypsin-like enzyme was between pH 7.0 and 8.0; chymotrypsin was near pH 8.0; and maximal pepsin-like activity occurred between pH 1.0 and 2.0. Regurgitate from fed adult females displayed enzyme activity consistent with the proteolytic enzymes detected in crop gut fluids. Enzymes in regurgitate were not derived from food sources based on assays of bovine liver samples. These latter observations suggest that adult flies release fluids from foregut when encountering dry foods, potentially as a means to initiate extra-oral digestion. Copyright © 2014 Elsevier Ltd. All rights reserved.

Piezo1 in Smooth Muscle Cells Is Involved in Hypertension-Dependent Arterial Remodeling.

PubMed

Retailleau, Kevin; Duprat, Fabrice; Arhatte, Malika; Ranade, Sanjeev Sumant; Peyronnet, Rémi; Martins, Joana Raquel; Jodar, Martine; Moro, Céline; Offermanns, Stefan; Feng, Yuanyi; Demolombe, Sophie; Patel, Amanda; Honoré, Eric

2015-11-10

The mechanically activated non-selective cation channel Piezo1 is a determinant of vascular architecture during early development. Piezo1-deficient embryos die at midgestation with disorganized blood vessels. However, the role of stretch-activated ion channels (SACs) in arterial smooth muscle cells in the adult remains unknown. Here, we show that Piezo1 is highly expressed in myocytes of small-diameter arteries and that smooth-muscle-specific Piezo1 deletion fully impairs SAC activity. While Piezo1 is dispensable for the arterial myogenic tone, it is involved in the structural remodeling of small arteries. Increased Piezo1 opening has a trophic effect on resistance arteries, influencing both diameter and wall thickness in hypertension. Piezo1 mediates a rise in cytosolic calcium and stimulates activity of transglutaminases, cross-linking enzymes required for the remodeling of small arteries. In conclusion, we have established the connection between an early mechanosensitive process, involving Piezo1 in smooth muscle cells, and a clinically relevant arterial remodeling. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Application of activity-based protein profiling to study enzyme function in adipocytes.

PubMed

Galmozzi, Andrea; Dominguez, Eduardo; Cravatt, Benjamin F; Saez, Enrique

2014-01-01

Activity-based protein profiling (ABPP) is a chemical proteomics approach that utilizes small-molecule probes to determine the functional state of enzymes directly in native systems. ABPP probes selectively label active enzymes, but not their inactive forms, facilitating the characterization of changes in enzyme activity that occur without alterations in protein levels. ABPP can be a tool superior to conventional gene expression and proteomic profiling methods to discover new enzymes active in adipocytes and to detect differences in the activity of characterized enzymes that may be associated with disorders of adipose tissue function. ABPP probes have been developed that react selectively with most members of specific enzyme classes. Here, using as an example the serine hydrolase family that includes many enzymes with critical roles in adipocyte physiology, we describe methods to apply ABPP analysis to the study of adipocyte enzymatic pathways. © 2014 Elsevier Inc. All rights reserved.

Effects of non-starch polysaccharides enzymes on pancreatic and small intestinal digestive enzyme activities in piglet fed diets containing high amounts of barley.

PubMed

Li, Wei-Fen; Feng, Jie; Xu, Zi-Rong; Yang, Cai-Mei

2004-03-15

To investigate effects of non-starch polysaccharides(NSP) enzymes on pancreatic and small intestinal digestive enzyme activities in piglet fed diets containing high amounts of barley. Sixty crossbred piglets averaging 13.5 kg were randomly assigned to two treatment groups with three replications (pens) based on sex and mass. Each group was fed on the diet based on barley with or without added NSP enzymes (0.15%) for a 40-d period. At the end of the experiment the pigs were weighed. Three piglets of each group were chosen and slaughtered. Pancreas, digesta from the distal end of the duodenum and jejunal mucosa were collected for determination. Activities of the digestive enzymes trypsin, chymotrypsin, amylase and lipase were determined in the small intestinal sections as well as in homogenates of pancreatic tissue. Maltase, sucrase, lactase and gamma-glutamyl transpeptidase (gamma-GT) activities were analyzed in jejunal mucosa. Supplementation with NSP enzymes improved growth performance of piglets. It showed that NSP enzymes had no effect on digestive enzyme activities in pancreas, but decreased the activities of proteolytic enzyme, trypsin, amylase and lipase in duodenal contents by 57.56%, 76.08%, 69.03% and 40.22%(P<0.05) compared with control, and increased gamma-GT activities in jejunal mucosa by 118.75%(P<0.05). Supplementation with NSP enzymes in barley based diets could improve piglets' growth performance, decrease activities of proteolytic enzyme, trypsin, amylase and lipase in duodenal contents and increase gamma-GT activities in jejunal mucosa.

A New Versatile Microarray-based Method for High Throughput Screening of Carbohydrate-active Enzymes*

PubMed Central

Vidal-Melgosa, Silvia; Pedersen, Henriette L.; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B.; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G. T.

2015-01-01

Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths. PMID:25657012

[Effect of altitude chronic hypoxia on liver enzymes and its correlation with ACE/ACE2 in yak and migrated cattle].

PubMed

Liu, Feng-yun; Hu, Lin; Li, Yu-xian; Liu, Shi-ming; Tang, Yong-ping; Qi, Sheng-gui; Yang, Lei; Wu, Tian-yi

2015-05-01

To investigate the difference of liver enzyme levels and its correlation with serum ACE/ACE2 among yak and cattle on Qinghai-Tibetan plateau, and to further explore the biochemical mechanism of their liver of altitude adaptation. The serum samples of yak were collected at 3,000 m, 3,500 m, 4,000 m and 4,300 m respectively, meanwhile the serum samples of migrated cattle on plateau (2,500 m) and lowland cattle (1,300 m) were also collected. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), cholinesterase (CHE), gamma glutamyl transferase (GGT), alkaline phosphatase (ALP), serum lipase (LPS), angiotensin converting enzyme(ACE), angiotensin converting enzyme-2 (ACE2) in serum were measured by using fully automatic blood biochemcal analyzer. We analysed the differences of the above enzymes and its correlation with ACE/ACE2. We used one way analysis of variance (ANOVA). The levels of ALT in 4,000 m group and 4,300 m group of yak increased significantly compared with other groups, there were no statistically significant differences in AST, CHE, GGT, ACE/ACE2 levels of yaks at different altitudes. As compared to lowland cattle, the serum levels of AST and CHE were increased, the level of LPS and ACE was decreased significantly, respectively, and especially, the ratio of ACE/ACE2 of migranted cattle reduced nearly two times. The levels of LPS were significantly correlated to the ratio of ACE/ACE2 in yak (r = 0.357, P < 0.01), and a high correlation between ALP and ACE/ACE2 in lowland cattle( r = 0.418, P < 0.05), But the biggest contribution rate of the ratio of ACE/ACE2 was only 17.5% for the changes of the levels of liver enzyme. The results indicated that with the altitude increased did not significantly influence the changes of liver enzymes' activities in mountainous yaks but not in cattle. However, all above these changes weren't actually correlated to the ratio of ACE/ACE2.

Protease activity, localization and inhibition in the human hair follicle.

PubMed

Bhogal, R K; Mouser, P E; Higgins, C A; Turner, G A

2014-02-01

In humans, the process of hair shedding, referred to as exogen, is believed to occur independently of the other hair cycle phases. Although the actual mechanisms involved in hair shedding are not fully known, it has been hypothesized that the processes leading to the final step of hair shedding may be driven by proteases and/or protease inhibitor activity. In this study, we investigated the presence of proteases and protease activity in naturally shed human hairs and assessed enzyme inhibition activity of test materials. We measured enzyme activity using a fluorescence-based assay and protein localization by indirect immunohistochemistry (IHC). We also developed an ex vivo skin model for measuring the force required to pull hair fibres from skin. Our data demonstrate the presence of protease activity in the tissue material surrounding club roots. We also demonstrated the localization of specific serine protease protein expression in human hair follicle by IHC. These data provide evidence demonstrating the presence of proteases around the hair club roots, which may play a role during exogen. We further tested the hypothesis that a novel protease inhibitor system (combination of Trichogen) and climbazole) could inhibit protease activity in hair fibre club root extracts collected from a range of ethnic groups (U.K., Brazil, China, first-generation Mexicans in the U.S.A., Thailand and Turkey) in both males and females. Furthermore, we demonstrated that this combination is capable of increasing the force required to remove hair in an ex vivo skin model system. These studies indicate the presence of proteolytic activity in the tissue surrounding the human hair club root and show that it is possible to inhibit this activity with a combination of Trichogen and climbazole. This technology may have potential to reduce excessive hair shedding. © 2013 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Effects of triazole fungicides on androgenic disruption and CYP3A4 enzyme activity.

PubMed

Lv, Xuan; Pan, Liumeng; Wang, Jiaying; Lu, Liping; Yan, Weilin; Zhu, Yanye; Xu, Yiwen; Guo, Ming; Zhuang, Shulin

2017-03-01

Triazole fungicides are widely used as broad-spectrum fungicides, non-steroidal antiestrogens and for various industrial applications. Their residues have been frequently detected in multiple environmental and human matrices. The increasingly reported toxicity incidents have led triazole fungicides as emerging contaminants of environmental and public health concern. However, whether triazole fungicides behave as endocrine disruptors by directly mimicking environmental androgens/antiandrogens or exerting potential androgenic disruption indirectly through the inhibition of cytochrome P450 (CYP450) enzyme activity is yet an unresolved question. We herein evaluated five commonly used triazole fungicides including bitertanol, hexaconazole, penconazole, tebuconazole and uniconazole for the androgenic and anti-androgenic activity using two-hybrid recombinant human androgen receptor (AR) yeast bioassay and comparatively evaluated their effects on enzymatic activity of CYP3A4 by P450-Glo™ CYP3A4 bioassay. All five fungicides showed moderate anti-androgenic activity toward human AR with the IC 50 ranging from 9.34 μM to 79.85 μM. The anti-androgenic activity remained no significant change after the metabolism mediated by human liver microsomes. These fungicides significantly inhibited the activity of CYP3A4 at the environmental relevant concentrations and the potency ranks as tebuconazole > uniconazole > hexaconazole > penconazole > bitertanol with the corresponding IC 50 of 0.81 μM, 0.93 μM, 1.27 μM, 2.22 μM, and 2.74 μM, respectively. We found that their anti-androgenic activity and the inhibition potency toward CYP3A4 inhibition was significantly correlated (R 2 between 0.83 and 0.97, p < 0.001). Our results indicated that the risk assessment of triazole pesticides and structurally similar chemicals should fully consider potential androgenic disrupting effects and the influences on the activity of CYP450s. Copyright © 2016 Elsevier Ltd. All rights reserved.

Enzymes produced by halotolerant spore-forming gram-positive bacterial strains isolated from a resting habitat (Restinga de Jurubatiba) in Rio de Janeiro, Brazil: focus on proteases.

PubMed

D Santos, Anderson Fragoso; Pacheco, Clarissa Almeida; Valle, Roberta D Santos; Seldin, Lucy; D Santos, André Luis Souza

2014-12-01

The screening for hydrolases-producing, halotolerant, and spore-forming gram-positive bacteria from the root, rhizosphere, and non-rhizosphere soil of Blutaparon portulacoides, a plant found in the Restinga de Jurubatiba located at the northern region of Rio de Janeiro State, Brazil, resulted in the isolation of 22 strains. These strains were identified as Halobacillus blutaparonensis (n = 2), Oceanobacillus picturae (n = 5), and Oceanobacillus iheyensis (n = 15), and all showed the ability to produce different extracellular enzymes. A total of 20 isolates (90.9 %) showed activity for protease, 5 (22.7 %) for phytase, 3 (13.6 %) for cellulase, and 2 (9.1 %) for amylase. Some bacterial strains were capable of producing three (13.6 %) or two (9.1 %) distinct hydrolytic enzymes. However, no bacterial strain with ability to produce esterase and DNase was observed. The isolate designated M9, belonging to the species H. blutaparonensis, was the best producer of protease and also yielded amylase and phytase. This strain was chosen for further studies regarding its protease activity. The M9 strain produced similar amounts of protease when grown either without or with different NaCl concentrations (from 0.5 to 10 %). A simple inspection of the cell-free culture supernatant by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of three major alkaline proteases of 40, 50, and 70 kDa, which were fully inhibited by phenylmethylsulfonyl fluoride (PMSF) and tosyl-L-phenylalanine chloromethyl ketone (TPCK) (two classical serine protease inhibitors). The secreted proteases were detected in a wide range of temperature (from 4 to 45 °C) and their hydrolytic activities were stimulated by NaCl (up to 10 %). The serine proteases produced by the M9 strain cleaved gelatin, casein, albumin, and hemoglobin, however, in different extensions. Collectively, these results suggest the potential use of the M9 strain in biotechnological and/or industrial processes.

Quantitative High-Throughput Screen Identifies Inhibitors of the Schistosoma mansoni Redox Cascade

PubMed Central

Simeonov, Anton; Jadhav, Ajit; Sayed, Ahmed A.; Wang, Yuhong; Nelson, Michael E.; Thomas, Craig J.; Inglese, James; Williams, David L.; Austin, Christopher P.

2008-01-01

Schistosomiasis is a tropical disease associated with high morbidity and mortality, currently affecting over 200 million people worldwide. Praziquantel is the only drug used to treat the disease, and with its increased use the probability of developing drug resistance has grown significantly. The Schistosoma parasites can survive for up to decades in the human host due in part to a unique set of antioxidant enzymes that continuously degrade the reactive oxygen species produced by the host's innate immune response. Two principal components of this defense system have been recently identified in S. mansoni as thioredoxin/glutathione reductase (TGR) and peroxiredoxin (Prx) and as such these enzymes present attractive new targets for anti-schistosomiasis drug development. Inhibition of TGR/Prx activity was screened in a dual-enzyme format with reducing equivalents being transferred from NADPH to glutathione via a TGR-catalyzed reaction and then to hydrogen peroxide via a Prx-catalyzed step. A fully automated quantitative high-throughput (qHTS) experiment was performed against a collection of 71,028 compounds tested as 7- to 15-point concentration series at 5 µL reaction volume in 1536-well plate format. In order to generate a robust data set and to minimize the effect of compound autofluorescence, apparent reaction rates derived from a kinetic read were utilized instead of end-point measurements. Actives identified from the screen, along with previously untested analogues, were subjected to confirmatory experiments using the screening assay and subsequently against the individual targets in secondary assays. Several novel active series were identified which inhibited TGR at a range of potencies, with IC50s ranging from micromolar to the assay response limit (∼25 nM). This is, to our knowledge, the first report of a large-scale HTS to identify lead compounds for a helminthic disease, and provides a paradigm that can be used to jump-start development of novel therapeutics for other neglected tropical diseases. PMID:18235848

What is the role of the second “structural” NADP+-binding site in human glucose 6-phosphate dehydrogenase?

PubMed Central

Wang, Xiao-Tao; Chan, Ting Fai; Lam, Veronica M.S.; Engel, Paul C.

2008-01-01

Human glucose 6-phosphate dehydrogenase, purified after overexpression in E. coli, was shown to contain one molecule/subunit of acid-extractable “structural” NADP+ and no NADPH. This tightly bound NADP+ was reduced by G6P, presumably following migration to the catalytic site. Gel-filtration yielded apoenzyme, devoid of bound NADP+ but, surprisingly, still fully active. Mr of the main component of “stripped” enzyme by gel filtration was ∼100,000, suggesting a dimeric apoenzyme (subunit Mr = 59,000). Holoenzyme also contained tetramer molecules and, at high protein concentration, a dynamic equilibrium gave an apparent intermediate Mr of 150 kDa. Fluorescence titration of the stripped enzyme gave the K d for structural NADP+ as 37 nM, 200-fold lower than for “catalytic” NADP+. Structural NADP+ quenches 91% of protein fluorescence. At 37°C, stripped enzyme, much less stable than holoenzyme, inactivated irreversibly within 2 d. Inactivation at 4°C was partially reversed at room temperature, especially with added NADP+. Apoenzyme was immediately active, without any visible lag, in rapid-reaction studies. Human G6PD thus forms active dimer without structural NADP+. Apparently, the true role of the second, tightly bound NADP+ is to secure long-term stability. This fits the clinical pattern, G6PD deficiency affecting the long-lived non-nucleate erythrocyte. The K d values for two class I mutants, G488S and G488V, were 273 nM and 480 nM, respectively (seven- and 13-fold elevated), matching the structural prediction of weakened structural NADP+ binding, which would explain decreased stability and consequent disease. Preparation of native apoenzyme and measurement of K d constant for structural NADP+ will now allow quantitative assessment of this defect in clinical G6PD mutations. PMID:18493020

Improving colorimetric assays through protein enzyme-assisted gold nanoparticle amplification.

PubMed

Xie, Xiaoji; Xu, Wei; Liu, Xiaogang

2012-09-18

The discovery of the DNA-mediated assembly of gold nanoparticles was a great moment in the history of science; this understanding and chemical control enabled the rational design of functional nanomaterials as novel probes in biodetection. In contrast with conventional probes such as organic dyes, gold nanoparticles exhibit high photostability and unique size-dependent optical properties. Because of their high extinction coefficients and strong distance dependent optical properties, these nanoparticles have emerged over the past decade as a promising platform for rapid, highly sensitive colorimetric assays that allow for the visual detection of low concentrations of metal ions, small molecules, and biomacromolecules. These discoveries have deepened our knowledge of biological phenomena and facilitated the development of many new diagnostic and therapeutic tools. Despite these many advances and continued research efforts, current nanoparticle-based colorimetric detection systems still suffer from several drawbacks, such as limited sensitivity and selectivity. This Account describes the recent development of colorimetric assays based on protein enzyme-assisted gold nanoparticle amplification. The benefits of such detection systems include significantly improved detection sensitivity and selectivity. First, we discuss the general design of enzyme-modified nanoparticle systems in colorimetric assays. We show that a quantitative understanding of the unique properties of different enzymes is paramount for effective biological assays. We then examine the assays for nucleic acid detection based on different types of enzymes, including endonucleases, ligases, and polymerases. For each of these assays, we identify the underlying principles that contribute to the enhanced detection capability of nanoparticle systems and illustrate them with selected examples. Furthermore, we demonstrate that the combination of gold nanoparticles and specific enzymes can probe enzyme dynamics and function with high specificity, offering substantial advantages in both sensitivity and specificity over conventional detection methods. The screening of nuclease, methyltransferase, protease, and kinase activities can be colorimetrically performed in a straightforward manner. Finally, we discuss examples of colorimetric assays for metal ions and small molecules that constitute important advances toward visual monitoring of enzyme catalytic functions and gene expression. Although these enzyme-assisted assay methods hold great promise for myriad applications in biomedicine and bioimaging, the application of the described techniques in vivo faces formidable challenges. In addition, researchers do not fully understand the interactions of gold nanoparticles with enzyme molecules. This understanding will require the development of new techniques to probe enzyme substrate dynamics at the particle interface with higher spatial resolution and chemical specificity.

Effects of epithalon on activities gastrointestinal enzymes in young and old rats.

PubMed

Khavinson, V Kh; Malinin, V V; Timofeeva, N M; Egorova, V V; Nikitina, A A

2002-03-01

Peroral administration of Epithalon (Ala-Glu-Asp-Gly) to male and female Wistar rats aging 3 and 11 months changed activity of enzymes hydrolyzing carbohydrates, proteins, and phosphoric acid esters in various portions of the gastrointestinal tract. The most pronounced activation of enzymes was observed in 11-month-old animals. This effect diminished the differences in enzyme activities between young and old rats (compared to untreated animals). Our results indicate that Epithalon modulates activity of gastrointestinal enzymes during aging.

Co-immobilization of multiple enzymes by metal coordinated nucleotide hydrogel nanofibers: improved stability and an enzyme cascade for glucose detection.

PubMed

Liang, Hao; Jiang, Shuhui; Yuan, Qipeng; Li, Guofeng; Wang, Feng; Zhang, Zijie; Liu, Juewen

2016-03-21

Preserving enzyme activity and promoting synergistic activity via co-localization of multiple enzymes are key topics in bionanotechnology, materials science, and analytical chemistry. This study reports a facile method for co-immobilizing multiple enzymes in metal coordinated hydrogel nanofibers. Specifically, four types of protein enzymes, including glucose oxidase, Candida rugosa lipase, α-amylase, and horseradish peroxidase, were respectively encapsulated in a gel nanofiber made of Zn(2+) and adenosine monophosphate (AMP) with a simple mixing step. Most enzymes achieved quantitative loading and retained full activity. At the same time, the entrapped enzymes were more stable against temperature variation (by 7.5 °C), protease attack, extreme pH (by 2-fold), and organic solvents. After storing for 15 days, the entrapped enzyme still retained 70% activity while the free enzyme nearly completely lost its activity. Compared to nanoparticles formed with AMP and lanthanide ions, the nanofiber gels allowed much higher enzyme activity. Finally, a highly sensitive and selective biosensor for glucose was prepared using the gel nanofiber to co-immobilize glucose oxidase and horseradish peroxidase for an enzyme cascade system. A detection limit of 0.3 μM glucose with excellent selectivity was achieved. This work indicates that metal coordinated materials using nucleotides are highly useful for interfacing with biomolecules.

[Enzymatic degradation of organophosphorus insecticide chlorpyrifos by fungus WZ-I].

PubMed

Xie, Hui; Zhu, Lu-sheng; Wang, Jun; Wang, Xiu-guo; Liu, Wei; Qian, Bo; Wang, Qian

2005-11-01

Degradation characteristics of chlorpyrifos insecticides was determined by the crude enzyme extracted from the isolated strain WZ-I ( Fusarium LK. ex Fx). The best separating condition and the degrading characteristic of chlorpyrifos were studied. Rate of degradation for chlorpyrifos by its intracellular enzyme, extracellular enzyme and cell fragment was 60.8%, 11.3% and 48%, respectively. The degrading enzyme was extracted after this fungus was incubated for 8 generations in the condition of noninducement, and its enzymic activity lost less, the results show that this enzyme is an intracellular and connatural enzyme. The solubility protein of the crude enzyme was determined with Albumin (bovine serum) as standard protein and the solubility protein of the crude enzyme was 3.36 mg x mL(-1). The pH optimum for crude enzyme was 6.8 for enzymatic degradation of chlorpyrifos, and it had comparatively high activity in the range of pH 6.0 - 9.0. The optimum temperature for enzymatic activity was at 40 degrees C, it still had comparatively high activity in the range of temperature 20-50 degrees C, the activity of enzyme rapidly reduced at 55 degrees C, its activity was 41% of the maximal activity. The crude enzyme showed Km value for chlorpyrifos of 1.049 26 mmol x L(-1), and the maximal enzymatic degradation rate was 0.253 5 micromol x (mg x min)(-1). Additional experimental evidence suggests that the enzyme had the stability of endure for temperature and pH, the crude enzyme of fungus WZ-I could effectively degrade chlorpyrifos.

The deubiquitinating enzyme DUBAI stabilizes DIAP1 to suppress Drosophila apoptosis

PubMed Central

Yang, C-S; Sinenko, S A; Thomenius, M J; Robeson, A C; Freel, C D; Horn, S R; Kornbluth, S

2014-01-01

Deubiquitinating enzymes (DUBs) counteract ubiquitin ligases to modulate the ubiquitination and stability of target signaling molecules. In Drosophila, the ubiquitin–proteasome system has a key role in the regulation of apoptosis, most notably, by controlling the abundance of the central apoptotic regulator DIAP1. Although the mechanism underlying DIAP1 ubiquitination has been extensively studied, the precise role of DUB(s) in controlling DIAP1 activity has not been fully investigated. Here we report the identification of a DIAP1-directed DUB using two complementary approaches. First, a panel of putative Drosophila DUBs was expressed in S2 cells to determine whether DIAP1 could be stabilized, despite treatment with death-inducing stimuli that would induce DIAP1 degradation. In addition, RNAi fly lines were used to detect modifiers of DIAP1 antagonist-induced cell death in the developing eye. Together, these approaches identified a previously uncharacterized protein encoded by CG8830, which we named DeUBiquitinating-Apoptotic-Inhibitor (DUBAI), as a novel DUB capable of preserving DIAP1 to dampen Drosophila apoptosis. DUBAI interacts with DIAP1 in S2 cells, and the putative active site of its DUB domain (C367) is required to rescue DIAP1 levels following apoptotic stimuli. DUBAI, therefore, represents a novel locus of apoptotic regulation in Drosophila, antagonizing cell death signals that would otherwise result in DIAP1 degradation. PMID:24362437

Effects of arginine on rabbit muscle creatine kinase and salt-induced molten globule-like state.

PubMed

Ou, Wen-bin; Wang, Ri-Sheng; Lu, Jie; Zhou, Hai-Meng

2003-11-03

The arginine (Arg)-induced unfolding and the salt-induced folding of creatine kinase (CK) have been studied by measuring enzyme activity, fluorescence emission spectra, native polyacrylamide gel electrophoresis and size exclusion chromatography (SEC). The results showed that Arg caused inactivation and unfolding of CK, but there was no aggregation during CK denaturation. The kinetics of CK unfolding followed a one-phase process. At higher concentrations of Arg (>160 mM), the CK dimers were fully dissociated, the alkali characteristic of Arg mainly led to the dissociation of dimers, but not denaturation effect of Arg's guanidine groups on CK. The inactivation of CK occurred before noticeable conformational changes of the whole molecules. KCl induced monomeric and dimeric molten globule-like states of CK denatured by Arg. These results suggest that as a protein denaturant, the effect of Arg on CK differed from that of guanidine and alkali, its denaturation for protein contains the double effects, which acts not only as guanidine hydrochloride but also as alkali. The active sites of CK have more flexibility than the whole enzyme conformation. Monomeric and dimeric molten globule-like states of CK were formed by the salt inducing in 160 and 500 mM Arg H(2)O solutions, respectively. The molten globule-like states indicate that monomeric and dimeric intermediates exist during CK folding. Furthermore, these results also proved the orderly folding model of CK.

Molecular architectures and functions of radical enzymes and their (re)activating proteins.

PubMed

Shibata, Naoki; Toraya, Tetsuo

2015-10-01

Certain proteins utilize the high reactivity of radicals for catalysing chemically challenging reactions. These proteins contain or form a radical and therefore named 'radical enzymes'. Radicals are introduced by enzymes themselves or by (re)activating proteins called (re)activases. The X-ray structures of radical enzymes and their (re)activases revealed some structural features of these molecular apparatuses which solved common enigmas of radical enzymes—i.e. how the enzymes form or introduce radicals at the active sites, how they use the high reactivity of radicals for catalysis, how they suppress undesired side reactions of highly reactive radicals and how they are (re)activated when inactivated by extinction of radicals. This review highlights molecular architectures of radical B12 enzymes, radical SAM enzymes, tyrosyl radical enzymes, glycyl radical enzymes and their (re)activating proteins that support their functions. For generalization, comparisons of the recently reported structures of radical enzymes with those of canonical radical enzymes are summarized here. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Iodothyronine 5'-deiodinase activity and thyroid hormone content in brown adipose tissue during the breeding cycle of the rat.

PubMed Central

Viñas, O; Giralt, M; Obregón, M J; Iglesias, R; Villarroya, F; Mampel, T

1988-01-01

Brown adipose tissue iodothyronine 5'-deiodinase activity is significantly lower in 17-day pregnant rats compared with virgin controls and remains low during late pregnancy and lactation. It fully recovers with abrupt weaning, but only partially with spontaneous weaning. Even though this profile of changes is remarkably in step with the known pattern of modifications in brown fat thermogenesis during the breeding cycle, the lowered iodothyronine 5'-deiodinase activity appearing between days 15 and 17 of pregnancy occurs earlier than the reduction in brown adipose tissue thermogenesis. Brown fat 3,3',5-tri-iodothyronine content is also reduced in late pregnant, early and mid-lactating rats, most probably as a consequence of the lowered 5'-deiodination of thyroxine in situ. Acute insulin treatment increases brown fat iodothyronine 5'-deiodinase activity in virgin animals as well as in late-pregnant and lactating rats, despite the lowered basal enzyme activity levels in the latter groups. Thus an impaired response to insulin in brown fat does not appear to be a factor leading to the lowered iodothyronine 5'-deiodinase activity during late pregnancy and lactation. PMID:3060112

Defying the activity-stability trade-off in enzymes: taking advantage of entropy to enhance activity and thermostability.

PubMed

Siddiqui, Khawar Sohail

2017-05-01

The biotechnological applications of enzymes are limited due to the activity-stability trade-off, which implies that an increase in activity is accompanied by a concomitant decrease in protein stability. This premise is based on thermally adapted homologous enzymes where cold-adapted enzymes show high intrinsic activity linked to enhanced thermolability. In contrast, thermophilic enzymes show low activity around ambient temperatures. Nevertheless, genetically and chemically modified enzymes are beginning to show that the activity-stability trade-off can be overcome. In this review, the origin of the activity-stability trade-off, the thermodynamic basis for enhanced activity and stability, and various approaches for escaping the activity-stability trade-off are discussed. The role of entropy in enhancing both the activity and the stability of enzymes is highlighted with a special emphasis placed on the involvement of solvent water molecules. This review is concluded with suggestions for further research, which underscores the implications of these findings in the context of productivity curves, the Daniel-Danson equilibrium model, catalytic antibodies, and life on cold planets.

Effects of Nanoparticle Size on Multilayer Formation and Kinetics of Tethered Enzymes.

PubMed

Lata, James P; Gao, Lizeng; Mukai, Chinatsu; Cohen, Roy; Nelson, Jacquelyn L; Anguish, Lynne; Coonrod, Scott; Travis, Alexander J

2015-09-16

Despite numerous applications, we lack fundamental understanding of how variables such as nanoparticle (NP) size influence the activity of tethered enzymes. Previously, we showed that biomimetic oriented immobilization yielded higher specific activities versus nonoriented adsorption or carboxyl-amine binding. Here, we standardize NP attachment strategy (oriented immobilization via hexahistidine tags) and composition (Ni-NTA coated gold NPs), to test the impact of NP size (⌀5, 10, 20, and 50 nm) on multilayer formation, activity, and kinetic parameters (kcat, KM, kcat/KM) of enzymes representing three different classes: glucose-6-phosphate isomerase (GPI), an isomerase; Glyceraldehyde-3-phosphate dehydrogenase S (GAPDHS), an oxidoreductase; and pyruvate kinase (PK), a transferase. Contrary to other reports, we observed no trend in kinetic parameters for individual enzymes when found in monolayers (<100% enzyme coverage), suggesting an advantage for oriented immobilization versus other attachment strategies. Saturating the NPs to maximize activity per NP resulted in enzyme multilayer formation. Under these conditions, total activity per NP increased with increasing NP size. Conversely, specific activity for all three enzymes was highest when tethered to the smallest NPs, retaining a remarkable 73-94% of the activity of free/untethered enzymes. Multilayer formations caused a clear trend of kcat decreasing with increasing NP size, yet negligible change in KM. Understanding the fundamental relationships between NP size and tethered enzyme activity enables optimized design of various applications, maximizing activity per NP or activity per enzyme molecule.

[Seasonal variations of soil enzyme activities in typical plant communities in the Ebinur Lake wetland, China].

PubMed

Zhu, Hai Qiang; Li, Yan Hong; Li, Fa Dong

2017-04-18

In this study, the soil catalase, phosphatase and urease activities of typical plant communities of reed (Phragmites australis) and tamarisk (Tamarix ramosissima) and their influencing factors were investigated in Ebinur Lake wetland. The results showed that three soil enzyme activities of reed and tamarisk had seasonal dynamic characteristics during different growth periods. For the reed community, the peak concentrations of soil catalase, phosphatase and urease appeared at vigorous stage with 3.26, 0.60 and 0.33 mg·g -1 , respectively, and the minimum value occurred at budding stage and leaf-expansion stage. For the tamarisk community, the peak values of three soil enzyme activities appeared at withered stage with values of 6.33, 0.58 and 0.21 mg·g -1 , respectively, and the valley values were observed at flowering and vigorous stages. Urease was stable during different growth periods, and it could be used as an indicator to identify the differences of soil enzyme activities in the wetlands. The enzyme activities of reed and tamarisk had significant positive correlation with soil organic matter and total P in all growth periods, while there was no significant relationship between enzyme activities and soil water content. The enzyme activities of reed had significant positive correlation with ammonium nitrogen in the rapid growth period. There were no significant relationships between enzyme activities and soil salinity in both communities. The soil enzyme activities of reed and tamarisk were controlled by many factors. Soil organic matter, soil water and soil temperature were the main factors influencing the enzyme activities in the Ebinur Lake wetland.

Assessment of digestive enzymes activity during the fry development of the endangered Caspian brown trout Salmo caspius.

PubMed

Zamani, A; Hajimoradloo, A; Madani, R; Farhangi, M

2009-09-01

The study of digestive enzymes activity at Salmo caspius fry showed that enzymes were available at the moment of mouth opening on the first day post hatching (dph) and the activity of enzymes showed no significant difference from the hatching day 28 dph. An increased activity was seen between 32 and 43 dph and this activity was significantly higher than the activity during the first 28 days. In the primary stages after yolk sac resorption (43-58 dph), enzymes activity showed an increased profile, however none of them showed a significant difference between 43 and 58 dph.

Temperature and UV light affect the activity of marine cell-free enzymes

NASA Astrophysics Data System (ADS)

Thomson, Blair; Hepburn, Christopher David; Lamare, Miles; Baltar, Federico

2017-09-01

Microbial extracellular enzymatic activity (EEA) is the rate-limiting step in the degradation of organic matter in the oceans. These extracellular enzymes exist in two forms: cell-bound, which are attached to the microbial cell wall, and cell-free, which are completely free of the cell. Contrary to previous understanding, cell-free extracellular enzymes make up a substantial proportion of the total marine EEA. Little is known about these abundant cell-free enzymes, including what factors control their activity once they are away from their sites (cells). Experiments were run to assess how cell-free enzymes (excluding microbes) respond to ultraviolet radiation (UVR) and temperature manipulations, previously suggested as potential control factors for these enzymes. The experiments were done with New Zealand coastal waters and the enzymes studied were alkaline phosphatase (APase), β-glucosidase, (BGase), and leucine aminopeptidase (LAPase). Environmentally relevant UVR (i.e. in situ UVR levels measured at our site) reduced cell-free enzyme activities by up to 87 % when compared to controls, likely a consequence of photodegradation. This effect of UVR on cell-free enzymes differed depending on the UVR fraction. Ambient levels of UV radiation were shown to reduce the activity of cell-free enzymes for the first time. Elevated temperatures (15 °C) increased the activity of cell-free enzymes by up to 53 % when compared to controls (10 °C), likely by enhancing the catalytic activity of the enzymes. Our results suggest the importance of both UVR and temperature as control mechanisms for cell-free enzymes. Given the projected warming ocean environment and the variable UVR light regime, it is possible that there could be major changes in the cell-free EEA and in the enzymes contribution to organic matter remineralization in the future.

Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

NASA Astrophysics Data System (ADS)

Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

2016-02-01

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

PubMed Central

Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

2016-01-01

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology. PMID:26861509

Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion.

PubMed

Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W; Liu, Yan; Walter, Nils G; Yan, Hao

2016-02-10

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

A new versatile microarray-based method for high throughput screening of carbohydrate-active enzymes.

PubMed

Vidal-Melgosa, Silvia; Pedersen, Henriette L; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G T

2015-04-03

Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Measuring the Enzyme Activity of Arabidopsis Deubiquitylating Enzymes.

PubMed

Kalinowska, Kamila; Nagel, Marie-Kristin; Isono, Erika

2016-01-01

Deubiquitylating enzymes, or DUBs, are important regulators of ubiquitin homeostasis and substrate stability, though the molecular mechanisms of most of the DUBs in plants are not yet understood. As different ubiquitin chain types are implicated in different biological pathways, it is important to analyze the enzyme characteristic for studying a DUB. Quantitative analysis of DUB activity is also important to determine enzyme kinetics and the influence of DUB binding proteins on the enzyme activity. Here, we show methods to analyze DUB activity using immunodetection, Coomassie Brilliant Blue staining, and fluorescence measurement that can be useful for understanding the basic characteristic of DUBs.

Enzyme activities in plasma, kidney, liver, and muscle of five avian species

USGS Publications Warehouse

Franson, J.C.; Murray, H.C.; Bunck, C.

1985-01-01

Activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatine phosphokinase (CPK), and lactate dehydrogenase (LDH) were determined in plasma, kidney, liver, and muscle from five species of captive birds. Few differences occurred in plasma activities between sexes but considerable differences occurred between species. All five enzymes were detected in each of the tissues sampled. Relative enzyme activities in liver, kidney, and muscle were similar for each species. CPK activity was much higher in muscle than in liver or kidney and, of the five enzymes studied, may be the best indicator of muscle damage. Most of the other enzymes were more evenly distributed among the three tissues, and no organ-specific enzyme could be identified for liver or kidney. Because of interspecific variations in plasma enzyme activities, it is important to establish baseline values for each species to ensure accurate interpretation of results.

The molecular basis of the effect of temperature on enzyme activity.

PubMed

Daniel, Roy M; Peterson, Michelle E; Danson, Michael J; Price, Nicholas C; Kelly, Sharon M; Monk, Colin R; Weinberg, Cristina S; Oudshoorn, Matthew L; Lee, Charles K

2009-12-23

Experimental data show that the effect of temperature on enzymes cannot be adequately explained in terms of a two-state model based on increases in activity and denaturation. The Equilibrium Model provides a quantitative explanation of enzyme thermal behaviour under reaction conditions by introducing an inactive (but not denatured) intermediate in rapid equilibrium with the active form. The temperature midpoint (Teq) of the rapid equilibration between the two forms is related to the growth temperature of the organism, and the enthalpy of the equilibrium (DeltaHeq) to its ability to function over various temperature ranges. In the present study, we show that the difference between the active and inactive forms is at the enzyme active site. The results reveal an apparently universal mechanism, independent of enzyme reaction or structure, based at or near the active site, by which enzymes lose activity as temperature rises, as opposed to denaturation which is global. Results show that activity losses below Teq may lead to significant errors in the determination of DeltaG*cat made on the basis of the two-state ('Classical') model, and the measured kcat will then not be a true indication of an enzyme's catalytic power. Overall, the results provide a molecular rationale for observations that the active site tends to be more flexible than the enzyme as a whole, and that activity losses precede denaturation, and provide a general explanation in molecular terms for the effect of temperature on enzyme activity.

A review on the effects of supercritical carbon dioxide on enzyme activity.

PubMed

Wimmer, Zdenek; Zarevúcka, Marie

2010-01-19

Different types of enzymes such as lipases, several phosphatases, dehydrogenases, oxidases, amylases and others are well suited for the reactions in SC-CO(2). The stability and the activity of enzymes exposed to carbon dioxide under high pressure depend on enzyme species, water content in the solution and on the pressure and temperature of the reaction system. The three-dimensional structure of enzymes may be significantly altered under extreme conditions, causing their denaturation and consequent loss of activity. If the conditions are less adverse, the protein structure may be largely retained. Minor structural changes may induce an alternative active protein state with altered enzyme activity, specificity and stability.

A Review on the Effects of Supercritical Carbon Dioxide on Enzyme Activity

PubMed Central

Wimmer, Zdeněk; Zarevúcka, Marie

2010-01-01

Different types of enzymes such as lipases, several phosphatases, dehydrogenases, oxidases, amylases and others are well suited for the reactions in SC-CO2. The stability and the activity of enzymes exposed to carbon dioxide under high pressure depend on enzyme species, water content in the solution and on the pressure and temperature of the reaction system. The three-dimensional structure of enzymes may be significantly altered under extreme conditions, causing their denaturation and consequent loss of activity. If the conditions are less adverse, the protein structure may be largely retained. Minor structural changes may induce an alternative active protein state with altered enzyme activity, specificity and stability. PMID:20162013

Mechanobiocatalysis: Modulating Enzymatic Activity with Mechanical Force

DTIC Science & Technology

2015-09-28

displayed by enzymes and other materials. It was demonstrated that the application of forces to enzymes properly outfitted with polymers resulted in...distortions at the active sites of the corresponding enzymes . For example, polymer-protein composites were found to display photophysical properties that...intrinsic activities displayed by enzymes and other materials. It was demonstrated that the application of forces to enzymes properly outfitted with polymers

Biochemistry of Mitochondrial Coenzyme Q Biosynthesis.

PubMed

Stefely, Jonathan A; Pagliarini, David J

2017-10-01

Coenzyme Q (CoQ, ubiquinone) is a redox-active lipid produced across all domains of life that functions in electron transport and oxidative phosphorylation and whose deficiency causes human diseases. Yet, CoQ biosynthesis has not been fully defined in any organism. Several proteins with unclear molecular functions facilitate CoQ biosynthesis through unknown means, and multiple steps in the pathway are catalyzed by currently unidentified enzymes. Here we highlight recent progress toward filling these knowledge gaps through both traditional biochemistry and cutting-edge 'omics' approaches. To help fill the remaining gaps, we present questions framed by the recently discovered CoQ biosynthetic complex and by putative biophysical barriers. Mapping CoQ biosynthesis, metabolism, and transport pathways has great potential to enhance treatment of numerous human diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Study on relationship between effective components and soil enzyme activity in different growth patterns of Panax ginseng].

PubMed

Yang, Yan-Wen; Jiang, Yuan-Tong

2016-08-01

Study on 5 effective components and 6 soil enzyme activities of 2 different growth patterns, analyse the dates with the canonical correlation analysis, In order to reveal the relations between the effective components and soil enzyme activities. The result showed that they had a great relation between the effective components and soil enzyme activities, the activity of the same enzyme in humus soil was higher than that in farmland soil. Growth pattern of farmland soil, if the invertase and phosphatase activity were too high, which would inhibit the accumulation of total ginsenoside, water-miscible total proteins and total amino acid; Growth pattern of humus soil, if the invertase, urease and phosphatase activity were too high, which would inhibit the accumulation of total ginsenoside and the total essential oils. Integral soil enzyme activity can be used as a index of soil quality, which, together with other growth factors. The appropriate enzyme activity can accelerate the circulation and transformation of all kinds of material in the soil, improve effectively components accumulation. Copyright© by the Chinese Pharmaceutical Association.

The effect of RAAS blockade on the progression of diabetic nephropathy.

PubMed

Roscioni, Sara S; Heerspink, Hiddo J Lambers; de Zeeuw, Dick

2014-02-01

The renin-angiotensin-aldosterone system (RAAS) has a key role in the regulation of blood pressure, sodium and water balance, and cardiovascular and renal homeostasis. In diabetic nephropathy, excessive activation of the RAAS results in progressive renal damage. RAAS blockade using angiotensin-converting-enzyme inhibitors or angiotensin-receptor blockers is the cornerstone of treatment of diabetic renal disease. Alternative RAAS-blockade strategies include renin inhibition and aldosterone blockade. Data from small initial studies of these agents are promising. However, single-agent interventions do not fully block the RAAS and patients treated with these therapies remain at high residual renal risk. Approaches to optimize drug responses include dietary changes and increasing dosages. The theoretically attractive option of combining different RAAS interventions has also been tested in clinical trials but long-term outcomes were disappointing. However, dual RAAS blockade might represent a good therapeutic option for specific patients. A better knowledge of the pathophysiology of the RAAS is crucial to fully understand the mechanisms of action of RAAS blockers and to exploit their renoprotective effects. Moreover, lifestyle interventions or diagnostic tools might be used to optimize RAAS blockade and identify those patients who are most likely to benefit from the therapy.

Dynamic Perturbation of the Active Site Determines Reversible Thermal Inactivation in Glycoside Hydrolase Family 12.

PubMed

Jiang, Xukai; Li, Wen; Chen, Guanjun; Wang, Lushan

2017-02-27

The temperature dependence of enzyme catalysis is highly debated. Specifically, how high temperatures induce enzyme inactivation has broad implications for both fundamental and applied science. Here, we explored the mechanism of the reversible thermal inactivation in glycoside hydrolase family 12 (GH12) using comparative molecular dynamics simulations. First, we investigated the distribution of structural flexibility over the enzyme and found that the active site was the general thermal-sensitive region in GH12 cellulases. The dynamic perturbation of the active site before enzyme denaturation was explored through principal-component analysis, which indicated that variations in the collective motion and conformational ensemble of the active site may precisely correspond to enzyme transition from its active form to the inactive form. Furthermore, the degree of dynamic perturbation of the active site was found to be negatively correlated with the melting temperatures of GH12 enzymes, further proving the importance of the dynamic stability of the active site. Additionally, analysis of the residue-interaction network revealed that the active site in thermophilic enzyme was capable of forming additional contacts with other amino acids than those observed in the mesophilic enzyme. These interactions are likely the key mechanisms underlying the differences in rigidity of the active site. These findings provide further biophysical insights into the reversible thermal inactivation of enzymes and potential applications in future protein engineering.

Sulfonyl 3-alkynyl pantetheinamides as mechanism-based crosslinkers of ACP dehydratase

PubMed Central

Ishikawa, Fumihiro; Haushalter, Robert W.; Lee, D. John; Finzel, Kara; Burkart, Michael D.

2013-01-01

The acyl carrier protein (ACP) plays a central function in acetate biosynthetic pathways, serving as a tether for substrates and growing intermediates. Activity and structural studies have highlighted the complexities of this role, and its protein-protein interactions have recently come under scrutiny as a regulator of catalysis. As existing methods to interrogate these interactions have fallen short, we have sought to develop new tools to aid their study. Here we describe the design, synthesis, and application of pantetheinamides capable of crosslinking ACPs with catalytic β-hydroxyacyl carrier protein dehydratase (DH) domains based upon a 3-alkynyl sulfone warhead. We demonstrate this process by application to the Escherichia coli fatty acid synthase and apply it to probe protein-protein interactions with non-cognate carrier proteins. Finally, we use solution phase protein NMR to demonstrate that sulfonyl-3-alkynyl pantetheinamide is fully sequestered by the ACP, indicating that the crypto-ACP closely mimics the natural DH substrate. This crosslinking technology offers immediate potential to lock these biosynthetic enzymes in their native binding states by providing access to mechanistically-crosslinked enzyme complexes, presenting a solution to ongoing structural challenges. PMID:23718183

4-Hydroxyphenylpyruvate Dioxygenase Catalysis

PubMed Central

Raspail, Corinne; Graindorge, Matthieu; Moreau, Yohann; Crouzy, Serge; Lefèbvre, Bertrand; Robin, Adeline Y.; Dumas, Renaud; Matringe, Michel

2011-01-01

4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxyphenylpyruvate (HPP) into homogentisate. HPPD is the molecular target of very effective synthetic herbicides. HPPD inhibitors may also be useful in treating life-threatening tyrosinemia type I and are currently in trials for treatment of Parkinson disease. The reaction mechanism of this key enzyme in both plants and animals has not yet been fully elucidated. In this study, using site-directed mutagenesis supported by quantum mechanical/molecular mechanical theoretical calculations, we investigated the role of catalytic residues potentially interacting with the substrate/intermediates. These results highlight the following: (i) the central role of Gln-272, Gln-286, and Gln-358 in HPP binding and the first nucleophilic attack; (ii) the important movement of the aromatic ring of HPP during the reaction, and (iii) the key role played by Asn-261 and Ser-246 in C1 hydroxylation and the final ortho-rearrangement steps (numbering according to the Arabidopsis HPPD crystal structure 1SQD). Furthermore, this study reveals that the last step of the catalytic reaction, the 1,2 shift of the acetate side chain, which was believed to be unique to the HPPD activity, is also catalyzed by a structurally unrelated enzyme. PMID:21613226

Extremophiles and biotechnology: current uses and prospects

PubMed Central

Coker, James A.

2016-01-01

Biotechnology has almost unlimited potential to change our lives in very exciting ways. Many of the chemical reactions that produce these products can be fully optimized by performing them at extremes of temperature, pressure, salinity, and pH for efficient and cost-effective outcomes. Fortunately, there are many organisms (extremophiles) that thrive in extreme environments found in nature and offer an excellent source of replacement enzymes in lieu of mesophilic ones currently used in these processes. In this review, I discuss the current uses and some potential new applications of extremophiles and their products, including enzymes, in biotechnology. PMID:27019700

Correlation Among Soil Enzyme Activities, Root Enzyme Activities, and Contaminant Removal in Two-Stage In Situ Constructed Wetlands Purifying Domestic Wastewater.

PubMed

Ni, Lixiao; Xu, Jiajun; Chu, Xianglin; Li, Shiyin; Wang, Peifang; Li, Yiping; Li, Yong; Zhu, Liang; Wang, Chao

2016-07-01

Two-stage in situ wetlands (two vertical flow constructed wetlands in parallel and a horizontal flow constructed wetland) were constructed for studying domestic wastewater purification and the correlations between contaminant removal and plant and soil enzyme activities. Results indicated the removal efficiency of NH4 (+) and NO3 (-) were significantly correlated with both urease and protease activity, and the removal of total phosphorus was significantly correlated with phosphatase activity. Chemical oxygen demand removal was not correlated with enzyme activity in constructed wetlands. Plant root enzyme (urease, phosphatase, protease and cellulose) activity correlation was apparent with all contaminant removal in the two vertical flow constructed wetlands. However, the correlation between the plant root enzyme activity and contaminant removal was poor in horizontal flow constructed wetlands. Results indicated that plant roots clearly played a role in the removal of contaminants.

Heparin/heparan sulfate 6-O-sulfatase from Flavobacterium heparinum: integrated structural and biochemical investigation of enzyme active site and substrate specificity.

PubMed

Myette, James R; Soundararajan, Venkataramanan; Shriver, Zachary; Raman, Rahul; Sasisekharan, Ram

2009-12-11

Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this enzyme in Escherichia coli in a soluble, active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the enzyme and mode of action through an analysis of the active-site topology of the enzyme including identifying key enzyme-substrate interactions and assigning key amino acids within the active site of the enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the active site of the enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of enzymes.

Stabilizing effect of biochar on soil extracellular enzymes after a denaturing stress.

PubMed

Elzobair, Khalid A; Stromberger, Mary E; Ippolito, James A

2016-01-01

Stabilizing extracellular enzymes may maintain enzymatic activity while protecting enzymes from proteolysis and denaturation. A study determined whether a fast pyrolysis hardwood biochar (CQuest™) would reduce evaporative losses, subsequently stabilizing soil extracellular enzymes and prohibiting potential enzymatic activity loss following a denaturing stress (microwaving). Soil was incubated in the presence of biochar (0%, 1%, 2%, 5%, or 10% by wt.) for 36 days and then exposed to microwave energies (0, 400, 800, 1600, or 3200 J g(-1) soil). Soil enzymes (β-glucosidase, β-d-cellobiosidase, N-acetyl-β-glucosaminidase, phosphatase, leucine aminopeptidase, β-xylosidase) were analyzed by fluorescence-based assays. Biochar amendment reduced leucine aminopeptidase and β-xylosidase potential activity after the incubation period and prior to stress exposure. The 10% biochar rate reduced soil water loss at the lowest stress level (400 J microwave energy g(-1) soil). Enzyme stabilization was demonstrated for β-xylosidase; intermediate biochar application rates prevented a complete loss of this enzyme's potential activity after soil was exposed to 400 (1% biochar treatment) or 1600 (5% biochar treatment) J microwave energy g(-1) soil. Remaining enzyme potential activities were not affected by biochar, and activities decreased with increasing stress levels. We concluded that biochar has the potential to reduce evaporative soil water losses and stabilize certain extracellular enzymes where activity is maintained after a denaturing stress; this effect was biochar rate and enzyme dependent. While biochar may reduce the potential activity of certain soil extracellular enzymes, this phenomenon was not universal as the majority of enzymes assayed in this study were unaffected by exposure to biochar. Copyright © 2015 Elsevier Ltd. All rights reserved.

Incorporating unnatural amino acids to engineer biocatalysts for industrial bioprocess applications.

PubMed

Ravikumar, Yuvaraj; Nadarajan, Saravanan Prabhu; Hyeon Yoo, Tae; Lee, Chong-Soon; Yun, Hyungdon

2015-12-01

The bioprocess engineering with biocatalysts broadly spans its development and actual application of enzymes in an industrial context. Recently, both the use of bioprocess engineering and the development and employment of enzyme engineering techniques have been increasing rapidly. Importantly, engineering techniques that incorporate unnatural amino acids (UAAs) in vivo has begun to produce enzymes with greater stability and altered catalytic properties. Despite the growth of this technique, its potential value in bioprocess applications remains to be fully exploited. In this review, we explore the methodologies involved in UAA incorporation as well as ways to synthesize these UAAs. In addition, we summarize recent efforts to increase the yield of UAA engineered proteins in Escherichia coli and also the application of this tool in enzyme engineering. Furthermore, this protein engineering tool based on the incorporation of UAA can be used to develop immobilized enzymes that are ideal for bioprocess applications. Considering the potential of this tool and by exploiting these engineered enzymes, we expect the field of bioprocess engineering to open up new opportunities for biocatalysis in the near future. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of impaired processing of neuropeptides in the brains of endoprotease knockout mice.

PubMed

Beinfeld, Margery C

2011-01-01

With the development of mice in which individual proteolytic enzymes have been inactivated, it has been of great interest to see how loss of these enzymes alters the processing of neuropeptides. In the course of studying changes in the peptide cholecystokinin (CCK) and other neuropeptides in several of these knockout mice, it has become clear that neuropeptide processing is complex and regionally specific. The enzyme responsible for processing in one part of the brain may not be involved in other parts of the brain. It is essential to do a detailed dissection of the brain and analyze peptide levels in many brain regions to fully understand the role of the enzymes. Because loss of these proteases may trigger compensatory mechanisms which involve expression of the neuropeptides being studied or other proteases or accessory proteins, it is also important to examine how loss of an enzyme alters expression of the neuropeptides being studied as well as other proteins thought to be involved in neuropeptide processing. By determining how loss of an enzyme alters the molecular form(s) of the peptide that are made, additional mechanistic information can be obtained. This review will describe established methods to achieve these research goals.

The immunological capacity in the larvae of Pacific oyster Crassostrea gigas.

PubMed

Song, Xiaorui; Wang, Hao; Xin, Lusheng; Xu, Jiachao; Jia, Zhihao; Wang, Lingling; Song, Linsheng

2016-02-01

As the immune system has not fully developed during early developmental stages, bivalve larvae are more susceptible for pathogens, which frequently leads to the significant mortality in hatcheries. In the present study, the development of immune system and its response against bacteria challenge were investigated in order to characterize the repertoire of immunological capacity of Pacific oyster Crassostrea gigas during the ontogenesis. The phagocytosis was firstly observed in the early D-veliger larvae (17 hpf), especially in their velum site, which indicated the appearance of functional hemocytes during early D-veliger larvae stage. The whole-mount immunofluorescence assay of three pattern recognition receptors (integrin β-1, caspase-3 and C-type lectin 3) and one immune effector gene (IL17-5) was performed in blastula, early D-veliger and umbo larvae, suggested that velum and digestive gland were the potential sites of immune system in the larvae. The lowest activities of antioxidant enzymes (superoxide dismutase and catalase) and hydrolytic enzyme (lysozyme), as well as descended expression levels of 12 immune genes at the transition between embryogenesis and planktonic, indicated that the larvae at hatching (9 hpf) were in hypo-immunity. While the ascending activities of enzymes and expression levels of seven immune genes during the trochophore stage (15 hpf) suggested the initiation of immune system. The steadily increasing trend of all the 12 candidate genes at the early umbo larvae (120 h) hinted that the immune system was well developed at this stage. After bacterial challenge, some immune recognition (TLR4) and immune effector (IL17-5 and defh2) genes were activated in blastula stage (4 hpf), and other immune genes were up regulated in D-veliger larvae, indicating that the zygotic immune system could respond earlier against the bacterial challenge during its development. These results indicated that the cellular and humoral immune components appeared at trochophore stage, and the cellular immune system was activated with its occurrence, while the humoral immune system executed until the early umbo larval stage. The immune system emerged earlier to aid larvae in defending bacterial challenge during the early stages of oyster development. Copyright © 2016 Elsevier Ltd. All rights reserved.

Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism.

PubMed

Dickey, Deborah M; Edmund, Aaron B; Otto, Neil M; Chaffee, Thomas S; Robinson, Jerid W; Potter, Lincoln R

2016-05-20

C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound (125)I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The significance of low substrate concentration measurements for mechanistic interpretation in cholinesterases.

PubMed

Stojan, Jure

2013-03-25

Cholinesterases do not follow the Michaelis-Menten kinetics. In the past, many reaction schemes were suggested to explain their complex interactions during the substrate turnover. Covalent catalysis was recognized very early and therefore, double intermediate traditional reaction scheme for the hydrolysis of good substrates at low concentrations was postulated. However, at intermediate and high substrate concentrations homotropic pseudocooperative effects take place in all cholinesterases, due to the nature of their buried active center. In this study, the significance and usefulness of experimental data obtained at low substrate concentrations, where only one substrate molecule accesses the active site at a time, are to be specified for the overall mechanistic evaluations. Indeed, different interpretations are expected when data are processed with equations derived from different reaction schemes. Consequently, the scheme with two substrate binding sites which comprises the structurally evidenced fully occupied active site as ultimate cause for substantially decreased cholinesterase activity at extremely high substrate concentrations is considered here. A special emphasis is put on butyrylcholinesterase, the enzyme with the largest active site among cholinesterases, where the pseudocooperative effects appear at much higher concentrations than in acetylcholinesterases. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Rapid intranasal delivery of chloramphenicol acetyltransferase in the active form to different brain regions as a model for enzyme therapy in the CNS.

PubMed

Appu, Abhilash P; Arun, Peethambaran; Krishnan, Jishnu K S; Moffett, John R; Namboodiri, Aryan M A

2016-02-01

The blood brain barrier (BBB) is critical for maintaining central nervous system (CNS) homeostasis by restricting entry of potentially toxic substances. However, the BBB is a major obstacle in the treatment of neurotoxicity and neurological disorders due to the restrictive nature of the barrier to many medications. Intranasal delivery of active enzymes to the brain has therapeutic potential for the treatment of numerous CNS enzyme deficiency disorders and CNS toxicity caused by chemical threat agents. The aim of this work is to provide a sensitive model system for analyzing the rapid delivery of active enzymes into various regions of the brain with therapeutic bioavailability. We tested intranasal delivery of chloramphenicol acetyltransferase (CAT), a relatively large (75kD) enzyme, in its active form into different regions of the brain. CAT was delivered intranasally to anaesthetized rats and enzyme activity was measured in different regions using a highly specific High Performance Thin Layer Chromatography (HP-TLC)-radiometry coupled assay. Active enzyme reached all examined areas of the brain within 15min (the earliest time point tested). In addition, the yield of enzyme activity in the brain was almost doubled in the brains of rats pre-treated with matrix metalloproteinase-9 (MMP-9). Intranasal administration of active enzymes in conjunction with MMP-9 to the CNS is both rapid and effective. The present results suggest that intranasal enzyme therapy is a promising method for counteracting CNS chemical threat poisoning, as well as for treating CNS enzyme deficiency disorders. Published by Elsevier B.V.

Calcium in the Mechanism of Ammonia-Induced Astrocyte Swelling

PubMed Central

Jayakumar, A.R.; Rao, K.V. Rama; Tong, X.Y; Norenberg, M.D.

2016-01-01

Brain edema, due largely to astrocyte swelling, is an important clinical problem in patients with acute liver failure. While mechanisms underlying astrocyte swelling in this condition are not fully understood, ammonia and associated oxidative/nitrosative stress (ONS) appear to be involved. Mechanisms responsible for the increase in reactive oxygen/nitrogen species (RONS) and their role in ammonia-induced astrocyte swelling, however, are poorly understood. Recent studies have demonstrated a transient increase in intracellular Ca2+ in cultured astrocytes exposed to ammonia. As Ca2+ is a known inducer of RONS, we investigated potential mechanisms by which Ca2+ may be responsible for the production of RONS and cell swelling in cultured astrocytes after treatment with ammonia. Exposure of cultured astrocytes to ammonia (5 mM) increased the formation of free radicals, including nitric oxide, and such increase was significantly diminished by treatment with the Ca2+ chelator BAPTA-AM. We then examined the activity of Ca2+-dependent enzymes that are known to generate RONS and found that ammonia significantly increased the activities of NADPH oxidase (NOX), constitutive nitric oxide synthase (cNOS) and phospholipase A2 (PLA2) and such increases in activity were significantly diminished by BAPTA. Pretreatment of cultures with 7-nitroindazole, apocyanin and quinacrine, respective inhibitors of cNOS, NOX and PLA2, all significantly diminished RONS production. Additionally, treatment of cultures with BAPTA or with inhibitors of cNOS, NOX and PLA2 reduced ammonia-induced astrocyte swelling. These studies suggest that the ammonia-induced rise in intracellular Ca2+ activates free radical producing enzymes that ultimately contribute to the mechanism of astrocyte swelling. PMID:19393035

Microbial responses to multi-factor climate change: effects on soil enzymes.

PubMed

Steinweg, J Megan; Dukes, Jeffrey S; Paul, Eldor A; Wallenstein, Matthew D

2013-01-01

The activities of extracellular enzymes, the proximate agents of decomposition in soils, are known to depend strongly on temperature, but less is known about how they respond to changes in precipitation patterns, and the interaction of these two components of climate change. Both enzyme production and turnover can be affected by changes in temperature and soil moisture, thus it is difficult to predict how enzyme pool size may respond to altered climate. Soils from the Boston-Area Climate Experiment (BACE), which is located in an old field (on abandoned farmland), were used to examine how climate variables affect enzyme activities and microbial biomass carbon (MBC) in different seasons and in soils exposed to a combination of three levels of precipitation treatments (ambient, 150% of ambient during growing season, and 50% of ambient year-round) and four levels of warming treatments (unwarmed to ~4°C above ambient) over the course of a year. Warming, precipitation and season had very little effect on potential enzyme activity. Most models assume that enzyme dynamics follow microbial biomass, because enzyme production should be directly controlled by the size and activity of microbial biomass. We observed differences among seasons and treatments in mass-specific potential enzyme activity, suggesting that this assumption is invalid. In June 2009, mass-specific potential enzyme activity, using chloroform fumigation-extraction MBC, increased with temperature, peaking under medium warming and then declining under the highest warming. This finding suggests that either enzyme production increased with temperature or turnover rates decreased. Increased maintenance costs associated with warming may have resulted in increased mass-specific enzyme activities due to increased nutrient demand. Our research suggests that allocation of resources to enzyme production could be affected by climate-induced changes in microbial efficiency and maintenance costs.

Mechanism of Enhanced HIV Restriction by Virion Coencapsidated Cytidine Deaminases APOBEC3F and APOBEC3G.

PubMed

Ara, Anjuman; Love, Robin P; Follack, Tyson B; Ahmed, Khawaja A; Adolph, Madison B; Chelico, Linda

2017-02-01

The APOBEC3 (A3) enzymes, A3G and A3F, are coordinately expressed in CD4 + T cells and can become coencapsidated into HIV-1 virions, primarily in the absence of the viral infectivity factor (Vif). A3F and A3G are deoxycytidine deaminases that inhibit HIV-1 replication by inducing guanine-to-adenine hypermutation through deamination of cytosine to form uracil in minus-strand DNA. The effect of the simultaneous presence of both A3G and A3F on HIV-1 restriction ability is not clear. Here, we used a single-cycle infectivity assay and biochemical analyses to determine if coencapsidated A3G and A3F differ in their restriction capacity from A3G or A3F alone. Proviral DNA sequencing demonstrated that compared to each A3 enzyme alone, A3G and A3F, when combined, had a coordinate effect on hypermutation. Using size exclusion chromatography, rotational anisotropy, and in vitro deamination assays, we demonstrate that A3F promotes A3G deamination activity by forming an A3F/G hetero-oligomer in the absence of RNA which is more efficient at deaminating cytosines. Further, A3F caused the accumulation of shorter reverse transcripts due to decreasing reverse transcriptase efficiency, which would leave single-stranded minus-strand DNA exposed for longer periods of time, enabling more deamination events to occur. Although A3G and A3F are known to function alongside each other, these data provide evidence for an A3F/G hetero-oligomeric A3 with unique properties compared to each individual counterpart. The APOBEC3 enzymes APOBEC3F and APOBEC3G act as a barrier to HIV-1 replication in the absence of the HIV-1 Vif protein. After APOBEC3 enzymes are encapsidated into virions, they deaminate cytosines in minus-strand DNA, which forms promutagenic uracils that induce transition mutations or proviral DNA degradation. Even in the presence of Vif, footprints of APOBEC3-catalyzed deaminations are found, demonstrating that APOBEC3s still have discernible activity against HIV-1 in infected individuals. We undertook a study to better understand the activity of coexpressed APOBEC3F and APOBEC3G. The data demonstrate that an APOBEC3F/APOBEC3G hetero-oligomer can form that has unique properties compared to each APOBEC3 alone. This hetero-oligomer has increased efficiency of virus hypermutation, raising the idea that we still may not fully realize the antiviral mechanisms of endogenous APOBEC3 enzymes. Hetero-oligomerization may be a mechanism to increase their antiviral activity in the presence of Vif. Copyright © 2017 American Society for Microbiology.

Allosteric regulation of epigenetic modifying enzymes.

PubMed

Zucconi, Beth E; Cole, Philip A

2017-08-01

Epigenetic enzymes including histone modifying enzymes are key regulators of gene expression in normal and disease processes. Many drug development strategies to target histone modifying enzymes have focused on ligands that bind to enzyme active sites, but allosteric pockets offer potentially attractive opportunities for therapeutic development. Recent biochemical studies have revealed roles for small molecule and peptide ligands binding outside of the active sites in modulating the catalytic activities of histone modifying enzymes. Here we highlight several examples of allosteric regulation of epigenetic enzymes and discuss the biological significance of these findings. Copyright © 2017 Elsevier Ltd. All rights reserved.

DGAT enzymes are required for triacylglycerol synthesis and lipid droplets in adipocytes[S

PubMed Central

Harris, Charles A.; Haas, Joel T.; Streeper, Ryan S.; Stone, Scot J.; Kumari, Manju; Yang, Kui; Han, Xianlin; Brownell, Nicholas; Gross, Richard W.; Zechner, Rudolf; Farese, Robert V.

2011-01-01

The total contribution of the acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, to mammalian triacylglycerol (TG) synthesis has not been determined. Similarly, whether DGAT enzymes are required for lipid droplet (LD) formation is unknown. In this study, we examined the requirement for DGAT enzymes in TG synthesis and LDs in differentiated adipocytes with genetic deletions of DGAT1 and DGAT2. Adipocytes with a single deletion of either enzyme were capable of TG synthesis and LD formation. In contrast, adipocytes with deletions of both DGATs were severely lacking in TG and did not have LDs, indicating that DGAT1 and DGAT2 account for nearly all TG synthesis in adipocytes and appear to be required for LD formation during adipogenesis. DGAT enzymes were not absolutely required for LD formation in mammalian cells, however; macrophages deficient in both DGAT enzymes were able to form LDs when incubated with cholesterol-rich lipoproteins. Although adipocytes lacking both DGATs had no TG or LDs, they were fully differentiated by multiple criteria. Our findings show that DGAT1 and DGAT2 account for the vast majority of TG synthesis in mice, and DGAT function is required for LDs in adipocytes, but not in all cell types. PMID:21317108

DGAT enzymes are required for triacylglycerol synthesis and lipid droplets in adipocytes.

PubMed

Harris, Charles A; Haas, Joel T; Streeper, Ryan S; Stone, Scot J; Kumari, Manju; Yang, Kui; Han, Xianlin; Brownell, Nicholas; Gross, Richard W; Zechner, Rudolf; Farese, Robert V

2011-04-01

The total contribution of the acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, to mammalian triacylglycerol (TG) synthesis has not been determined. Similarly, whether DGAT enzymes are required for lipid droplet (LD) formation is unknown. In this study, we examined the requirement for DGAT enzymes in TG synthesis and LDs in differentiated adipocytes with genetic deletions of DGAT1 and DGAT2. Adipocytes with a single deletion of either enzyme were capable of TG synthesis and LD formation. In contrast, adipocytes with deletions of both DGATs were severely lacking in TG and did not have LDs, indicating that DGAT1 and DGAT2 account for nearly all TG synthesis in adipocytes and appear to be required for LD formation during adipogenesis. DGAT enzymes were not absolutely required for LD formation in mammalian cells, however; macrophages deficient in both DGAT enzymes were able to form LDs when incubated with cholesterol-rich lipoproteins. Although adipocytes lacking both DGATs had no TG or LDs, they were fully differentiated by multiple criteria. Our findings show that DGAT1 and DGAT2 account for the vast majority of TG synthesis in mice, and DGAT function is required for LDs in adipocytes, but not in all cell types.

Activity-based proteomics of enzyme superfamilies: serine hydrolases as a case study.

PubMed

Simon, Gabriel M; Cravatt, Benjamin F

2010-04-09

Genome sequencing projects have uncovered thousands of uncharacterized enzymes in eukaryotic and prokaryotic organisms. Deciphering the physiological functions of enzymes requires tools to profile and perturb their activities in native biological systems. Activity-based protein profiling has emerged as a powerful chemoproteomic strategy to achieve these objectives through the use of chemical probes that target large swaths of enzymes that share active-site features. Here, we review activity-based protein profiling and its implementation to annotate the enzymatic proteome, with particular attention given to probes that target serine hydrolases, a diverse superfamily of enzymes replete with many uncharacterized members.

Mechanism of activation of glycogen phosphorylase by fructose in the liver. Stimulation of phosphorylase kinase related to the consumption of adenosine triphosphate.

PubMed

Van de Werve, G; Hers, H G

1979-01-15

1. A dose-dependent activation of phosphorylase and consumption of ATP was observed in isolated hepatocytes incubated in the presence of fructose; histone kinase and phosphorylase kinase activities were unchanged at doses of this sugar that were fully effective on phosphorylase. The activation of phosphorylase by fructose was also observed in cells incubated in a Ca2+-free medium as well as in the livers of rats in vivo. 2. In a liver high-speed supernatant, fructose, tagatose and sorbose stimulated the activity of phosphorylase kinase; this effect was dependent on the presence of K+ ions, which are required for the activity of fructokinase; it was accompanied by the transformation of ATP into ADP. In the presence of hexokinase, glucose also stimulated phosphorylase kinase, both in an Na+ or a K+ medium. 3. The activities of partially purified muscle or liver phosphorylase kinase were unchanged in the presence of fructose. 4. Some properties of liver phosphorylase kinase are described, including a high molecular weight and an inhibition at ATP/Mg ratios above 0.5, as well as an effect of ATP concentration on the hysteretic behaviour of this enzyme. 5. The effect of fructose on the activation of phosphorylase is discussed in relation to the comsumption of ATP.

Profiling the orphan enzymes.

PubMed

Sorokina, Maria; Stam, Mark; Médigue, Claudine; Lespinet, Olivier; Vallenet, David

2014-06-06

The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called "orphan enzymes". The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to "local orphan enzymes" that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new activities.

Targeting homeostasis in drug delivery using bioresponsive hydrogel microforms.

PubMed

Wilson, A Nolan; Guiseppi-Elie, Anthony

2014-01-30

A drug delivery platform comprising a biocompatible, bioresponsive hydrogel and possessing a covalently tethered peptide-drug conjugate was engineered to achieve stasis, via a closed control loop, of the external biochemical activity of the actuating protease. The delivery platform contains a peptide-drug conjugate covalently tethered to the hydrogel matrix, which in the presence of the appropriate protease, was cleaved and the drug released into the bathing environment. This platform was developed and investigated in silico using a finite element modeling (FEM) approach. Firstly, the primary governing phenomena guiding drug release profiles were investigated, and it was confirmed that under transport-limited conditions, the diffusion of the enzyme within the hydrogel and the coupled enzyme kinetics accurately model the system and are in agreement with published results. Secondly, the FEM model was used to investigate the release of a competitive protease inhibitor, MAG283, via cleavage of Acetyl-Pro-Leu-Gly|Leu-MAG-283 by MMP9 in order to achieve targeted homeostasis of MMP-9 activity, such as in the pathophysiology of chronic wounds, via closed-loop feedback control. The key engineering parameters for the delivery device are the radii of the hydrogel microspheres and the concentration of the peptide-inhibitor conjugate. Homeostatic drug delivery, where the focus turns away from the drug release rate and turns toward achieving targeted control of biochemical activity within a biochemical pathway, is an emerging approach in drug delivery methodologies for which the potential has not yet been fully realized. Copyright © 2013 Elsevier B.V. All rights reserved.

Competitive protein tyrosine phosphatase 1B (PTP1B) inhibitors, prenylated caged xanthones from Garcinia hanburyi and their inhibitory mechanism.

PubMed

Tan, Xue Fei; Uddin, Zia; Park, Chanin; Song, Yeong Hun; Son, Minky; Lee, Keun Woo; Park, Ki Hun

2017-04-15

Protein tyrosine phosphatase 1B (PTP1B) plays important role in diabetes, obesity and cancer. The methanol extract of the gum resin of Garcinia hanburyi (G. hanburyi) showed potent PTP1B inhibition at 10µg/ml. The active compounds were identified as prenylated caged xanthones (1-9) which inhibited PTP1B in dose-dependent manner. Carboxybutenyl group within caged motif (A ring) was found to play a critical role in enzyme inhibition such as 1-6 (IC 50 s=0.47-4.69µM), whereas compounds having hydroxymethylbutenyl 7 (IC 50 =70.25µM) and methylbutenyl 8 (IC 50 >200µM) showed less activity. The most potent inhibitor, gambogic acid 1 (IC 50 =0.47µM) showed 30-fold more potency than ursolic acid (IC 50 =15.5µM), a positive control. In kinetic study, all isolated xanthones behaved as competitive inhibitors which were fully demonstrated with K m , V max and K ik /K iv ratio. It was also proved that inhibitor 1 operated under the enzyme isomerization model having k 5 =0.0751µM - 1 S - 1 , k 6 =0.0249µM - 1 S - 1 and K i app =0.499µM. To develop a pharmacophore model, we explored the binding sites of compound 1 and 7 in PTP1B. These modeling results were in agreement with our findings, which revealed that the inhibitory activities are tightly related to caged motif and prenyl group in A ring. Copyright © 2017 Elsevier Ltd. All rights reserved.

The relationship of proline content and metabolism on the productivity of maize plants

PubMed Central

Špoljarević, Marija; Agić, Dejan; Lisjak, Miroslav; Gumze, Andrej; Wilson, Ian D; Hancock, John T

2011-01-01

The free proline content in maize ear-leaves, silk and pollen were analyzed in field grown plants which had matured to the pollination stage. Using maize hybrids PR34F02, PR35P12 and PR36B08 field trials were set up at two locations in eastern Croatia in two different years. Two enzymes of proline metabolism were analyzed in the same leaf samples and specific activities of synthetase (P5CS) and proline dehydrogenase (PDH). Plant productivity was evaluated at harvest by the estimation of total and fully developed grain number per ear and per plant, the mean single grain mass, and the mass of grain per plant. The year in which the plants were grown had a very significant effect on the free proline content in the leaf and pollen, as well as on the enzyme activities assayed. The differences between the plants from the two localities were very significant in all tested parameters of plant grain productivity. There was a significant genotype effect on proline content and P5CS total activity in leaf and on all the productivity parameters. Some of the correlations established suggest that the rate of proline synthesis and degradation in maize ear-leaf at pollination might contribute to the final grain production of the maize plant. Multiple regression analyses was used to further analyze the relationship between proline and grain productivity, but it is clear that future work should include other environmental conditions, plant species and organs such as roots. PMID:21415600

Three-dimensional HepaRG model as an attractive tool for toxicity testing.

PubMed

Leite, Sofia B; Wilk-Zasadna, Iwona; Zaldivar, Jose M; Airola, Elodie; Reis-Fernandes, Marcos A; Mennecozzi, Milena; Guguen-Guillouzo, Christiane; Chesne, Christopher; Guillou, Claude; Alves, Paula M; Coecke, Sandra

2012-11-01

The culture of HepaRG cells as three dimensional (3D) structures in the spinner-bioreactor may represent added value as a hepatic system for toxicological purposes. The use of a cost-effective commercially available bioreactor, which is compatible with high-throughput cell analysis, constitutes an attractive approach for routine use in the drug testing industry. In order to assess specific aspects of the biotransformation capacity of the bioreactor-based HepaRG system, the induction of CYP450 enzymes (i.e., CYP1A2, 2B6, 2C9, and 3A4) and the activity of the phase II enzyme, uridine diphosphate glucuronoltransferase (UGT), were tested. The long-term functionality of the system was demonstrated by 7-week stable profiles of albumin secretion, CYP3A4 induction, and UGT activities. Immunofluorescence-based staining showed formation of tissue-like arrangements including bile canaliculi-like structures and polar distribution of transporters. The use of in silico models to analyze the in vitro data related to hepatotoxic activity of acetaminophen (APAP) demonstrated the advantage of the integration of kinetic and dynamic aspects for a better understanding of the in vitro cell behavior. The bioactivation of APAP and its related cytotoxicity was assessed in a system compatible to high-throughput screening. The approach also proved to be a good strategy to reduce the time necessary to obtain fully differentiated cell cultures. In conclusion, HepaRG cells cultured in 3D spinner-bioreactors are an attractive tool for toxicological studies, showing a liver-like performance and demonstrating a practical applicability for toxicodynamic approaches.

Aqueous Solutions of the Ionic Liquid 1-butyl-3-methylimidazolium Chloride Denature Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baker, Gary A; Heller, William T

2009-01-01

As we advance our understanding, ionic liquids (ILs) are finding ever broader scope within the chemical sciences including, most recently, pharmaceutical, enzymatic, and bioanalytical applications. With examples of enzymatic activity reported in both neat ILs and in IL/water mixtures, enzymes are frequently assumed to adopt a quasi-native conformation, even if little work has been carried out to date toward characterizing the conformation, dynamics, active-site perturbation, cooperativity of unfolding transitions, free energy of stabilization, or aggregation/oligomerization state of enzymes in the presence of an IL solvent component. In this study, human serum albumin and equine heart cytochrome c were characterized inmore » aqueous solutions of the fully water-miscible IL 1-butyl-3-methylimidazolium chloride, [bmim]Cl, by small-angle neutron and X-ray scattering. At [bmim]Cl concentrations up to 25 vol.%, these two proteins were found to largely retain their higher-order structures whereas both proteins become highly denatured at the highest IL concentration studied here (i.e., 50 vol.% [bmim]Cl). The response of these proteins to [bmim]Cl is analogous to their behavior in the widely studied denaturants guanidine hydrochloride and urea which similarly lead to random coil conformations at excessive molar concentrations. Interestingly, human serum albumin dimerizes in response to [bmim]Cl, whereas cytochrome c remains predominantly in monomeric form. These results have important implications for enzymatic studies in aqueous IL media, as they suggest a facile pathway through which biocatalytic activity can be altered in these nascent and potentially green electrolyte systems.« less

Study on the Correlation between Gene Expression and Enzyme Activity of Seven Key Enzymes and Ginsenoside Content in Ginseng in Over Time in Ji'an, China.

PubMed

Yin, Juxin; Zhang, Daihui; Zhuang, Jianjian; Huang, Yi; Mu, Ying; Lv, Shaowu

2017-12-11

Panax ginseng is a traditional medicine. Fresh ginseng is one of the most important industries related to ginseng development, and fresh ginseng of varying ages has different medicinal properties. Previous research has not systematically reported the correlation between changes in key enzyme activity with changes in ginsenoside content in fresh ginseng over time. In this study, for the first time, we use ginseng samples of varying ages in Ji'an and systematically reported the changes in the activity of seven key enzymes (HMGR, FPS, SS, SE, DS, CYP450, and GT). We investigated the content of ginsenoside and gene expression of these key enzymes. Ginsenoside content was measured using HPLC. HPLC, GC-MS, and LC-MS were combined to measure the enzyme activity of the key enzymes. Quantitative PCR was used in the investigation of gene expression. By analyzing the correlation between the enzyme activity and the transcription level of the key enzymes with ginsenoside content, we found that DS and GT enzyme activities are significantly correlated with the ginsenoside content in different ages of ginseng. Our findings might provide a new strategy to discriminate between ginseng of different years. Meanwhile, this research provides important information for the in-depth study of ginsenoside biosynthesis.

Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers.

PubMed

Yuan, Lin; Wang, Mingfa; Zhang, Xiaotu; Wang, Zhixiang

2017-01-01

Three hundred one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets containing non-starch polysaccharide (NSP) enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI) and average daily gain (ADG) were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05). Feed-to-gain ratio (FGR) was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05). Apparent digestibility of crude protein (ADCP) was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05). Cholecystokinin (CCK) level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05), but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05), respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05). However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05). The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05). Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased pancreatic trypsin mRNA levels by 40%, 44% and 28%, respectively. Supplementation with NSP enzyme and 160 mg/kg protease decreased pancreatic trypsin mRNA levels by 13%. Pancreatic lipase and amylase mRNA expression were significantly elevated in treated animals compared to the control group (p<0.05). These results suggest that the amount of NSP enzyme and acid protease in the diet significantly affects digestive function, endogenous digestive-enzyme activity and mRNA expression in broilers.

Oxidation of indole-3-acetic acid to oxindole-3-acetic acid by an enzyme preparation from Zea mays

NASA Technical Reports Server (NTRS)

Reinecke, D. M.; Bandurski, R. S.

1988-01-01

Indole-3-acetic acid is oxidized to oxindole-3-acetic acid by Zea mays tissue extracts. Shoot, root, and endosperm tissues have enzyme activities of 1 to 10 picomoles per hour per milligram protein. The enzyme is heat labile, is soluble, and requires oxygen for activity. Cofactors of mixed function oxygenase, peroxidase, and intermolecular dioxygenase are not stimulatory to enzymic activity. A heat-stable, detergent-extractable component from corn enhances enzyme activity 6- to 10-fold. This is the first demonstration of the in vitro enzymic oxidation of indole-3-acetic acid to oxindole-3-acetic acid in higher plants.

Activation of immobilized enzymes by acoustic wave resonance oscillation.

PubMed

Nishiyama, Hiroshi; Watanabe, Tomoya; Inoue, Yasunobu

2014-12-01

Acoustic wave resonance oscillation has been used successfully in the development of methods to activate immobilized enzyme catalysts. In this study, resonance oscillation effects were demonstrated for enzyme reactions on galactose oxidase (GAD), D-amino acid oxidase (DAAO), and L-amino acid oxidase (LAAO), all of which were immobilized covalently on a ferroelectric lead zirconate titanate (PZT) device that could generate thickness-extensional resonance oscillations (TERO) of acoustic waves. For galactose oxidation on immobilized GAD in a microreactor, TERO generation immediately increased enzyme activity 2- to 3-fold. Eliminating TERO caused a slight decrease in the activity, with ∼90% of the enhanced activity retained while the reaction proceeded. Contact of the enhanced enzyme with a galactose-free solution caused almost complete reversion of the activity to the original low level before TERO generation, indicating that, not only TERO-induced GAD activation, but also preservation of the increased activity, required a galactose substrate. Similar activity changes with TERO were observed for enzyme reactions on DAAO and LAAO. Kinetic analysis demonstrated that TERO helped strengthen the interactions of the immobilized enzyme with the reactant substrate and promoted formation of an activation complex. Copyright © 2014 Elsevier Inc. All rights reserved.

Diffusional correlations among multiple active sites in a single enzyme.

PubMed

Echeverria, Carlos; Kapral, Raymond

2014-04-07

Simulations of the enzymatic dynamics of a model enzyme containing multiple substrate binding sites indicate the existence of diffusional correlations in the chemical reactivity of the active sites. A coarse-grain, particle-based, mesoscopic description of the system, comprising the enzyme, the substrate, the product and solvent, is constructed to study these effects. The reactive and non-reactive dynamics is followed using a hybrid scheme that combines molecular dynamics for the enzyme, substrate and product molecules with multiparticle collision dynamics for the solvent. It is found that the reactivity of an individual active site in the multiple-active-site enzyme is reduced substantially, and this effect is analyzed and attributed to diffusive competition for the substrate among the different active sites in the enzyme.

Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein

PubMed Central

Chen, Huizhong; Hopper, Sherryll L.; Cerniglia, Carl E.

2018-01-01

Azo dyes are a predominant class of colourants used in tattooing, cosmetics, foods and consumer products. A gene encoding NADPH-flavin azoreductase (Azo1) from the skin bacterium Staphylococcus aureus ATCC 25923 was identified and overexpressed in Escherichia coli. RT-PCR results demonstrated that the azo1 gene was constitutively expressed at the mRNA level in S. aureus. Azo1 was found to be a tetramer with a native molecular mass of 85 kDa containing four non-covalently bound FMN. Azo1 requires NADPH, but not NADH, as an electron donor for its activity. The enzyme was resolved to dimeric apoprotein by removing the flavin prosthetic groups using hydrophobic-interaction chromatography. The dimeric apoprotein was reconstituted on-column and in free stage with FMN, resulting in the formation of a fully functional native-like tetrameric enzyme. The enzyme cleaved the model azo dye 2-[4-(dimethylamino)phenylazo]benzoic acid (Methyl Red) into N,N-dimethyl-p-phenylenediamine and 2-aminobenzoic acid. The apparent Km values for NADPH and Methyl Red substrates were 0·;074 and 0·057 mM, respectively. The apparent Vmax was 0·4 µM min−1 (mg protein)−1. Azo1 was also able to metabolize Orange II, Amaranth, Ponceau BS and Ponceau S azo dyes. Azo1 represents the first azoreductase to be identified and characterized from human skin microflora. PMID:15870453

Recent Progress on the Characterization of Aldonolactone Oxidoreductases

PubMed Central

Aboobucker, Siddique I; Lorence, Argelia

2015-01-01

l-Ascorbic acid (ascorbate, AsA, vitamin C) is essential for animal and plant health. Despite our dependence on fruits and vegetables to fulfill our requirement for this vitamin, the metabolic network leading to its formation in plants is just being fully elucidated. There is evidence supporting the operation of at least four biosynthetic pathways leading to AsA formation in plants. These routes use d-mannose/l-Galactose, l-gulose, d-galacturonate, and myo-inositol as the main precursors. This review focuses on aldonolactone oxidoreductases, a subgroup of the vanillyl alcohol oxidase (VAO; EC 1.1.3.38) superfamily, enzymes that catalyze the terminal step in AsA biosynthesis in bacteria, protozoa, animals, and plants. In this report, we review the properties of well characterized aldonolactone oxidoreductases to date. A shared feature in these proteins is the presence of a flavin cofactor as well as a thiol group. The flavin cofactor in many cases is bound to the N terminus of the enzymes or to a recently discovered HWXK motif in the C terminus. The binding between the flavin moiety and the protein can be either covalent or non-covalent. Substrate specificity and subcellular localization differ among the isozymes of each kingdom. All oxidases among these enzymes possess dehydrogenase activity, however, exclusive dehydrogenases are also found. We also discuss recent evidence indicating that plants have both l-gulono-1,4-lactone oxidases and l-Galactono-1,4-lactone dehydrogenases involved in AsA biosynthesis. PMID:26696130

Fatty acid regulation of hepatic lipid metabolism

PubMed Central

Jump, Donald B.

2012-01-01

Purpose of review To discuss transcriptional mechanisms regulating hepatic lipid metabolism. Recent findings Humans who are obese or have diabetes (NIDDM) or metabolic syndrome (MetS) have low blood and tissue levels of C20–22 polyunsaturated fatty acids (PUFAs). Although the impact of low C20–22 PUFAs on disease progression in humans is not fully understood, studies with mice have provided clues suggesting that impaired PUFA metabolism may contribute to the severity of risk factors associated with NIDDM and MetS. High fat diets promote hyperglycemia, insulin resistance and fatty liver in C57BL/6J mice, an effect that correlates with suppressed expression of enzymes involved in PUFA synthesis and decreased hepatic C20–22 PUFA content. A/J mice, in contrast, are resistant to diet-induced obesity and diabetes; these mice have elevated expression of hepatic enzymes involved in PUFA synthesis and C20–22 PUFA content. Moreover, loss-of-function and gain-of-function studies have identified fatty acid elongase (Elovl5), a key enzyme involved in PUFA synthesis, as a regulator of hepatic lipid and carbohydrate metabolism. Elovl5 activity regulates hepatic C20–22 PUFA content, signaling pathways (Akt and PP2A) and transcription factors (SREBP-1, PPARα, FoxO1 and PGC1α) that control fatty acid synthesis and gluconeogenesis. Summary These studies may help define novel strategies to control fatty liver and hyperglycemia associated with NIDDM and MetS. PMID:21178610

Crystallographic insight into the evolutionary origins of xyloglucan endo-transglycosylases and endo-hydrolases

PubMed Central

McGregor, Nicholas; Yin, Victor; Tung, Ching-Chieh; Van Petegem, Filip; Brumer, Harry

2016-01-01

SUMMARY The xyloglucan endo-transglycosylase/hydrolase (XTH) gene family encodes enzymes of central importance to plant cell wall remodelling. The evolutionary history of plant XTH gene products is incompletely understood vis-à-vis the larger body of bacterial endo-glycanases in Glycoside Hydrolase Family 16 (GH16). To provide molecular insight into this issue, high-resolution X-ray crystal structures and detailed enzyme kinetics of an extant transitional plant endo-glucanase (EG) were determined. Functionally intermediate between plant XTH gene products and bacterial licheninases of GH16, Vitis vinifera EG16 (VvEG16) effectively catalyzes the hydrolysis of the backbones of two dominant plant cell wall matrix glycans, xyloglucan (XyG) and β(1,3)/β(1,4)-mixed-linkage glucan (MLG). Crystallographic complexes with extended oligosaccharide substrates reveal the structural basis for the accommodation of both unbranched, mixed-linked (MLG) and highly decorated, linear (XyG) polysaccharide chains in a broad, extended active-site cleft. Structural comparison with representative bacterial licheninases, a xyloglucan endo-tranglycosylase (XET), and a xyloglucan endo-hydrolase (XEH) outline the functional ramifications of key sequence deletions and insertions across the phylogenetic landscape of GH16. Although the biological role(s) of EG16 orthologs remains to be fully resolved, the present biochemical and tertiary structural characterization provides key insight into plant cell wall enzyme evolution, which will continue to inform genomic analyses and functional studies across species. PMID:27859885

Direct measurement of glutamate release in the brain using a dual enzyme-based electrochemical sensor.

PubMed

Hu, Y; Mitchell, K M; Albahadily, F N; Michaelis, E K; Wilson, G S

1994-10-03

The in vivo measurement of the rapid changes in the extracellular concentrations of L-glutamic acid in the mammalian brain during normal neuronal activity or following excessive release due to episodes of anoxia or ischemia has not been possible to this date. Current techniques for the measurement of the release of endogenous glutamate into the extracellular space of the central nervous system are relatively slow and do not measure the actual concentration of free glutamate in the extracellular space. An enzyme-based electrode with rapid response times (about 1 s) and high degree of sensitivity (less than 2 microM) and selectivity for L-glutamic acid is described in this paper. This electrode has both L-glutamate and ascorbate oxidase immobilized on its surface. The latter enzyme removes almost completely any interferences produced by the high levels of extracellular ascorbate present in brain tissue. The response of the electrode to glutamate and other potentially interfering substances was fully characterized in vitro and its selectivity, sensitivity and rapidity in responding to a rise in extracellular glutamate concentrations was also demonstrated in vivo. Placement of the electrode in the dentate gyrus of the hippocampus led to the detection of both KCl-induced release of L-glutamic acid and the release induced by stimulation of the axons in the perforant pathway. The development of this selective, sensitive and rapidly responding glutamate sensor should make it now possible to measure the dynamic events associated with glutamate neurotransmission in the central nervous system.

Physics-based enzyme design: predicting binding affinity and catalytic activity.

PubMed

Sirin, Sarah; Pearlman, David A; Sherman, Woody

2014-12-01

Computational enzyme design is an emerging field that has yielded promising success stories, but where numerous challenges remain. Accurate methods to rapidly evaluate possible enzyme design variants could provide significant value when combined with experimental efforts by reducing the number of variants needed to be synthesized and speeding the time to reach the desired endpoint of the design. To that end, extending our computational methods to model the fundamental physical-chemical principles that regulate activity in a protocol that is automated and accessible to a broad population of enzyme design researchers is essential. Here, we apply a physics-based implicit solvent MM-GBSA scoring approach to enzyme design and benchmark the computational predictions against experimentally determined activities. Specifically, we evaluate the ability of MM-GBSA to predict changes in affinity for a steroid binder protein, catalytic turnover for a Kemp eliminase, and catalytic activity for α-Gliadin peptidase variants. Using the enzyme design framework developed here, we accurately rank the most experimentally active enzyme variants, suggesting that this approach could provide enrichment of active variants in real-world enzyme design applications. © 2014 Wiley Periodicals, Inc.

Stability improvement of immobilized lactoperoxidase using polyaniline polymer.

PubMed

Jafary, Fariba; Kashanian, Soheila; Sharieat, Ziadin Samsam; Jafary, Farzaneh; Omidfar, Kobra; Paknejad, Maliheh

2012-12-01

Enzyme engineering via immobilization techniques is perfectly compatible against the other chemical or biological approximate to improve enzyme functions and stability. In this study lactoperoxidase was immobilized onto polyaniline polymer activated with glutaraldehyde as a bifunctional agent, to improve enzyme properties. Polyaniline polymer was used due its unique physical and chemical properties to immobilize lactoperoxidase (LPO). The optimum activity of immobilized LPO was observed at pH 6 and 55 °C, which has been increased about 10 °C for the immobilized enzyme. The immobilized enzyme maintained absolutely active for 60 days whereas the native enzyme lost 80 % of its initial activity within this period of time. Moreover, the immobilized enzyme can be reused for several times without loss of activity. The kinetic parameter studies showed slight differences between free and immobilized enzymes. The K(m) and K(m.app) were calculated to be 0.6 and 0.4; also V(max) and V(max.app) were 1.3 and 0.9 respectively.

Engineering a hyper-catalytic enzyme by photo-activated conformation modulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agarwal, Pratul K

2012-01-01

Enzyme engineering for improved catalysis has wide implications. We describe a novel chemical modification of Candida antarctica lipase B that allows modulation of the enzyme conformation to promote catalysis. Computational modeling was used to identify dynamical enzyme regions that impact the catalytic mechanism. Surface loop regions located distal to active site but showing dynamical coupling to the reaction were connected by a chemical bridge between Lys136 and Pro192, containing a derivative of azobenzene. The conformational modulation of the enzyme was achieved using two sources of light that alternated the azobenzene moiety in cis and trans conformations. Computational model predicted thatmore » mechanical energy from the conformational fluctuations facilitate the reaction in the active-site. The results were consistent with predictions as the activity of the engineered enzyme was found to be enhanced with photoactivation. Preliminary estimations indicate that the engineered enzyme achieved 8-52 fold better catalytic activity than the unmodulated enzyme.« less

Dipeptidyl peptidase IV in angiotensin-converting enzyme inhibitor associated angioedema.

PubMed

Byrd, James Brian; Touzin, Karine; Sile, Saba; Gainer, James V; Yu, Chang; Nadeau, John; Adam, Albert; Brown, Nancy J

2008-01-01

Angioedema is a potentially life-threatening adverse effect of angiotensin-converting enzyme inhibitors. Bradykinin and substance P, substrates of angiotensin-converting enzyme, increase vascular permeability and cause tissue edema in animals. Studies indicate that amino-terminal degradation of these peptides, by aminopeptidase P and dipeptidyl peptidase IV, may be impaired in individuals with angiotensin-converting enzyme inhibitor-associated angioedema. This case-control study tested the hypothesis that dipeptidyl peptidase IV activity and antigen are decreased in sera of patients with a history of angiotensin-converting enzyme inhibitor-associated angioedema. Fifty subjects with a history of angiotensin-converting enzyme inhibitor-associated angioedema and 176 angiotensin-converting enzyme inhibitor-exposed control subjects were ascertained. Sera were assayed for angiotensin-converting enzyme activity, aminopeptidase P activity, aminopeptidase N activity, dipeptidyl peptidase IV activity, and antigen and the ex vivo degradation half-lives of bradykinin, des-Arg(9)-bradykinin, and substance P in a subset. The prevalence of smoking was increased and of diabetes decreased in case versus control subjects. Overall, dipeptidyl peptidase IV activity (26.6+/-7.8 versus 29.6+/-7.3 nmol/mL per minute; P=0.026) and antigen (465.8+/-260.8 versus 563.1+/-208.6 ng/mL; P=0.017) were decreased in sera from individuals with angiotensin-converting enzyme inhibitor-associated angioedema compared with angiotensin-converting enzyme inhibitor-exposed control subjects without angioedema. Dipeptidyl peptidase IV activity (21.5+/-4.9 versus 29.8+/-6.7 nmol/mL per minute; P=0.001) and antigen (354.4+/-124.7 versus 559.8+/-163.2 ng/mL; P=0.003) were decreased in sera from cases collected during angiotensin-converting enzyme inhibition but not in the absence of angiotensin-converting enzyme inhibition. The degradation half-life of substance P correlated inversely with dipeptidyl peptidase IV antigen during angiotensin-converting enzyme inhibition. Environmental or genetic factors that reduce dipeptidyl peptidase IV activity may predispose individuals to angioedema.

Dipeptidyl Peptidase IV in Angiotensin-Converting Enzyme Inhibitor–Associated Angioedema

PubMed Central

Byrd, James Brian; Touzin, Karine; Sile, Saba; Gainer, James V.; Yu, Chang; Nadeau, John; Adam, Albert; Brown, Nancy J.

2009-01-01

Angioedema is a potentially life-threatening adverse effect of angiotensin-converting enzyme inhibitors. Bradykinin and substance P, substrates of angiotensin-converting enzyme, increase vascular permeability and cause tissue edema in animals. Studies indicate that amino-terminal degradation of these peptides, by aminopeptidase P and dipeptidyl peptidase IV, may be impaired in individuals with angiotensin-converting enzyme inhibitor–associated angioedema. This case-control study tested the hypothesis that dipeptidyl peptidase IV activity and antigen are decreased in sera of patients with a history of angiotensin-converting enzyme inhibitor–associated angioedema. Fifty subjects with a history of angiotensin-converting enzyme inhibitor–associated angioedema and 176 angiotensin-converting enzyme inhibitor–exposed control subjects were ascertained. Sera were assayed for angiotensin-converting enzyme activity, aminopeptidase P activity, aminopeptidase N activity, dipeptidyl peptidase IV activity, and antigen and the ex vivo degradation half-lives of bradykinin, des-Arg9-bradykinin, and substance P in a subset. The prevalence of smoking was increased and of diabetes decreased in case versus control subjects. Overall, dipeptidyl peptidase IV activity (26.6±7.8 versus 29.6±7.3 nmol/mL per minute; P=0.026) and antigen (465.8±260.8 versus 563.1±208.6 ng/mL; P=0.017) were decreased in sera from individuals with angiotensin-converting enzyme inhibitor–associated angioedema compared with angiotensin-converting enzyme inhibitor–exposed control subjects without angioedema. Dipeptidyl peptidase IV activity (21.5±4.9 versus 29.8±6.7 nmol/mL per minute; P=0.001) and antigen (354.4±124.7 versus 559.8±163.2 ng/mL; P=0.003) were decreased in sera from cases collected during angiotensin-converting enzyme inhibition but not in the absence of angiotensin-converting enzyme inhibition. The degradation half-life of substance P correlated inversely with dipeptidyl peptidase IV antigen during angiotensin-converting enzyme inhibition. Environmental or genetic factors that reduce dipeptidyl peptidase IV activity may predispose individuals to angioedema. PMID:18025295

ORENZA: a web resource for studying ORphan ENZyme activities

PubMed Central

Lespinet, Olivier; Labedan, Bernard

2006-01-01

Background Despite the current availability of several hundreds of thousands of amino acid sequences, more than 36% of the enzyme activities (EC numbers) defined by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) are not associated with any amino acid sequence in major public databases. This wide gap separating knowledge of biochemical function and sequence information is found for nearly all classes of enzymes. Thus, there is an urgent need to explore these sequence-less EC numbers, in order to progressively close this gap. Description We designed ORENZA, a PostgreSQL database of ORphan ENZyme Activities, to collate information about the EC numbers defined by the NC-IUBMB with specific emphasis on orphan enzyme activities. Complete lists of all EC numbers and of orphan EC numbers are available and will be periodically updated. ORENZA allows one to browse the complete list of EC numbers or the subset associated with orphan enzymes or to query a specific EC number, an enzyme name or a species name for those interested in particular organisms. It is possible to search ORENZA for the different biochemical properties of the defined enzymes, the metabolic pathways in which they participate, the taxonomic data of the organisms whose genomes encode them, and many other features. The association of an enzyme activity with an amino acid sequence is clearly underlined, making it easy to identify at once the orphan enzyme activities. Interactive publishing of suggestions by the community would provide expert evidence for re-annotation of orphan EC numbers in public databases. Conclusion ORENZA is a Web resource designed to progressively bridge the unwanted gap between function (enzyme activities) and sequence (dataset present in public databases). ORENZA should increase interactions between communities of biochemists and of genomicists. This is expected to reduce the number of orphan enzyme activities by allocating gene sequences to the relevant enzymes. PMID:17026747

Remote enzyme activation using gold coated magnetite as antennae for radio frequency fields

NASA Astrophysics Data System (ADS)

Collins, Christian B.; Ackerson, Christopher J.

2018-02-01

The emerging field of remote enzyme activation, or the ability to remotely turn thermophilic increase enzyme activity, could be a valuable tool for understanding cellular processes. Through exploitation of the temperature dependence of enzymatic processes and high thermal stability of thermophilic enzymes these experiments utilize nanoparticles as `antennae' that convert radiofrequency (RF) radiation into local heat, increasing activity of the enzymes without increasing the temperature of the surrounding bulk solution. To investigate this possible tool, thermolysin, a metalloprotease was covalently conjugated to 4nm gold coated magnetite particles via peptide bond formation with the protecting ligand shell. RF stimulated protease activity at 17.76 MHz in a solenoid shaped antenna, utilizing both electric and magnetic field interactions was investigated. On average 40 percent higher protease activity was observed in the radio frequency fields then when bulk heating the sample to the same temperature. This is attributed to electrophoretic motion of the nanoparticle enzyme conjugates and local regions of heat generated by the relaxation of the magnetite cores with the oscillating field. Radio frequency local heating of nanoparticles conjugated to enzymes as demonstrated could be useful in the activation of specific enzymes in complex cellular environments.

High activity CAZyme cassette for improving biomass degradation in thermophiles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brunecky, Roman; Chung, Daehwan; Sarai, Nicholas S.

Currently, Thermophilic microorganisms and their enzymes offer several advantages for industrial application over their mesophilic counterparts. For example, a hyperthermophilic anaerobe, Caldicellulosiruptor bescii, was recently isolated from hot springs in Kamchatka, Siberia, and shown to have very high cellulolytic activity. Additionally, it is one of a few microorganisms being considered as viable candidates for consolidated bioprocessing applications. Moreover, C. bescii is capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This ability is accomplished by the production and secretion of free, multi-modular and multi-functional enzymes, one of which, CbCel9A/Cel48A also secretion of free, multi-modular and multi-functional enzymes, one ofmore » which, CbCel9A/Cel48A also known as CelA, is able to outperform enzymes found in commercial enzyme preparations. Furthermore, the complete C. bescii exoproteome is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Understanding the functional diversity of enzymes in the C. bescii exoproteome and how inter-molecular synergy between them confers C. bescii with its high cellulolytic activity is an important endeavor to enable the production more efficient biomass degrading enzyme formulations and in turn, better cellulolytic industrial microorganisms. We found that the combination of three or four of the most highly expressed enzymes in the C. bescii exoproteome exhibits such synergistic activity. For example, some discrete combinations of these enzymes mimic and even improve upon the activity of the exoproteome, even though some of the enzymes lack significant activity on their own. We have demonstrated that it is possible to replicate the cellulolytic activity of the native C. bescii exoproteome utilizing a minimal gene set, and that these minimal gene sets are more active than the whole exoproteome. In the future, this may lead to more simplified and efficient cellulolytic enzyme preparations or yield improvements when these enzymes are expressed in microorganisms engineered for consolidated bioprocessing.« less

High activity CAZyme cassette for improving biomass degradation in thermophiles

DOE PAGES

Brunecky, Roman; Chung, Daehwan; Sarai, Nicholas S.; ...

2018-02-01

Currently, Thermophilic microorganisms and their enzymes offer several advantages for industrial application over their mesophilic counterparts. For example, a hyperthermophilic anaerobe, Caldicellulosiruptor bescii, was recently isolated from hot springs in Kamchatka, Siberia, and shown to have very high cellulolytic activity. Additionally, it is one of a few microorganisms being considered as viable candidates for consolidated bioprocessing applications. Moreover, C. bescii is capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This ability is accomplished by the production and secretion of free, multi-modular and multi-functional enzymes, one of which, CbCel9A/Cel48A also secretion of free, multi-modular and multi-functional enzymes, one ofmore » which, CbCel9A/Cel48A also known as CelA, is able to outperform enzymes found in commercial enzyme preparations. Furthermore, the complete C. bescii exoproteome is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Understanding the functional diversity of enzymes in the C. bescii exoproteome and how inter-molecular synergy between them confers C. bescii with its high cellulolytic activity is an important endeavor to enable the production more efficient biomass degrading enzyme formulations and in turn, better cellulolytic industrial microorganisms. We found that the combination of three or four of the most highly expressed enzymes in the C. bescii exoproteome exhibits such synergistic activity. For example, some discrete combinations of these enzymes mimic and even improve upon the activity of the exoproteome, even though some of the enzymes lack significant activity on their own. We have demonstrated that it is possible to replicate the cellulolytic activity of the native C. bescii exoproteome utilizing a minimal gene set, and that these minimal gene sets are more active than the whole exoproteome. In the future, this may lead to more simplified and efficient cellulolytic enzyme preparations or yield improvements when these enzymes are expressed in microorganisms engineered for consolidated bioprocessing.« less

Programmable formation of catalytic RNA triangles and squares by assembling modular RNA enzymes.

PubMed

Oi, Hiroki; Fujita, Daisuke; Suzuki, Yuki; Sugiyama, Hiroshi; Endo, Masayuki; Matsumura, Shigeyoshi; Ikawa, Yoshiya

2017-05-01

RNA is a biopolymer that is attractive for constructing nano-scale objects with complex structures. Three-dimensional (3D) structures of naturally occurring RNAs often have modular architectures. The 3D structure of a group I (GI) ribozyme from Tetrahymena has a typical modular architecture, which can be separated into two structural modules (ΔP5 and P5abc). The fully active ribozyme can be reconstructed by assembling the two separately prepared modules through highly specific and strong assembly between ΔP5 ribozyme and P5abc RNA. Such non-covalent assembly of the two modules allows the design of polygonal RNA nano-structures. Through rational redesign of the parent GI ribozyme, we constructed variant GI ribozymes as unit RNAs for polygonal-shaped (closed) oligomers with catalytic activity. Programmed trimerization and tetramerization of the unit RNAs afforded catalytically active nano-sized RNA triangles and squares, the structures of which were directly observed by atomic force microscopy (AFM). © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Regulation of hepatic energy metabolism by the nuclear receptor PXR.

PubMed

Hakkola, Jukka; Rysä, Jaana; Hukkanen, Janne

2016-09-01

The pregnane X receptor (PXR) is a nuclear receptor that is traditionally thought to be specialized for sensing xenobiotic exposure. In concurrence with this feature PXR was originally identified to regulate drug-metabolizing enzymes and transporters. During the last ten years it has become clear that PXR harbors broader functions. Evidence obtained both in experimental animals and humans indicate that ligand-activated PXR regulates hepatic glucose and lipid metabolism and affects whole body metabolic homeostasis. Currently, the consequences of PXR activation on overall metabolic health are not yet fully understood and varying results on the effect of PXR activation or knockout on metabolic disorders and weight gain have been published in mouse models. Rifampicin and St. John's wort, the prototypical human PXR agonists, impair glucose tolerance in healthy volunteers. Chronic exposure to PXR agonists could potentially represent a risk factor for diabetes and metabolic syndrome. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie. Copyright © 2016 Elsevier B.V. All rights reserved.

Optimization of process variables by central composite design for the immobilization of urease enzyme on functionalized gold nanoparticles for various applications.

PubMed

Talat, Mahe; Singh, Ashwani Kumar; Srivastava, O N

2011-08-01

In the present study, enzyme urease has been immobilized on amine-functionalized gold nanoparticles (AuNPs). AuNPs were synthesized using natural precursor, i.e., clove extract and amine functionalized through 0.004 M L: -cysteine. Enzyme (urease) was extracted and purified from the vegetable waste, i.e., seeds of pumpkin to apparent homogeneity (sp. activity 353 U/mg protein). FTIR spectroscopy and transmission electron microscopy was used to characterize the immobilized enzyme. The immobilized enzyme exhibited enhanced activity as compared with the enzyme in the solution, especially, at lower enzyme concentration. Based on the evaluation of activity assay of the immobilized enzyme, it was found that the immobilized enzyme was quite stable for about a month and could successfully be used even after eight cycles having enzyme activity of about 47%. In addition to this central composite design (CCD) with the help of MINITAB version 15 Software was utilized to optimize the process variables viz., pH and temperature affecting the enzyme activity upon immobilization on AuNPs. The results predicted by the design were found in good agreement (R2 = 96.38%) with the experimental results indicating the applicability of proposed model. The multiple regression analysis and ANOVA showed the individual and cumulative effect of pH and temperature on enzyme activity indicating that the activity increased with the increase of pH up to 7.5 and temperature 75 °C. The effects of each variables represented by main effect plot, 3D surface plot, isoresponse contour plot and optimized plot were helpful in predicting results by performing a limited set of experiments.

Metagenomics as a Tool for Enzyme Discovery: Hydrolytic Enzymes from Marine-Related Metagenomes.

PubMed

Popovic, Ana; Tchigvintsev, Anatoly; Tran, Hai; Chernikova, Tatyana N; Golyshina, Olga V; Yakimov, Michail M; Golyshin, Peter N; Yakunin, Alexander F

2015-01-01

This chapter discusses metagenomics and its application for enzyme discovery, with a focus on hydrolytic enzymes from marine metagenomic libraries. With less than one percent of culturable microorganisms in the environment, metagenomics, or the collective study of community genetics, has opened up a rich pool of uncharacterized metabolic pathways, enzymes, and adaptations. This great untapped pool of genes provides the particularly exciting potential to mine for new biochemical activities or novel enzymes with activities tailored to peculiar sets of environmental conditions. Metagenomes also represent a huge reservoir of novel enzymes for applications in biocatalysis, biofuels, and bioremediation. Here we present the results of enzyme discovery for four enzyme activities, of particular industrial or environmental interest, including esterase/lipase, glycosyl hydrolase, protease and dehalogenase.

Ectomycorrhizal Fungal Communities and Enzymatic Activities Vary across an Ecotone between a Forest and Field.

PubMed

Rúa, Megan A; Moore, Becky; Hergott, Nicole; Van, Lily; Jackson, Colin R; Hoeksema, Jason D

2015-08-28

Extracellular enzymes degrade macromolecules into soluble substrates and are important for nutrient cycling in soils, where microorganisms, such as ectomycorrhizal (ECM) fungi, produce these enzymes to obtain nutrients. Ecotones between forests and fields represent intriguing arenas for examining the effect of the environment on ECM community structure and enzyme activity because tree maturity, ECM composition, and environmental variables may all be changing simultaneously. We studied the composition and enzymatic activity of ECM associated with loblolly pine (Pinus taeda) across an ecotone between a forest where P. taeda is established and an old field where P. taeda saplings had been growing for <5 years. ECM community and environmental characteristics influenced enzyme activity in the field, indicating that controls on enzyme activity may be intricately linked to the ECM community, but this was not true in the forest. Members of the Russulaceae were associated with increased phenol oxidase activity and decreased peroxidase activity in the field. Members of the Atheliaceae were particularly susceptible to changes in their abiotic environment, but this did not mediate differences in enzyme activity. These results emphasize the complex nature of factors that dictate the distribution of ECM and activity of their enzymes across a habitat boundary.

Inhibition by derivatives of diguanidines of cell proliferation in Ehrlich ascites cells grown in cultures.

PubMed Central

Alhonen-Hongisto, L; Pösö, H; Jänne, J

1980-01-01

The anti-proliferative effects of 1,1'-[(methylethanediylidene)dinitrilo]diguanidine [methylglyoxal bis(guanylhydrazone)] and 1,1'-[(metHYLETHANEDIYLIDENE)dinitrilo]bis-(3-aminoguaNIDINE) HAVE BEEN STUDIED IN Ehrlich ascites carcinoma cells grown in suspension cultures. Both compounds are potent inhibitors of S-adenosyl-L-methionine decarboxylase from the tumour cells. In the presence of putrescine (but not in its absence), the inhibition produced by 1,1'-[methylethanediylidene)dinitrilo]bis-(3-aminoguanadine) was apparently irreversible, as judged by persistent depression of the enzyme activity even after extensive dialysis. The two compounds produced similar increases in adenosylmethionine decarboxylase activity, which resulted from a striking stabilization of the enzyme in cells grown in the presence of the drugs. The inhibitory effect of the two diguanidine derivatives on the synthesis of DNA and protein became evident after an exposure of 4--8 h. At that time, the only change seen in tumour polyamines in cells grown in the presence of the inhibitors was an increase in cellular putrescine. To find out whether the compounds initially interfered with the energy production of the tumour cells, the cultures were grown in the presence of uniformly labelled glucose, and the formation of lactate, as well as the oxidation of the sugar into CO2, were measured. The activation of glycolysis upon dilution of the tumour cells with fresh medium and the subsequent formation of labelled CO2 were siliar in control cells and in cells exposed to methylglyoxal bis(buanylhydrazone), 1,1'-[(methylethanediylidene)dinitrilo]bis-(3-aminoguanidine) or diaminopropanol. Only a marginal decrease in the cellular content of ATP was found in cells exposed to the inhibitors for 24 h. The diguanidine-induced growth inhibition was fully reversed by low concentrations of exogenous polyamines. However, the possibility remained that the reversal by polyamines was due to a decrease of intracellular diguanidine concentration. Our results indicate that the mode of action of 1,1'-[(methylethanediylidene)dinitrilo]bis-(3-aminoguanidine) is fully comparable to that of methylglyoxal bis(guanylhydrazone), as regards stabilization of adenosylmethionine decarboxylase and the appearance of growth inhibition in Ehrlich ascites cells. The data tend to support the view that both compounds apparently have an early anti-proliferative effect unrelated to polyamine metabolism. PMID:7396877

Overproduction in Escherichia coli and Characterization of a Soybean Ferric Leghemoglobin Reductase.

PubMed Central

Ji, L.; Becana, M.; Sarath, G.; Shearman, L.; Klucas, R. V.

1994-01-01

We previously cloned and sequenced a cDNA encoding soybean ferric leghemoglobin reductase (FLbR), an enzyme postulated to play an important role in maintaining leghemoglobin in a functional ferrous state in nitrogen-fixing root nodules. This cDNA was sub-cloned into an expression plasmid, pTrcHis C, and overexpressed in Escherichia coli. The recombinant FLbR protein, which was purified by two steps of column chromatography, was catalytically active and fully functional. The recombinant FLbR cross-reacted with antisera raised against native FLbR purified from soybean root nodules. The recombinant FLbR, the native FLbR purified from soybean (Glycine max L.) root nodules, and dihydrolipoamide dehydrogenases from pig heart and yeast had similar but not identical ultraviolet-visible absorption and fluorescence spectra, cofactor binding, and kinetic properties. FLbR shared common structural features in the active site and prosthetic group binding sites with other pyridine nucleotide-disulfide oxidoreductases such as dihydrolipoamide dehydrogenases, but displayed different microenvironments for the prosthetic groups. PMID:12232320

Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism.

PubMed

Liberato, Marcelo V; Silveira, Rodrigo L; Prates, Érica T; de Araujo, Evandro A; Pellegrini, Vanessa O A; Camilo, Cesar M; Kadowaki, Marco A; Neto, Mario de O; Popov, Alexander; Skaf, Munir S; Polikarpov, Igor

2016-04-01

Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.

Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism

NASA Astrophysics Data System (ADS)

Liberato, Marcelo V.; Silveira, Rodrigo L.; Prates, Érica T.; de Araujo, Evandro A.; Pellegrini, Vanessa O. A.; Camilo, Cesar M.; Kadowaki, Marco A.; Neto, Mario De O.; Popov, Alexander; Skaf, Munir S.; Polikarpov, Igor

2016-04-01

Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.

Calcium signaling through CaMKII regulates hepatic glucose production in fasting and obesity

PubMed Central

Ozcan, Lale; Wong, Catherine C.L.; Li, Gang; Xu, Tao; Pajvani, Utpal; Park, Sung Kyu Robin; Wronska, Anetta; Chen, Bi-Xing; Marks, Andrew R.; Fukamizu, Akiyoshi; Backs, Johannes; Singer, Harold A.; Yates, John R.; Accili, Domenico; Tabas, Ira

2012-01-01

SUMMARY Hepatic glucose production (HGP) is crucial for glucose homeostasis, but the underlying mechanisms have not been fully elucidated. Here we show that a calcium-sensing enzyme, CaMKII, is activated in a calcium- and IP3R-dependent manner by cAMP and glucagon in primary HCs and by glucagon and fasting in vivo. Genetic deficiency or inhibition of CaMKII blocks nuclear translocation of FoxO1 by affecting its phosphorylation, impairs fasting- and glucagon/cAMP-induced glycogenolysis and gluconeogenesis, and lowers blood glucose levels, while constitutively active CaMKII has the opposite effects. Importantly, the suppressive effect of CaMKII deficiency on glucose metabolism is abrogated by transduction with constitutively nuclear FoxO1, indicating that the effect of CaMKII deficiency requires nuclear exclusion of FoxO1. This same pathway is also involved in excessive HGP in the setting of obesity. These results reveal a calcium-mediated signaling pathway involved in FoxO1 nuclear localization and hepatic glucose homeostasis. PMID:22503562

Production and characterization of a PEGylated derivative of recombinant human deoxyribonuclease I for cystic fibrosis therapy.

PubMed

Guichard, Marie-Julie; Patil, Harshad P; Koussoroplis, Salome Juliette; Wattiez, Ruddy; Leal, Teresinha; Vanbever, Rita

2017-05-30

Recombinant human deoxyribonuclease I (rhDNase) is the mucolytic agent most widely used for the treatment of respiratory disease in cystic fibrosis. However, rhDNase is rapidly cleared from the lungs which implies a high dosing frequency and limited patient adherence. The aim of this study was to produce a long-acting PEGylated derivative of rhDNase presenting a preserved enzymatic activity. Site-specific PEGylation on the N-terminal (N-ter) leucine residue of rhDNase was achieved by reductive alkylation at acidic pH using linear 20kDa, linear 30kDa or two-arm 40kDa polyethylene glycol (PEG) propionaldehydes. Yields of mono-PEGylated products ranged between 45% and 61%. Conjugation to PEG fully preserved the secondary structure and the in vitro enzymatic activity of the native protein. These properties offer interesting perspectives for in vivo inhalation studies of the PEGylated enzyme. Copyright © 2017 Elsevier B.V. All rights reserved.

Immobilization of alkaline phosphatase on solid surface through self-assembled monolayer and by active-site protection.

PubMed

Gao, En-Feng; Kang, Kyung Lhi; Kim, Jeong Hee

2014-06-01

Retaining biological activity of a protein after immobilization is an important issue and many studies reported to enhance the activity of proteins after immobilization. We recently developed a new immobilization method of enzyme using active-site protection and minimization of the cross-links between enzyme and surface with a DNA polymerase as a model system. In this study, we extended the new method to an enzyme with a small mono-substrate using alkaline phosphatase (AP) as another model system. A condition to apply the new method is that masking agents, in this case its own substrate needs to stay at the active-site of the enzyme to be immobilized in order to protect the active-site during the harsh immobilization process. This could be achieved by removal of essential divalent ion, Zn2+ that is required for full enzyme activity of AP from the masking solution while active-site of AP was protected with p-nitrophenyl phosphate (pNPP). Approximately 40% of the solution-phase activity was acquired with active-site protected immobilized AP. In addition to protection active-site of AP, the number of immobilization links was kinetically controlled. When the mole fraction of the activated carboxyl group of the linker molecule in self-assembled monolayer (SAM) of 12-mercaptododecanoic acid and 6-mercapto-1-ethanol was varied, 10% of 12-mercaptododecanoic acid gave the maximum enzyme activity. Approximately 51% increase in enzyme activity of the active-site protected AP was observed compared to that of the unprotected group. It was shown that the concept of active-site protection and kinetic control of the number of covalent immobilization bonds can be extended to enzymes with small mono-substrates. It opens the possibility of further extension of the new methods of active-site protection and kinetic control of immobilization bond to important enzymes used in research and industrial fields.

Membraneless glucose/oxygen enzymatic fuel cells using redox hydrogel films containing carbon nanotubes.

PubMed

MacAodha, Domhnall; Ó Conghaile, Peter; Egan, Brenda; Kavanagh, Paul; Leech, Dónal

2013-07-22

Co-immobilisation of three separate multiple blue copper oxygenases, a Myceliophthora thermophila laccase, a Streptomyces coelicolor laccase and a Myrothecium verrucaria bilirubin oxidase, with an [Os(2,2'-bipyridine)2 (polyvinylimidazole)10Cl](+/2+) redox polymer in the presence of multi-walled carbon nanotubes (MWCNTs) on graphite electrodes results in enzyme electrodes that produce current densities above 0.5 mA cm(-2) for oxygen reduction at an applied potential of 0 V versus Ag/AgCl. Fully enzymatic membraneless fuel cells are assembled with the oxygen-reducing enzyme electrodes connected to glucose-oxidising anodes based on co-immobilisation of glucose oxidase or a flavin adenine dinucleotide-dependent glucose dehydrogenase with an [Os(4,4'-dimethyl-2,2'-bipyridine)2(polyvinylimidazole)10Cl](+/2+) redox polymer in the presence of MWCNTs on graphite electrodes. These fuel cells can produce power densities of up to 145 μW cm(-2) on operation in pH 7.4 phosphate buffer solution at 37 °C containing 150 mM NaCl, 5 mM glucose and 0.12 mM O2. The fuel cells based on Myceliophthora thermophila laccase enzyme electrodes produce the highest power density if combined with glucose oxidase-based anodes. Although the maximum power density of a fuel cell of glucose dehydrogenase and Myceliophthora thermophila laccase enzyme electrodes decreases from 110 μW cm(-2) in buffer to 60 μW cm(-2) on testing in artificial plasma, it provides the highest power output reported to date for a fully enzymatic glucose-oxidising, oxygen-reducing fuel cell in artificial plasma. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of 2-Thioxo-2,3,5,6,7,8-hexahydropyrimido[4,5-d]pyrimidin-4(1H)-one analogues as GAA Activators

PubMed Central

Marugan, Juan J.; Zheng, Wei; Motabar, Omid; Southall, Noel; Goldin, Ehud; Sidransky, Ellen; Aungst, Ronald A.; Liu, Ke; Sadhukhan, Subir Kumar; Austin, Christopher P.

2010-01-01

Pompe disease is a lysosomal storage disease (LSD) caused by a deficiency in the lysosomal enzyme acid α-glucosidase. In several LSDs, enzyme inhibitors have been used as small molecule chaperones to facilitate and increase the translocation of mutant protein from the endoplasmic reticulum to the lysosome. Enzyme activators with chaperone activity would be even more desirable as they would not inhibit the enzyme after translocation and might potentiate the activity of the enzyme that is successfully translocated. Herein we report our initial findings of a new series of acid α-glucosidase activators. PMID:20206419

Evaluation of 2-thioxo-2,3,5,6,7,8-hexahydropyrimido[4,5-d]pyrimidin-4(1H)-one analogues as GAA activators.

PubMed

Marugan, Juan J; Zheng, Wei; Motabar, Omid; Southall, Noel; Goldin, Ehud; Sidransky, Ellen; Aungst, Ronald A; Liu, Ke; Sadhukhan, Subir Kumar; Austin, Christopher P

2010-05-01

Pompe disease is a lysosomal storage disease (LSD) caused by a deficiency in the lysosomal enzyme acid alpha-glucosidase. In several LSDs, enzyme inhibitors have been used as small molecule chaperones to facilitate and increase the translocation of mutant protein from the endoplasmic reticulum to the lysosome. Enzyme activators with chaperone activity would be even more desirable as they would not inhibit the enzyme after translocation and might potentiate the activity of the enzyme that is successfully translocated. Herein we report our initial findings of a new series of acid alpha-glucosidase activators.

Calpain activation induced by glucose deprivation is mediated by oxidative stress and contributes to neuronal damage.

PubMed

Páramo, Blanca; Montiel, Teresa; Hernández-Espinosa, Diego R; Rivera-Martínez, Marlene; Morán, Julio; Massieu, Lourdes

2013-11-01

The mechanisms leading to neuronal death during glucose deprivation have not been fully elucidated, but a role of oxidative stress has been suggested. In the present study we have investigated whether the production of reactive oxygen species during glucose deprivation, contributes to the activation of calpain, a calcium-dependent protease involved in neuronal injury associated with brain ischemia and cerebral trauma. We have observed a rapid activation of calpain, as monitored by the cleavage of the cytoskeletal protein α-spectrin, after glucose withdrawal, which is reduced by inhibitors of xanthine oxidase, phospholipase A2 and NADPH oxidase. Results suggest that phospholipase A2 and NADPH oxidase contribute to the early activation of calpain after glucose deprivation. In particular NOX2, a member of the NADPH oxidase family is involved, since reduced stimulation of calpain activity is observed after glucose deprivation in hippocampal slices from transgenic mice lacking a functional NOX2. We observed an additive effect of the inhibitors of xanthine oxidase and phospholipase A2 on both ROS production and calpain activity, suggesting a synergistic action of these two enzymes. The present results provide new evidence showing that reactive oxygen species stimulate calpain activation during glucose deprivation and that this mechanism is involved in neuronal death. Copyright © 2013 Elsevier Ltd. All rights reserved.

Zinc regulates iNOS-derived nitric oxide formation in endothelial cells.

PubMed

Cortese-Krott, Miriam M; Kulakov, Larissa; Opländer, Christian; Kolb-Bachofen, Victoria; Kröncke, Klaus-D; Suschek, Christoph V

2014-01-01

Aberrant production of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathogenesis of endothelial dysfunction and vascular disease. Mechanisms responsible for the fine-tuning of iNOS activity in inflammation are still not fully understood. Zinc is an important structural element of NOS enzymes and is known to inhibit its catalytical activity. In this study we aimed to investigate the effects of zinc on iNOS activity and expression in endothelial cells. We found that zinc down-regulated the expression of iNOS (mRNA+protein) and decreased cytokine-mediated activation of the iNOS promoter. Zinc-mediated regulation of iNOS expression was due to inhibition of NF-κB transactivation activity, as determined by a decrease in both NF-κB-driven luciferase reporter activity and expression of NF-κB target genes, including cyclooxygenase 2 and IL-1β. However, zinc did not affect NF-κB translocation into the nucleus, as assessed by Western blot analysis of nuclear and cytoplasmic fractions. Taken together our results demonstrate that zinc limits iNOS-derived high output NO production in endothelial cells by inhibiting NF-κB-dependent iNOS expression, pointing to a role of zinc as a regulator of iNOS activity in inflammation.

Auranofin, as an anti-rheumatic gold compound suppresses LPS-induced homodimerization of TLR4

PubMed Central

Youn, Hyung S.; Lee, Joo Y.; Saitoh, Shin I.; Miyake, Kensuke; Hwang, Daniel H.

2009-01-01

Toll-like receptors (TLRs), which are activated by invading microorganisms or endogenous molecules, evoke immune and inflammatory responses. TLR activation is closely linked to the development of many chronic inflammatory diseases including rheumatoid arthritis. Auranofin, an Au(I) compound, is a well-known and long-used anti-rheumatic drug. However, the mechanism as to how auranofin relieves the symptom of rheumatoid arthritis has not been fully clarified. Our results demonstrated that auranofin suppressed TLR4-mediated activation of transcription factors, NF-κB and IRF3 and expression of COX-2, a pro-inflammatory enzyme. This suppression was well correlated with the inhibitory effect of auranofin on the homodimerization of TLR4 induced by an agonist. Furthermore, auranofin inhibited NF-κ activation induced by MyD88-dependent downstream signaling components of TLR4, MyD88, IKKβ, and p65. IRF3 activation induced by MyD88-independent signaling components, TRIF and TBK1, was also downregulated by auranofin. Our results first demonstrate that auranofin suppresses the multiple steps in TLR4 signaling, especially the homodimerization of TLR4. The results suggest that the suppression of TLR4 activity by auranofin may be the molecular mechanism through which auranofin exerts anti-rheumatic activity. PMID:17034761

Zinc regulates iNOS-derived nitric oxide formation in endothelial cells

PubMed Central

Cortese-Krott, Miriam M.; Kulakov, Larissa; Opländer, Christian; Kolb-Bachofen, Victoria; Kröncke, Klaus-D.; Suschek, Christoph V.

2014-01-01

Aberrant production of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathogenesis of endothelial dysfunction and vascular disease. Mechanisms responsible for the fine-tuning of iNOS activity in inflammation are still not fully understood. Zinc is an important structural element of NOS enzymes and is known to inhibit its catalytical activity. In this study we aimed to investigate the effects of zinc on iNOS activity and expression in endothelial cells. We found that zinc down-regulated the expression of iNOS (mRNA+protein) and decreased cytokine-mediated activation of the iNOS promoter. Zinc-mediated regulation of iNOS expression was due to inhibition of NF-κB transactivation activity, as determined by a decrease in both NF-κB-driven luciferase reporter activity and expression of NF-κB target genes, including cyclooxygenase 2 and IL-1β. However, zinc did not affect NF-κB translocation into the nucleus, as assessed by Western blot analysis of nuclear and cytoplasmic fractions. Taken together our results demonstrate that zinc limits iNOS-derived high output NO production in endothelial cells by inhibiting NF-κB-dependent iNOS expression, pointing to a role of zinc as a regulator of iNOS activity in inflammation. PMID:25180171

Nanoarmoring of Enzymes by Interlocking in Cellulose Fibers With Poly(Acrylic Acid).

PubMed

Riccardi, Caterina M; Kasi, Rajeswari M; Kumar, Challa V

2017-01-01

A simple method for interlocking glucose oxidase (GOx) and horseradish peroxidase (HRP) in cellulose fibers using poly(acrylic acid) (PAA) as an armor around the enzyme, without any need for activation of the cellulose support, is reported here. The resulting enzyme paper is an inexpensive, stable, simple, wearable, and washable biosensor. PAA functions as a multifunctional tether to interlock the enzyme molecules around the paper fibers so that the enzymes are protected against thermal/chemical denaturation and not released from the paper when washed with a detergent. The decreased conformational entropy of the interlocked enzyme protected by the nanoarmor is likely responsible for increased enzyme stability to heat and chemical denaturants (retained ≥70 percent enzyme activity after washing with urea or SDS for 30min), and the polymer protects the enzyme against inactivation by proteases, bacteria, inhibitors, etc. The kinetics of the interlocked enzyme were similar to that of the enzyme in solution. The V max was 6(±0.5)mM per minute before washing, then increased slightly to 9(±1.4)mM per minute after washing with water. The K m was 22(±6.4mM), which was slightly higher compared to GOx in solution (25-27mM). Because the surface area of the paper does not limit the enzyme loading, about 20% of enzyme was successfully loaded onto the paper (0.2g enzyme per gram of paper), and ≥95% of the enzyme was retained after washing. Interlocking works with other enzymes such as laccase, where ≥60% of the enzyme activity is retained. This novel methodology provides a low cost, simple, modular approach of achieving high enzyme loadings in ordinary filter paper, not limited by cellulose surface area, and there has been no need for complex methods of enzyme engineering or toxic methods of activation of the solid support to prepare highly active biocatalysts. © 2017 Elsevier Inc. All rights reserved.

Evaluation of simultaneous binding of Chromomycin A3 to the multiple sites of DNA by the new restriction enzyme assay.

PubMed

Murase, Hirotaka; Noguchi, Tomoharu; Sasaki, Shigeki

2018-06-01

Chromomycin A3 (CMA3) is an aureolic acid-type antitumor antibiotic. CMA3 forms dimeric complexes with divalent cations, such as Mg 2+ , which strongly binds to the GC rich sequence of DNA to inhibit DNA replication and transcription. In this study, the binding property of CMA3 to the DNA sequence containing multiple GC-rich binding sites was investigated by measuring the protection from hydrolysis by the restriction enzymes, AccII and Fnu4HI, for the center of the CGCG site and the 5'-GC↓GGC site, respectively. In contrast to the standard DNase I footprinting method, the DNA substrates are fully hydrolyzed by the restriction enzymes, therefore, the full protection of DNA at all the cleavable sites indicates that CMA3 simultaneously binds to all the binding sites. The restriction enzyme assay has suggested that CMA3 has a high tendency to bind the successive CGCG sites and the CGG repeat. Copyright © 2018 Elsevier Ltd. All rights reserved.

Enhancement of β-Glucan Content in the Cultivation of Cauliflower Mushroom (Sparassis latifolia) by Elicitation.

PubMed

Park, Hyun; Ka, Kang-Hyeon; Ryu, Sung-Ryul

2014-03-01

The effectiveness of three kinds of enzymes (chitinase, β-glucuronidase, and lysing enzyme complex), employed as elicitors to enhance the β-glucan content in the sawdust-based cultivation of cauliflower mushroom (Sparassis latifolia), was examined. The elicitors were applied to the cauliflower mushroom after primordium formation, by spraying the enzyme solutions at three different levels on the sawdust-based medium. Mycelial growth was fully accomplished by the treatments, but the metabolic process during the growth of fruiting bodies was affected. The application of a lysing enzyme resulted in an increase in the β-glucan concentration by up to 31% compared to that of the control. However, the treatment resulted in a decrease in mushroom yield, which necessitated the need to evaluate its economic efficiency. Although we still need to develop a more efficient way for using elicitors to enhance functional metabolites in mushroom cultivation, the results indicate that the elicitation technique can be applied in the cultivation of medicinal/edible mushrooms.

Unfolding and inactivation during thermal denaturation of an enzyme that exhibits phytase and acid phosphatase activities.

PubMed

Wang, Xiao-Yun; Meng, Fan-Guo; Zhou, Hai-Meng

2004-03-01

The thermostability of an enzyme that exhibits phytase and acid phosphatase activities was studied. Kinetics of inactivation and unfolding during thermal denaturation of the enzyme were compared. The loss of phytase activity on thermal denaturation is most suggestive of a reversible process. As for acid phosphatase activities, an interesting phenomenon was observed; there are two phases in thermal inactivation: when the temperature was between 45 and 50 degrees C, the thermal inactivation could be characterized as an irreversible inactivation which had some residual activity and when the temperature was above 55 degrees C, the thermal inactivation could be characterized as an irreversible process which had no residual activity. The microscopic rate constants for the free enzyme and substrate-enzyme complex were determined by Tsou's method [Adv. Enzymol. Relat. Areas Mol. Biol. 61 (1988) 381]. Fluorescence analyses indicate that when the enzyme was treated at temperatures below 60 degrees C for 60 min, the conformation of the enzyme had no detectable change; when the temperatures were above 60 degrees C, some fluorescence red-shift could be observed with a decrease in emission intensity. The inactivation rates (k(+0)) of free enzymes were faster than those of conformational changes during thermal denaturation at the same temperature. The rapid inactivation and slow conformational changes of phytase during thermal denaturation suggest that inactivation occurs before significant conformational changes of the enzyme, and the active site of this enzyme is situated in a relatively fragile region which makes the active site more flexible than the molecule as a whole.

Developmental and hormone-induced changes of mitochondrial electron transport chain enzyme activities during the last instar larval development of maize stem borer, Chilo partellus (Lepidoptera: Crambidae).

PubMed

VenkatRao, V; Chaitanya, R K; Naresh Kumar, D; Bramhaiah, M; Dutta-Gupta, A

2016-12-01

The energy demand for structural remodelling in holometabolous insects is met by cellular mitochondria. Developmental and hormone-induced changes in the mitochondrial respiratory activity during insect metamorphosis are not well documented. The present study investigates activities of enzymes of mitochondrial electron transport chain (ETC) namely, NADH:ubiquinone oxidoreductase or complex I, Succinate: ubiquinone oxidoreductase or complex II, Ubiquinol:ferricytochrome c oxidoreductase or complex III, cytochrome c oxidase or complex IV and F 1 F 0 ATPase (ATPase), during Chilo partellus development. Further, the effect of juvenile hormone (JH) analog, methoprene, and brain and corpora-allata-corpora-cardiaca (CC-CA) homogenates that represent neurohormones, on the ETC enzyme activities was monitored. The enzymatic activities increased from penultimate to last larval stage and thereafter declined during pupal development with an exception of ATPase which showed high enzyme activity during last larval and pupal stages compared to the penultimate stage. JH analog, methoprene differentially modulated ETC enzyme activities. It stimulated complex I and IV enzyme activities, but did not alter the activities of complex II, III and ATPase. On the other hand, brain homogenate declined the ATPase activity while the injected CC-CA homogenate stimulated complex I and IV enzyme activities. Cumulatively, the present study is the first to show that mitochondrial ETC enzyme system is under hormone control, particularly of JH and neurohormones during insect development. Copyright © 2015 Elsevier Inc. All rights reserved.

LECITHINASE AND LYSOLECITHINASE ACTIVITY OF RAT INTESTINAL MUCOSA AFTER WHOLE-BODY X-IRRADIATION

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ottolenghi, A.; Bernheim, F.

1961-11-01

Twenty-four hours after whole-body x irradiation the lecithinase activity of rat intestinal mucosa has markedly decreased and the lysolecithinase activity has decreasecp to a lesser extent. Addition of normal mucosa or chyi'otrypsin to the irradiated mucosa restores the activity of both enzymes. This indicates that irradiation eithei produces an inhibitor or inactivates a mechanism necessarly to convert pro-enzymes into active enzymes. Since chymo trypsin can increase to some extent the activity of the enzymes in normal mucosa, the second possibility seems more probable. (auth)

Regulation of Hydrolytic Enzyme Activity in Aquatic Microbial Communities Hosted by Carnivorous Pitcher Plants.

PubMed

Young, Erica B; Sielicki, Jessica; Grothjan, Jacob J

2018-04-20

Carnivorous pitcher plants Sarracenia purpurea host diverse eukaryotic and bacterial communities which aid in insect prey digestion, but little is known about the functional processes mediated by the microbial communities. This study aimed to connect pitcher community diversity with functional nutrient transformation processes, identifying bacterial taxa, and measuring regulation of hydrolytic enzyme activity in response to prey and alternative nutrient sources. Genetic analysis identified diverse bacterial taxa known to produce hydrolytic enzyme activities. Chitinase, protease, and phosphatase activities were measured using fluorometric assays. Enzyme activity in field pitchers was positively correlated with bacterial abundance, and activity was suppressed by antibiotics suggesting predominantly bacterial sources of chitinase and protease activity. Fungi, algae, and rotifers observed could also contribute enzyme activity, but fresh insect prey released minimal chitinase activity. Activity of chitinase and proteases was upregulated in response to insect additions, and phosphatase activity was suppressed by phosphate additions. Particulate organic P in prey was broken down, appearing as increasing dissolved organic and inorganic P pools within 14 days. Chitinase and protease were not significantly suppressed by availability of dissolved organic substrates, though organic C and N stimulated bacterial growth, resulting in elevated enzyme activity. This comprehensive field and experimental study show that pitcher plant microbial communities dynamically regulate hydrolytic enzyme activity, to digest prey nutrients to simpler forms, mediating biogeochemical nutrient transformations and release of nutrients for microbial and host plant uptake.

Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

DOEpatents

Jarvis, Eric E.; Roessler, Paul G.

1999-01-01

The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities.

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in murine epidermis. Modulation of enzyme content and activation state by barrier requirements.

PubMed Central

Proksch, E; Elias, P M; Feingold, K R

1990-01-01

Epidermal cholesterol biosynthesis is regulated by barrier function. We quantitated the amount and activation state (phosphorylation-dephosphorylation) of the rate-limiting enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, in epidermis before and after barrier disruption. In murine epidermis we found high enzyme activity (1.75 +/- 0.02 nmol/min per mg protein). After acute barrier disruption, enzyme activity began to increase after 1.5 h, reaching a maximum increase by 2.5 h, and returned to normal by 15 h. Chronic barrier disruption increased total enzyme activity by 83%. In normal epidermis, measurement of HMG CoA reductase activity in microsomes isolated in NaF- vs. NaCl-containing buffers demonstrated that 46 +/- 2% of the enzyme was in the active form. After acute or chronic barrier disruption, a marked increase in the percentage of HMG CoA reductase in the active form was observed. Acute disruption increased enzyme activation state as early as 15 min, reaching a maximum after 2.5 h, with an increase still present at 15 h, indicating that changes in activation state had a close temporal relationship with barrier function. Increases in total HMG CoA reductase activity occurred only after profound barrier disruption, whereas changes in activation state occur with lesser degrees of barrier disruption. Artificial correction of barrier function prevented the increase in total HMG CoA reductase activity, and partially prevented the increase in enzyme activation. These results show that barrier requirements regulate epidermal cholesterol synthesis by modulating both the HMG CoA reductase amount and activation state. Images PMID:2312730

Functional Evolution of PLP-dependent Enzymes based on Active-Site Structural Similarities

PubMed Central

Catazaro, Jonathan; Caprez, Adam; Guru, Ashu; Swanson, David; Powers, Robert

2014-01-01

Families of distantly related proteins typically have very low sequence identity, which hinders evolutionary analysis and functional annotation. Slowly evolving features of proteins, such as an active site, are therefore valuable for annotating putative and distantly related proteins. To date, a complete evolutionary analysis of the functional relationship of an entire enzyme family based on active-site structural similarities has not yet been undertaken. Pyridoxal-5’-phosphate (PLP) dependent enzymes are primordial enzymes that diversified in the last universal ancestor. Using the Comparison of Protein Active Site Structures (CPASS) software and database, we show that the active site structures of PLP-dependent enzymes can be used to infer evolutionary relationships based on functional similarity. The enzymes successfully clustered together based on substrate specificity, function, and three-dimensional fold. This study demonstrates the value of using active site structures for functional evolutionary analysis and the effectiveness of CPASS. PMID:24920327

Functional evolution of PLP-dependent enzymes based on active-site structural similarities.

PubMed

Catazaro, Jonathan; Caprez, Adam; Guru, Ashu; Swanson, David; Powers, Robert

2014-10-01

Families of distantly related proteins typically have very low sequence identity, which hinders evolutionary analysis and functional annotation. Slowly evolving features of proteins, such as an active site, are therefore valuable for annotating putative and distantly related proteins. To date, a complete evolutionary analysis of the functional relationship of an entire enzyme family based on active-site structural similarities has not yet been undertaken. Pyridoxal-5'-phosphate (PLP) dependent enzymes are primordial enzymes that diversified in the last universal ancestor. Using the comparison of protein active site structures (CPASS) software and database, we show that the active site structures of PLP-dependent enzymes can be used to infer evolutionary relationships based on functional similarity. The enzymes successfully clustered together based on substrate specificity, function, and three-dimensional-fold. This study demonstrates the value of using active site structures for functional evolutionary analysis and the effectiveness of CPASS. © 2014 Wiley Periodicals, Inc.

Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers

PubMed Central

Wang, Mingfa; Zhang, Xiaotu; Wang, Zhixiang

2017-01-01

Three hundred one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets containing non-starch polysaccharide (NSP) enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI) and average daily gain (ADG) were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05). Feed-to-gain ratio (FGR) was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05). Apparent digestibility of crude protein (ADCP) was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05). Cholecystokinin (CCK) level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05), but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05), respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05). However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05). The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05). Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased pancreatic trypsin mRNA levels by 40%, 44% and 28%, respectively. Supplementation with NSP enzyme and 160 mg/kg protease decreased pancreatic trypsin mRNA levels by 13%. Pancreatic lipase and amylase mRNA expression were significantly elevated in treated animals compared to the control group (p<0.05). These results suggest that the amount of NSP enzyme and acid protease in the diet significantly affects digestive function, endogenous digestive-enzyme activity and mRNA expression in broilers. PMID:28323908

NMR solution structure of the activation domain of human procarboxypeptidase A2

PubMed Central

Jiménez, M. Angeles; Villegas, Virtudes; Santoro, Jorge; Serrano, Luis; Vendrell, Josep; Avilés, Francesc X.; Rico, Manuel

2003-01-01

The activation domain of human procarboxypeptidase A2, ADA2h, is an 81-residue globular domain released during the proteolytic activation of the proenzyme. The role of this and other similar domains as assistants of the correct folding of the enzyme is not fully understood. The folding pathway of ADA2h was characterized previously, and it was also observed that under certain conditions it may convert into amyloid fibrils in vitro. To gain insight into these processes, a detailed description of its three-dimensional structure in aqueous solution is required so that eventual changes could be properly monitored. A complete assignment of the 1H and 15N resonances of ADA2h was performed, and the solution structure, as derived from a set of 1688 nonredundant constraints, is very well defined (pairwise backbone RMSD = 0.67 ± 0.17 Å for residues 10–80). The structure is composed of two antiparallel α-helices comprising residues 19–32 and 58–69 packed on the same side of a three-stranded β-sheet spanning residues 10–15, 50–55, and 73–75. The global fold for the isolated human A2 activation domain is very similar to that of porcine carboxypeptidase B, as well as to the structure of the domain in the crystal of the intact human proenzyme. The observed structural differences relative to the intact human proenzyme are located at the interface between the activation domain and the enzyme and can be related with the activation mechanism. The backbone amide proton exchange behavior of ADA2h was also examined. The global free energy of unfolding obtained from exchange data of the most protected amide protons at pH 7.0 and 298K is 4.9 ± 0.3 kcal.mole−1, in good agreement with the values determined by thermal or denaturant unfolding studies. PMID:12538893

Hinokitiol Inhibits Migration of A549 Lung Cancer Cells via Suppression of MMPs and Induction of Antioxidant Enzymes and Apoptosis

PubMed Central

Jayakumar, Thanasekaran; Liu, Chao-Hong; Wu, Guan-Yi; Lee, Tzu-Yin; Manubolu, Manjunath; Hsieh, Cheng-Ying; Yang, Chih-Hao; Sheu, Joen-Rong

2018-01-01

Hinokitiol, a natural monoterpenoid from the heartwood of Calocedrus formosana, has been reported to have anticancer effects against various cancer cell lines. However, the detailed molecular mechanisms and the inhibiting roles of hinokitiol on adenocarcinoma A549 cells remain to be fully elucidated. Thus, the current study was designed to evaluate the effect of hinokitiol on the migration of human lung adenocarcinoma A549 cells in vitro. The data demonstrates that hinokitiol does not effectively inhibit the viability of A549 cells at up to a 10 µM concentration. When treated with non-toxic doses (1–5 µM) of hinokitiol, the cell migration is markedly suppressed at 5 µM. Hinokitiol significantly reduced p53 expression, followed by attenuation of Bax in A549 cells. A dose-dependent inhibition of activated caspase-9 and -3 was observed in the presence of hinokitiol. An observed increase in protein expression of matrix metalloproteinases (MMPs) -2/-9 in A549 cells was significantly inhibited by hinokitiol. Remarkably, when A549 cells were subjected to hinokitiol (1–5 µM), there was an increase in the activities of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) from the reduction in cells. In addition, the incubation of A549 cells with hinokitiol significantly activated the cytochrome c expression, which may be triggered by activation of caspase-9 followed by caspase-3. These observations indicate that hinokitiol inhibited the migration of lung cancer A549 cells through several mechanisms, including the activation of caspases-9 and -3, induction of p53/Bax and antioxidant CAT and SOD, and reduction of MMP-2 and -9 activities. It also induces cytochrome c expression. These findings demonstrate a new therapeutic potential for hinokitiol in lung cancer chemoprevention. PMID:29565268

Visualization of enzyme activities inside earthworm biopores by in situ soil zymography

NASA Astrophysics Data System (ADS)

Thu Duyen Hoang, Thi; Razavi, Bahar. S.; Blagodatskaya, Evgenia; Kuzyakov, Yakov

2015-04-01

Earthworms can strongly activate microorganisms, increase microbial and enzyme activities and consequently the turnover of native soil organic matter. In extremely dynamic microhabitats and hotspots as biopores made by earthworms, the in situ enzyme activities are a footprint of complex biotic interactions. The effect of earthworms on the alteration of enzyme activities inside biopores and the difference between bio-pores and earthworm-free soil was visualized by in situ soil zymography (Spohn and Kuzyakov, 2014). For the first time, we prepared quantitative imaging of enzyme activities in biopores. Furthermore, we developed the zymography technique by direct application of a substrate saturated membrane to the soil to obtain better spatial resolution. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). Simultaneously, maize seed was sown in the soil. Control soil box with maize and without earthworm was prepared in the same way. After two weeks when bio-pore systems were formed by earthworm, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine aminopeptidase) and phosphatase. Followed by non-destructive zymography, biopore samples and control soil were destructively collected to assay enzyme kinetics by fluorogenically labeled substrates method. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. These differences were further confirmed by fluorimetric microplate enzyme assay detected significant difference of Vmax in four above mentioned enzymes. Vmax of β-glucosidase, chitinase, xylanase and phosphatase in biopores is 68%, 108%, 50% and 49% higher than that of control soil. However, no difference in cellobiohydrolase and leucine aminopeptidase kinetics between biopores and control soil were detected. This indicated little effect of earthworms on protein and cellulose transformation in soil. In conclusion, earthworms contribute to the decomposition of carbohydrates through promoting enzyme activities involved in the C-cycle except for leucine aminopeptidase and cellobiohydrolase. References Spohn M, Kuzyakov Y. (2014) Spatial and temporal dynamics of hotspots of enzyme activity in soil as affected by living and dead roots - a soil zymography analysis, Plant Soil 379: 67-77

Optimization of Enzyme Co-Immobilization with Sodium Alginate and Glutaraldehyde-Activated Chitosan Beads.

PubMed

Gür, Sinem Diken; İdil, Neslihan; Aksöz, Nilüfer

2018-02-01

In this study, two different materials-alginate and glutaraldehyde-activated chitosan beads-were used for the co-immobilization of α-amylase, protease, and pectinase. Firstly, optimization of multienzyme immobilization with Na alginate beads was carried out. Optimum Na alginate and CaCl 2 concentration were found to be 2.5% and 0.1 M, respectively, and optimal enzyme loading ratio was determined as 2:1:0.02 for pectinase, protease, and α-amylase, respectively. Next, the immobilization of multiple enzymes on glutaraldehyde-activated chitosan beads was optimized (3% chitosan concentration, 0.25% glutaraldehyde with 3 h of activation and 3 h of coupling time). While co-immobilization was successfully performed with both materials, the specific activities of enzymes were found to be higher for the enzymes co-immobilized with glutaraldehyde-activated chitosan beads. In this process, glutaraldehyde was acting as a spacer arm. SEM and FTIR were used for the characterization of activated chitosan beads. Moreover, pectinase and α-amylase enzymes immobilized with chitosan beads were also found to have higher activity than their free forms. Three different enzymes were co-immobilized with these two materials for the first time in this study.

Degradation of ureidoglycolate in French bean (Phaseolus vulgaris) is catalysed by a ubiquitous ureidoglycolate urea-lyase.

PubMed

Muñoz, Alfonso; Raso, María José; Pineda, Manuel; Piedras, Pedro

2006-06-01

A ureidoglycolate-degrading activity was analysed in different tissues of French bean (Phaseolus vulgaris L.) plants during development. Activity was detected in all the tissues analysed, although values were very low in seeds before germination and in cotyledons. After radicle emergence, the activity increased due to high activity present in the axes. The highest levels of specific activity were found in developing fruits, from which the enzyme was purified and characterised. This is the first ureidoglycolate-degrading activity that has been purified to homogeneity from a ureide legume. The enzyme was purified 280 fold, and the specific activity for the pure enzyme was 4.4 units mg(-1), which corresponds to a turnover number of 1,055 min(-1). The native enzyme has a molecular mass of 240 kDa and consists of six identical or similar-sized subunits each of 38 kDa. The activity of the purified enzyme was completely dependent on manganese and asparagine. The enzyme exhibited hyperbolic, Michaelian kinetics for ureidoglycolate with a K(m) value of 3.9 mM. This enzyme has been characterised as a ureidoglycolate urea-lyase (EC 4.3.2.3).

Enzyme activity screening of thermophilic bacteria isolated from Dusun Tua Hot Spring, Malaysia

NASA Astrophysics Data System (ADS)

Msarah, Marwan; Ibrahim, Izyanti; Aqma, Wan Syaidatul

2018-04-01

Thermophilic bacteria have biotechnological importance due to the availability of unique enzymes which are stable in extreme circumstances. The aim of this study includes to isolate thermophilic bacteria from hot spring and screen for important enzyme activities. Water samples from the Dusun Tua Hot Spring were collected and the physiochemical characterisation of water was measured. Eight thermophilic bacteria were isolated and determined to have at least three strong enzyme activity including protease, lipase, amylase, cellulase, pectinase and xylanase. The results showed that HuluC2 displayed all the enzyme activities and can be further studied.

The Catalytic Function of Enzymes.

ERIC Educational Resources Information Center

Splittgerber, Allan G.

1985-01-01

Discusses: structure of the enzyme molecule; active site; reaction mechanism; transition state; factors affecting enzyme reaction rates, concentration of enzyme; concentration of substrate; product concentration; temperature effects and pH effects; factors causing a lowering of activation energy; proximity and orientation effects; substrate strain…

Structural prediction and comparative docking studies of psychrophilic β- Galactosidase with lactose, ONPG and PNPG against its counter parts of mesophilic and thermophilic enzymes.

PubMed

Kumar, Ponnada Suresh; Pulicherla, Kk; Ghosh, Mrinmoy; Kumar, Anmol; Rao, Krs Sambasiva

2011-01-01

Enzymes from psychrophiles catalyze the reactions at low temperatures with higher specific activity. Among all the psychrophilic enzymes produced, cold active β-galactosidase from marine psychrophiles revalorizes a new arena in numerous areas at industrial level. The hydrolysis of lactose in to glucose and galactose by cold active β-galactosidase offers a new promising approach in removal of lactose from milk to overcome the problem of lactose intolerance. Herein we propose, a 3D structure of cold active β-galactosidase enzyme sourced from Pseudoalteromonas haloplanktis by using Modeler 9v8 and best model was developed having 88% of favourable region in ramachandran plot. Modelling was followed by docking studies with the help of Auto dock 4.0 against the three substrates lactose, ONPG and PNPG. In addition, comparative docking studies were also performed for the 3D model of psychrophilic β-galactosidase with mesophilic and thermophilic enzymes. Docking studies revealed that binding affinity of enzyme towards the three different substrates is more for psychrophilic enzyme when compared with mesophilic and thermophilic enzymes. It indicates that the enzyme has high specific activity at low temperature when compared with mesophilic and thermophilic enzymes.

Profiling the orphan enzymes

PubMed Central

2014-01-01

The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called “orphan enzymes”. The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to “local orphan enzymes” that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new activities. Reviewers This article was reviewed by Michael Galperin, Daniel Haft and Daniel Kahn. PMID:24906382

Development of in vivo biotransformation enzyme assays for ecotoxicity screening: In vivo measurement of phases I and II enzyme activities in freshwater planarians.

PubMed

Li, Mei-Hui

2016-08-01

The development of a high-throughput tool is required for screening of environmental pollutants and assessing their impacts on aquatic animals. Freshwater planarians can be used in rapid and sensitive toxicity bioassays. Planarians are known for their remarkable regeneration ability but much less known for their metabolic and xenobiotic biotransformation abilities. In this study, the activities of different phase I and II enzymes were determined in vivo by directly measuring fluorescent enzyme substrate disappearance or fluorescent enzyme metabolite production in planarian culture media. For phase I enzyme activity, O-deethylation activities with alkoxyresorufin could not be detected in planarian culture media. By contrast, O-deethylation activities with alkoxycoumarin were detected in planarian culture media. Increases in 7-ethoxycoumarin O-deethylase (ECOD) activities was only observed in planarians exposed to 1μM, but not 10μM, β-naphthoflavone for 24h. ECOD activity was inhibited in planarians exposed to 10 and 100μM rifampicin or carbamazepine for 24h. For phase II enzyme activity, DT-diaphorase, arylsulfatases, uridine 5'-diphospho (UDP)-glucuronosyltransferase or catechol-O-methyltransferase activity was determined in culture media containing planarians. The results of this study indicate that freshwater planarians are a promising model organism to monitor exposure to environmental pollutants or assess their impacts through the in vivo measurement of phase I and II enzyme activities. Copyright © 2016. Published by Elsevier Inc.

Immobilization of an enzyme from a Fusarium fungus WZ-I for chlorpyrifos degradation.

PubMed

Xie, Hui; Zhu, Lusheng; Ma, Tingting; Wang, Jun; Wang, Jinhua; Su, Jun; Shao, Bo

2010-01-01

The free enzyme extracted from WZ-I, which was identified as Fusarium LK. ex Fx, could effectively degrade chlorpyrifos, an organophosphate insecticide. The methods of immobilizing this free enzyme and determined its degradation-related characteristics were investigated. The properties of the immobilized enzyme were compared with those of the free enzyme. The optimal immobilization of the enzyme was achieved in a solution of 30 g/L sodium alginate at 4 degrees C for 4-12 hr. The immobilized enzyme showed the maximal activity at pH 8.0, 45 degrees C. The maximum initial rate and the substrate concentration of the immobilized enzyme were less than that of the free enzyme. The immobilized enzyme, therefore, had a higher capacity to withstand a broader range of temperatures and pH conditions than the free enzyme. With varying pH and temperatures, the immobilized enzyme was more active than the free enzyme in the degradation reaction. In addition, the immobilized enzyme exhibited only a slight loss in its initial activity, even after three repeated uses. The results showed that the immobilized enzyme was more resistant to different environmental conditions, suggesting that it was viable for future practical use.

Lignocellulolytic enzyme production of Pleurotus ostreatus growth in agroindustrial wastes

PubMed Central

da Luz, José Maria Rodrigues; Nunes, Mateus Dias; Paes, Sirlaine Albino; Torres, Denise Pereira; de Cássia Soares da Silva, Marliane; Kasuya, Maria Catarina Megumi

2012-01-01

The mushroom Pleurotus ostreatus has nutritional and medicinal characteristics that depend on the growth substrate. In nature, this fungus grows on dead wood, but it can be artificially cultivated on agricultural wastes (coffee husks, eucalyptus sawdust, corncobs and sugar cane bagasse). The degradation of agricultural wastes involves some enzyme complexes made up of oxidative (laccase, manganese peroxidase and lignin peroxidase) and hydrolytic enzymes (cellulases, xylanases and tanases). Understanding how these enzymes work will help to improve the productivity of mushroom cultures and decrease the potential pollution that can be caused by inadequate discharge of the agroindustrial residues. The objective of this work was to assess the activity of the lignocellulolytic enzymes produced by two P. ostreatus strains (PLO 2 and PLO 6). These strains were used to inoculate samples of coffee husks, eucalyptus sawdust or eucalyptus bark add with or without 20 % rice bran. Every five days after substrate inoculation, the enzyme activity and soluble protein concentration were evaluated. The maximum activity of oxidative enzymes was observed at day 10 after inoculation, and the activity of the hydrolytic enzymes increased during the entire period of the experiment. The results show that substrate composition and colonization time influenced the activity of the lignocellulolytic enzymes. PMID:24031982

Application of carbohydrate arrays coupled with mass spectrometry to detect activity of plant-polysaccharide degradative enzymes from the fungus Aspergillus niger.

PubMed

van Munster, Jolanda M; Thomas, Baptiste; Riese, Michel; Davis, Adrienne L; Gray, Christopher J; Archer, David B; Flitsch, Sabine L

2017-02-21

Renewables-based biotechnology depends on enzymes to degrade plant lignocellulose to simple sugars that are converted to fuels or high-value products. Identification and characterization of such lignocellulose degradative enzymes could be fast-tracked by availability of an enzyme activity measurement method that is fast, label-free, uses minimal resources and allows direct identification of generated products. We developed such a method by applying carbohydrate arrays coupled with MALDI-ToF mass spectrometry to identify reaction products of carbohydrate active enzymes (CAZymes) of the filamentous fungus Aspergillus niger. We describe the production and characterization of plant polysaccharide-derived oligosaccharides and their attachment to hydrophobic self-assembling monolayers on a gold target. We verify effectiveness of this array for detecting exo- and endo-acting glycoside hydrolase activity using commercial enzymes, and demonstrate how this platform is suitable for detection of enzyme activity in relevant biological samples, the culture filtrate of A. niger grown on wheat straw. In conclusion, this versatile method is broadly applicable in screening and characterisation of activity of CAZymes, such as fungal enzymes for plant lignocellulose degradation with relevance to biotechnological applications as biofuel production, the food and animal feed industry.

Function-based classification of carbohydrate-active enzymes by recognition of short, conserved peptide motifs.

PubMed

Busk, Peter Kamp; Lange, Lene

2013-06-01

Functional prediction of carbohydrate-active enzymes is difficult due to low sequence identity. However, similar enzymes often share a few short motifs, e.g., around the active site, even when the overall sequences are very different. To exploit this notion for functional prediction of carbohydrate-active enzymes, we developed a simple algorithm, peptide pattern recognition (PPR), that can divide proteins into groups of sequences that share a set of short conserved sequences. When this method was used on 118 glycoside hydrolase 5 proteins with 9% average pairwise identity and representing four characterized enzymatic functions, 97% of the proteins were sorted into groups correlating with their enzymatic activity. Furthermore, we analyzed 8,138 glycoside hydrolase 13 proteins including 204 experimentally characterized enzymes with 28 different functions. There was a 91% correlation between group and enzyme activity. These results indicate that the function of carbohydrate-active enzymes can be predicted with high precision by finding short, conserved motifs in their sequences. The glycoside hydrolase 61 family is important for fungal biomass conversion, but only a few proteins of this family have been functionally characterized. Interestingly, PPR divided 743 glycoside hydrolase 61 proteins into 16 subfamilies useful for targeted investigation of the function of these proteins and pinpointed three conserved motifs with putative importance for enzyme activity. Furthermore, the conserved sequences were useful for cloning of new, subfamily-specific glycoside hydrolase 61 proteins from 14 fungi. In conclusion, identification of conserved sequence motifs is a new approach to sequence analysis that can predict carbohydrate-active enzyme functions with high precision.

Value of bilirubin oxidase and its mutants in the diagnosis of hyperbilirubinemia.

PubMed

Zhang, Lei; Zhang, Xiao; Luo, Zhi-Ying

2005-11-01

To elucidate the significance of the coordination amino acid residues in bilirubin oxidase (BO) and their kinetic characteristics, and evaluate whether BO mutants may serve as better diagnostic agent for hyperbilirubinemia. The BO mutants I402G and C457S were obtained by site-directed mutagenesis and confirmed by amino acid sequence analysis. Ru-incorporated C457S mutant was obtained by direct incubation of ruthenium compounds with the mutant. The electron paramagnetic resonance (EPR) spectra of the recombinant BO and the mutants were investigated, and the enzyme kinetics of the recombinant BO and I402G mutant were measured with bilirubin as the substrate at 25 degrees C. The BO mutants were expressed and purified successfully. The mutant I402G showed low enzyme activity, and had C457S virtually no enzyme activity. Nevertheless Ru-incorporation conferred higher enzyme activity to C457S mutant. The enzyme kinetic investigations revealed that the kinetic parameter k(cat) of the recombinant BO and I402G mutant was 235.8 min(-1) and 6.9 min(-1), respectively, suggesting higher enzyme activity of the recombinant BO. The coordinating amino acids have important significance in maintaining the integrity of active centers and enzyme activities of recombinant BO and its mutants. The enzyme activities of the mutants I402G and C457S are much lower than those of recombinant BO, therefore they are not appropriate for diagnostic purpose. Ru-incorporation facilitates the formation of a new intact active center in C457S mutant, which therefore acquires enzyme activity.

A meta-analysis of soil exoenzyme responses to simulated climate change

NASA Astrophysics Data System (ADS)

Gebhardt, M.; Espinosa, N. J.; Blankinship, J. C.; Gallery, R. E.

2017-12-01

Microorganisms produce extracellular enzymes to decompose plant matter and drive biogeochemical transformations in soils. Climate change factors, such as warming and altered precipitation patterns, can impact enzyme activity through both direct and indirect mechanisms. Although many individual studies have examined how soil exoenzyme activities respond to climate change manipulations, there is disagreement surrounding the direction of these responses. We performed a synthesis of published studies to examine the influence of warming and altered precipitation on microbial exoenzyme activity. We found that warming increased enzyme activity with a more pronounced effect for oxidative relative to hydrolytic enzymes. Reduced precipitation consistently decreased exoenzyme activity. These responses, however, varied by season, biome, and enzyme type. The majority of studies fitting our criteria (e.g., experiments lasting a minimum of one growing season, paired treatments and controls) were located in North America and Europe. Inferences from this analysis therefore exclude many important ecosystems such as hyper-arid, wetlands, and artic systems. Carbon degrading enzyme activities were less sensitive to climate change manipulations when compared to phosphorus and nitrogen degrading enzyme activities. Linking enzyme activity to biogeochemical processes requires concomitant measurements of organic and inorganic carbon pools, mineralogy, nutrients, microbial biomass and community structure, and heterotrophic respiration within individual studies. Furthermore, linking these parameters to climate and environmental factors will require a comprehensive and consistent inclusion of biotic and abiotic variables among researchers and experiments. Globally, soils contain the largest carbon pools. Understanding the impacts of large-scale perturbations on soil enzyme activity will help to constrain predictions on the fate of biogeochemical transformations and improve model projections.

Novel epoxy activated hydrogels for solving lactose intolerance.

PubMed

Elnashar, Magdy M M; Hassan, Mohamed E

2014-01-01

"Lactose intolerance" is a medical problem for almost 70% of the world population. Milk and dairy products contain 5-10% w/v lactose. Hydrolysis of lactose by immobilized lactase is an industrial solution. In this work, we succeeded to increase the lactase loading capacity to more than 3-fold to 36.3 U/g gel using epoxy activated hydrogels compared to 11 U/g gel using aldehyde activated carrageenan. The hydrogel's mode of interaction was proven by FTIR, DSC, and TGA. The high activity of the epoxy group was regarded to its ability to attach to the enzyme's -SH, -NH, and -OH groups, whereas the aldehyde group could only bind to the enzyme's -NH2 group. The optimum conditions for immobilization such as epoxy chain length and enzyme concentration have been studied. Furthermore, the optimum enzyme conditions were also deliberated and showed better stability for the immobilized enzyme and the Michaelis constants, K m and V max, were doubled. Results revealed also that both free and immobilized enzymes reached their maximum rate of lactose conversion after 2 h, albeit, the aldehyde activated hydrogel could only reach 63% of the free enzyme. In brief, the epoxy activated hydrogels are more efficient in immobilizing more enzymes than the aldehyde activated hydrogel.