Effects of benzo[a]pyrene dietary intake to antioxidative enzymes of Lymantria dispar (Lepidoptera: Lymantriidae) larvae from unpolluted and polluted forests.

PubMed

Gavrilović, Anja; Ilijin, Larisa; Mrdaković, Marija; Vlahović, Milena; Mrkonja, Aleksandra; Matić, Dragana; Perić-Mataruga, Vesna

2017-07-01

Anthropogenic activity in industrial development has imposed great threats to the environment and wildlife in the form of persistent organic pollutants. Polycyclic aromatic hydrocarbons (PAH) tend to accumulate in vegetation foliage which is the main food source of polyphagous insect species Lymantria dispar L. Origin and multigenerational adaptation of L. dispar population to environmental challenges strongly condition the enzymes' sensitivity to pollutants. In this study, our aim was to investigate response of the superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) to the chronic dietary exposure of benzo[a]pyrene in the midgut tissues and hemolymph of two L. dispar populations originating from unpolluted and polluted forest habitat. Midgut tissue of the larvae from the polluted forest showed significant increase in SOD, CAT and GST activity, while in unpolluted forest's larvae SOD and CAT showed elevated activities in hemolymph. L. dispar populations adapted to different level of pollution in their environment and expressed distinct tissue-dependent antioxidative enzyme sensitivity to benzo[a]pyrene diet, implying high potential for further elucidation of these enzymes as molecular biomarkers. Copyright © 2017 Elsevier Ltd. All rights reserved.

Coral Carbonic Anhydrases: Regulation by Ocean Acidification.

PubMed

Zoccola, Didier; Innocenti, Alessio; Bertucci, Anthony; Tambutté, Eric; Supuran, Claudiu T; Tambutté, Sylvie

2016-06-03

Global change is a major threat to the oceans, as it implies temperature increase and acidification. Ocean acidification (OA) involving decreasing pH and changes in seawater carbonate chemistry challenges the capacity of corals to form their skeletons. Despite the large number of studies that have investigated how rates of calcification respond to ocean acidification scenarios, comparatively few studies tackle how ocean acidification impacts the physiological mechanisms that drive calcification itself. The aim of our paper was to determine how the carbonic anhydrases, which play a major role in calcification, are potentially regulated by ocean acidification. For this we measured the effect of pH on enzyme activity of two carbonic anhydrase isoforms that have been previously characterized in the scleractinian coral Stylophora pistillata. In addition we looked at gene expression of these enzymes in vivo. For both isoforms, our results show (1) a change in gene expression under OA (2) an effect of OA and temperature on carbonic anhydrase activity. We suggest that temperature increase could counterbalance the effect of OA on enzyme activity. Finally we point out that caution must, thus, be taken when interpreting transcriptomic data on carbonic anhydrases in ocean acidification and temperature stress experiments, as the effect of these stressors on the physiological function of CA will depend both on gene expression and enzyme activity.

Structurally complex and highly active RNA ligases derived from random RNA sequences

NASA Technical Reports Server (NTRS)

Ekland, E. H.; Szostak, J. W.; Bartel, D. P.

1995-01-01

Seven families of RNA ligases, previously isolated from random RNA sequences, fall into three classes on the basis of secondary structure and regiospecificity of ligation. Two of the three classes of ribozymes have been engineered to act as true enzymes, catalyzing the multiple-turnover transformation of substrates into products. The most complex of these ribozymes has a minimal catalytic domain of 93 nucleotides. An optimized version of this ribozyme has a kcat exceeding one per second, a value far greater than that of most natural RNA catalysts and approaching that of comparable protein enzymes. The fact that such a large and complex ligase emerged from a very limited sampling of sequence space implies the existence of a large number of distinct RNA structures of equivalent complexity and activity.

Feasibility test of utilizing Saccharophagus degradans 2-40(T) as the source of crude enzyme for the saccharification of lignocellulose.

PubMed

Jung, Young Hoon; Kim, Hyun Kyung; Song, Du-Sup; Choi, In-Geol; Yang, Taek Ho; Lee, Hee Jong; Seung, Doyoung; Kim, Kyoung Heon

2014-04-01

In the conversion of lignocellulose into high-value products, including fuels and chemicals, the production of cellulase and the enzymatic hydrolysis for producing fermentable sugar are the largest contributors to the cost of production of the final products. The marine bacterium Saccharophagus degradans 2-40(T) can degrade more than ten different complex polysaccharides found in the ocean, including cellulose and xylan. Accordingly, S. degradans has been actively considered as a practical source of crude enzymes needed for the saccharification of lignocellulose to produce ethanol by others including a leading commercial company. However, the overall enzyme system of S. degradans for hydrolyzing cellulose and hemicellulose has not been quantitatively evaluated yet in comparison with commercial enzymes. In this study, the inductions and activities of cellulase and xylanase of cell-free lysate of S. degradans were investigated. The growth of S. degradans cells and the activities of cellulase and xylanase were promoted by adding 2 % of cellulose and xylan mixture (cellulose:xylan = 4:3 in mass ratio) to the aquarium salt medium supplemented with 0.2 % glucose. The specific cellulase activity of the cell-free lysate of S. degradans, as determined by the filter paper activity assay, was approximately 70 times lower than those of commercial cellulases, including Celluclast 1.5 L and Accellerase 1000. These results imply that significant improvement in the cellulase activity of S. degradans is needed for the industrial uses of S. degradans as the enzyme source.

Patterns of functional enzyme activity in fungus farming ambrosia beetles.

PubMed

De Fine Licht, Henrik H; Biedermann, Peter H W

2012-06-06

In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles (Curculionidae: Scolytinae and Platypodinae), wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow) and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i) 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii) four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-β-1,3(4)-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-β-1,4-xylanase activity was exclusively detected in larvae. Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles hardly degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily accessible hemicellulose components of the ray-parenchyma cells in the wood xylem. Furthermore, the detection of xylanolytic enzymes exclusively in larvae (which feed on fungus colonized wood) and not in adults (which feed only on fungi) indicates that only larvae (pre-) digest plant cell wall structures. This implies that in X. saxesenii and likely also in many other ambrosia beetles, adults and larvae do not compete for the same food within their nests - in contrast, larvae increase colony fitness by facilitating enzymatic wood degradation and fungus cultivation.

Prediction and identification of sequences coding for orphan enzymes using genomic and metagenomic neighbours

PubMed Central

Yamada, Takuji; Waller, Alison S; Raes, Jeroen; Zelezniak, Aleksej; Perchat, Nadia; Perret, Alain; Salanoubat, Marcel; Patil, Kiran R; Weissenbach, Jean; Bork, Peer

2012-01-01

Despite the current wealth of sequencing data, one-third of all biochemically characterized metabolic enzymes lack a corresponding gene or protein sequence, and as such can be considered orphan enzymes. They represent a major gap between our molecular and biochemical knowledge, and consequently are not amenable to modern systemic analyses. As 555 of these orphan enzymes have metabolic pathway neighbours, we developed a global framework that utilizes the pathway and (meta)genomic neighbour information to assign candidate sequences to orphan enzymes. For 131 orphan enzymes (37% of those for which (meta)genomic neighbours are available), we associate sequences to them using scoring parameters with an estimated accuracy of 70%, implying functional annotation of 16 345 gene sequences in numerous (meta)genomes. As a case in point, two of these candidate sequences were experimentally validated to encode the predicted activity. In addition, we augmented the currently available genome-scale metabolic models with these new sequence–function associations and were able to expand the models by on average 8%, with a considerable change in the flux connectivity patterns and improved essentiality prediction. PMID:22569339

Single molecule studies reveal new mechanisms for microtubule severing

NASA Astrophysics Data System (ADS)

Ross, Jennifer; Diaz-Valencia, Juan Daniel; Morelli, Margaret; Zhang, Dong; Sharp, David

2011-03-01

Microtubule-severing enzymes are hexameric complexes made from monomeric enzyme subunits that remove tubulin dimers from the microtubule lattice. Severing proteins are known to remodel the cytoskeleton during interphase and mitosis, and are required in proper axon morphology and mammalian bone and cartilage development. We have performed the first single molecule imaging to determine where and how severing enzymes act to cut microtubules. We have focused on the original member of the group, katanin, and the newest member, fidgetin to compare their biophysical activities in vitro. We find that, as expected, severing proteins localize to areas of activity. Interestingly, the association is very brief: they do not stay bound nor do they bind cooperatively at active sites. The association duration changes with the nucleotide content, implying that the state in the catalytic cycle dictates binding affinity with the microtubule. We also discovered that, at lower concentrations, both katanin and fidgetin can depolymerize taxol-stabilized microtubules by removing terminal dimers. These studies reveal the physical regulation schemes to control severing activity in cells, and ultimately regulate cytoskeletal architecture. This work is supported by the March of Dimes Grant #5-FY09-46.

Low-temperature-active and salt-tolerant β-mannanase from a newly isolated Enterobacter sp. strain N18.

PubMed

You, Jia; Liu, Jin-Feng; Yang, Shi-Zhong; Mu, Bo-Zhong

2016-02-01

A low-temperature-active and salt-tolerant β-mannanase produced by a novel mannanase-producer, Enterobacter sp. strain N18, was isolated, purified and then evaluated for its potential application as a gel-breaker in relation to viscosity reduction of guar-based hydraulic fracturing fluids used in oil field. The enzyme could lower the viscosity of guar gum solution by more than 95% within 10 min. The purified β-mannanase with molecular mass of 90 kDa displayed high activity in a broad range of pH and temperature: more than 70% of activity was retained in the pH range of 3.0-8.0 with the optimal pH 7.5, about 50% activity at 20°C with the optimal temperature 50°C. Furthermore, the enzyme retained >70% activity in the presence of 0.5-4.0 M NaCl. These properties implied that the enzyme from strain N18 had potential for serving as a gel-breaker for low temperature oil wells and other industrial fields, where chemical gel breakers were inactive due to low temperature. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Cloning and expression of a novel prolyl endopeptidase from Aspergillus oryzae and its application in beer stabilization.

PubMed

Kang, Chao; Yu, Xiao-Wei; Xu, Yan

2015-02-01

A novel prolyl endopeptidase gene from Aspergillus oryzae was cloned and expressed in Pichia pastoris. Amino acid sequence analysis of the prolyl endopeptidase from Aspergillus oryzae (AO-PEP) showed that this enzyme belongs to a class serine peptide S28 family. Expression, purification and characterization of AO-PEP were analyzed. The optimum pH and temperature were pH 5.0 and 40 °C, respectively. The enzyme was activated and stabilized by metal ion Ca(2+) and inhibited by Zn(2+), Mn(2+), Al(3+), and Cu(2+). The K m and k cat values of the purified enzyme for different substrates were evaluated. The results implied that the recombinant AO-PEP possessed higher affinity for the larger substrate. A fed-batch strategy was developed for the high-cell-density fermentation and the enzyme activity reached 1,130 U/l after cultivation in 7 l fermentor. After addition of AO-PEP during the fermentation phase of beer brewing, demonstrated the potential application of AO-PEP in the non-biological stability of beer, which favor further industrial development of this new enzyme in beer stabilization, due to its reducing operational costs, as well as no beer losses unlike regeneration process and beer lost with regenerated polyvinylpolypyrrolidone system.

Common themes and unique proteins for the uptake and trafficking of nickel, a metal essential for the virulence of Helicobacter pylori

PubMed Central

de Reuse, Hilde; Vinella, Daniel; Cavazza, Christine

2013-01-01

Nickel is a virulence determinant for the human gastric pathogen Helicobacter pylori. Indeed, H. pylori possesses two nickel-enzymes that are essential for in vivo colonization, [NiFe] hydrogenase and urease, an abundant virulence factor that contains 24 nickel ions per active complex. Because of these two enzymes, survival of H. pylori relies on an important supply of nickel, implying a tight control of its distribution and storage. In this review, we will present the pathways of activation of the nickel enzymes as well as original mechanisms found in H. pylori for the uptake, trafficking and distribution of nickel between the two enzymes. These include (i) an outer-membrane nickel uptake system, the FrpB4 TonB-dependent transporter, (ii) overlapping protein complexes and interaction networks involved in nickel trafficking and distribution between urease and hydrogenase and, (iii) Helicobacter specific nickel-binding proteins that are involved in nickel storage and can play the role of metallo-chaperones. Finally, we will discuss the implication of the nickel trafficking partners in virulence and propose them as novel therapeutic targets for treatments against H. pylori infection. PMID:24367767

The link between antioxidant enzymes catalase and glutathione S-transferase and physiological condition of a control population of terrestrial isopod (Porcellio scaber).

PubMed

Jemec, Anita; Lešer, Vladka; Drobne, Damjana

2012-05-01

The aim of this work was to investigate if the activities of catalase and glutathione S-transferase in a control population of terrestrial isopods (Porcellio scaber) are correlated with the physiological condition of the isopods. For this purpose, the activities of these enzymes were analysed in isopods from a stock population and in parallel, the physiological condition of the same specimens was assessed using a histological approach based on epithelial thickness and lipid droplets. We found a correlation between antioxidant enzymes and the physiological condition of the isopods. This implies that these enzymes could be used as predictive indicators of the physiological condition in a stock population before comprehensive toxicological studies are conducted and also in control group after the experiment. When a control group is found to be very heterogeneous in terms of physiological condition, the experiment should be repeated with a larger number of experimental animals. The findings of this study will contribute to more accurate experimental design of toxicity tests when using biomarkers. This should encourage other researchers to increase their effort to know the physiological state of their test organisms. Copyright © 2011 Elsevier Inc. All rights reserved.

Active-Site Residues of Escherichia coli DNA Gyrase Required in Coupling ATP Hydrolysis to DNA Supercoiling and Amino Acid Substitutions Leading to Novobiocin Resistance

PubMed Central

Gross, Christian H.; Parsons, Jonathan D.; Grossman, Trudy H.; Charifson, Paul S.; Bellon, Steven; Jernee, James; Dwyer, Maureen; Chambers, Stephen P.; Markland, William; Botfield, Martyn; Raybuck, Scott A.

2003-01-01

DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP · PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A2B2 gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli. PMID:12604539

Nitric oxide synthase during early embryonic development in silkworm Bombyx mori: Gene expression, enzyme activity, and tissue distribution.

PubMed

Kitta, Ryo; Kuwamoto, Marina; Yamahama, Yumi; Mase, Keisuke; Sawada, Hiroshi

2016-12-01

To elucidate the mechanism for embryonic diapause or the breakdown of diapause in Bombyx mori, we biochemically analyzed nitric oxide synthase (NOS) during the embryogenesis of B. mori. The gene expression and enzyme activity of B. mori NOS (BmNOS) were examined in diapause, non-diapause, and HCl-treated diapause eggs. In the case of HCl-treated diapause eggs, the gene expression and enzyme activity of BmNOS were induced by HCl treatment. However, in the case of diapause and non-diapause eggs during embryogenesis, changes in the BmNOS activity and gene expressions did not coincide except 48-60 h after oviposition in diapause eggs. The results imply that changes in BmNOS activity during the embryogenesis of diapause and non-diapause eggs are regulated not only at the level of transcription but also post-transcription. The distribution and localization of BmNOS were also investigated with an immunohistochemical technique using antibodies against the universal NOS; the localization of BmNOS was observed mainly in the cytoplasm of yolk cells in diapause eggs and HCl-treated diapause eggs. These data suggest that BmNOS has an important role in the early embryonic development of the B. mori. © 2016 Japanese Society of Developmental Biologists.

Crystal structure of the catalytic core domain of the family 6 cellobiohydrolase II, Cel6A, from Humicola insolens, at 1.92 A resolution.

PubMed

Varrot, A; Hastrup, S; Schülein, M; Davies, G J

1999-01-15

The three-dimensional structure of the catalytic core of the family 6 cellobiohydrolase II, Cel6A (CBH II), from Humicola insolens has been determined by X-ray crystallography at a resolution of 1.92 A. The structure was solved by molecular replacement using the homologous Trichoderma reesei CBH II as a search model. The H. insolens enzyme displays a high degree of structural similarity with its T. reesei equivalent. The structure features both O- (alpha-linked mannose) and N-linked glycosylation and a hexa-co-ordinate Mg2+ ion. The active-site residues are located within the enclosed tunnel that is typical for cellobiohydrolase enzymes and which may permit a processive hydrolysis of the cellulose substrate. The close structural similarity between the two enzymes implies that kinetics and chain-end specificity experiments performed on the H. insolens enzyme are likely to be applicable to the homologous T. reesei enzyme. These cast doubt on the description of cellobiohydrolases as exo-enzymes since they demonstrated that Cel6A (CBH II) shows no requirement for non-reducing chain-ends, as had been presumed. There is no crystallographic evidence in the present structure to support a mechanism involving loop opening, yet preliminary modelling experiments suggest that the active-site tunnel of Cel6A (CBH II) is too narrow to permit entry of a fluorescenyl-derivatized substrate, known to be a viable substrate for this enzyme.

Crystal structure of dUTP pyrophosphatase from feline immunodeficiency virus.

PubMed Central

Prasad, G. S.; Stura, E. A.; McRee, D. E.; Laco, G. S.; Hasselkus-Light, C.; Elder, J. H.; Stout, C. D.

1996-01-01

We have determined the crystal structure of dUTP pyrophosphatase (dUTPase) from feline immunodeficiency virus (FIV) at 1.9 A resolution. The structure has been solved by the multiple isomorphous replacement (MIR) method using a P6(3) crystal form. The results show that the enzyme is a trimer of 14.3 kDa subunits with marked structural similarity to E. coli dUTPase. In both enzymes the C-terminal strand of an anti-parallel beta-barrel participates in the beta-sheet of an adjacent subunit to form an interdigitated, biologically functional trimer. In the P6(3) crystal form one trimer packs on the 6(3) screw-axis and another on the threefold axis so that there are two independent monomers per asymmetric unit. A Mg2+ ion is coordinated by three asparate residues on the threefold axis of each trimer. Alignment of 17 viral, prokaryotic, and eukaryotic dUTPase sequences reveals five conserved motifs. Four of these map onto the interface between pairs of subunits, defining a putative active site region; the fifth resides in the C-terminal 16 residues, which is disordered in the crystals. Conserved motifs from all three subunits are required to create a given active site. With respect to viral protein expression, it is particularly interesting that the gene for dUTPase (DU) resides in the middle of the Pol gene, the enzyme cassette of the retroviral genome. Other enzymes encoded in the Pol polyprotein, including protease (PR), reverse transcriptase (RT), and most likely integrase (IN), are dimeric enzymes, which implies that the stoichiometry of expression of active trimeric dUTPase is distinct from the other Pol-encoded enzymes. Additionally, due to structural constraints, it is unlikely that dUTPase can attain an active form prior to cleavage from the polyprotein. PMID:8976551

The Arrhenius Law and Storage of Food in a Freezer

NASA Astrophysics Data System (ADS)

Leenson, I. A.

1999-04-01

This article contains a brief review of some "unconventional" applications of the Arrhenius law. One such example is proposed as a problem concerning the shelf-life of frozen food (Italian pizza) at temperatures ranging from 0 to -18 °C. The effective activation energy (180 kJ/mole) calculated from the information presented by the manufacturer implies that the most probable mechanism of pizza deterioration on storage is enzyme and microbial destruction.

The influence of phase transitions in phosphatidylethanolamine models on the activity of violaxanthin de-epoxidase.

PubMed

Vieler, Astrid; Scheidt, Holger A; Schmidt, Peter; Montag, Cindy; Nowoisky, Janine F; Lohr, Martin; Wilhelm, Christian; Huster, Daniel; Goss, Reimund

2008-04-01

In the present study, the influence of the phospholipid phase state on the activity of the xanthophyll cycle enzyme violaxanthin de-epoxidase (VDE) was analyzed using different phosphatidylethanolamine species as model lipids. By using (31)P NMR spectroscopy, differential scanning calorimetry and temperature dependent enzyme assays, VDE activity could directly be related to the lipid structures the protein is associated with. Our results show that the gel (L beta) to liquid-crystalline (L alpha) phase transition in these single lipid component systems strongly enhances both the solubilization of the xanthophyll cycle pigment violaxanthin in the membrane and the activity of the VDE. This phase transition has a significantly stronger impact on VDE activity than the transition from the L alpha to the inverted hexagonal (HII) phase. Especially at higher temperatures we found increased VDE reaction rates in the presence of the L alpha phase compared to those in the presence of HII phase forming lipids. Our data furthermore imply that the HII phase is better suited to maintain high VDE activities at lower temperatures.

Preparation of a dual-enzyme co-immobilized capillary microreactor and simultaneous screening of multiple enzyme inhibitors by capillary electrophoresis.

PubMed

Lin, Pingtan; Zhao, Shulin; Lu, Xin; Ye, Fanggui; Wang, Hengshan

2013-08-01

A CE method based on a dual-enzyme co-immobilized capillary microreactor was developed for the simultaneous screening of multiple enzyme inhibitors. The capillary microreactor was prepared by co-immobilizing adenosine deaminase and xanthine oxidase on the inner wall at the inlet end of the separation capillary. The enzymes were first immobilized on gold nanoparticles, and the functionalized gold nanoparticles were then assembled on the inner wall at the inlet end of the separation capillary treated with polyethyleneimine. With the developed CE method, the substrates and products were baseline separated within 3 min. The activity of the immobilized enzyme can be directly detected by measuring the peak height of the products. A statistical parameter Z' factor was recommended for evaluation of the accuracy of a drug screening system. In the present study, it was calculated to be larger than 0.5, implying a good accuracy. Finally, screening a small compound library containing two known enzyme inhibitors and 20 natural extracts by the proposed method was demonstrated. The known inhibitors were identified, and some natural extracts were found to be positive for two-enzyme inhibition by the present method. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of recombinant PPi-dependent 6-phosphofructokinases from Methylosinus trichosporium OB3b and Methylobacterium nodulans ORS 2060.

PubMed

Rozova, O N; Khmelenina, V N; Trotsenko, Y A

2012-03-01

The properties of the purified recombinant PPi-dependent 6-phosphofructokinases (PPi-PFKs) from the methanotroph Methylosinus trichosporium OB3b and rhizospheric phytosymbiont Methylobacterium nodulans ORS 2060 were determined. The dependence of activities of PPi-PFK-His(6)-tag from Ms. trichosporium OB3b (6 × 45 kDa) and PPi-PFK from Mb. nodulans ORS 2060 (4 × 43 kDa) on the concentrations of substrates of forward and reverse reactions conformed to Michaelis-Menten kinetics. Besides fructose-6-phosphate, the enzymes also phosphorylated sedoheptulose-7-phosphate. ADP or AMP (1 mM each) inhibited activity of the Ms. trichosporium PPi-PFK but did not affect the activity of the Mb. nodulans enzyme. Preference of PPi-PFKs to fructose-1,6-bisphosphate implied a predominant function of the enzymes in hexose phosphate synthesis in these bacteria. PPi-PFKs from the methylotrophs have low similarity of translated amino acid sequences (17% identity) and belong to different phylogenetic subgroups of type II 6-phosphofructokinases. The relationship of PPi-PFKs with microaerophilic character of Ms. trichosporium OB3b and adaptation of Mb. nodulans ORS 2060 to anaerobic phase of phytosymbiosis are discussed.

3,4-Dihydroxyphenylacetate 2,3-dioxygenase from Pseudomonas aeruginosa: An Fe(II)-containing enzyme with fast turnover

PubMed Central

Kamutira, Philaiwarong; Watthaisong, Pratchaya; Thotsaporn, Kittisak; Tongsook, Chanakan; Juttulapa, Maneerat; Nijvipakul, Sarayut; Chaiyen, Pimchai

2017-01-01

3,4-dihydroxyphenylacetate (DHPA) dioxygenase (DHPAO) from Pseudomonas aeruginosa (PaDHPAO) was overexpressed in Escherichia coli and purified to homogeneity. As the enzyme lost activity over time, a protocol to reactivate and conserve PaDHPAO activity has been developed. Addition of Fe(II), DTT and ascorbic acid or ROS scavenging enzymes (catalase or superoxide dismutase) was required to preserve enzyme stability. Metal content and activity analyses indicated that PaDHPAO uses Fe(II) as a metal cofactor. NMR analysis of the reaction product indicated that PaDHPAO catalyzes the 2,3-extradiol ring-cleavage of DHPA to form 5-carboxymethyl-2-hydroxymuconate semialdehyde (CHMS) which has a molar absorptivity of 32.23 mM-1cm-1 at 380 nm and pH 7.5. Steady-state kinetics under air-saturated conditions at 25°C and pH 7.5 showed a Km for DHPA of 58 ± 8 μM and a kcat of 64 s-1, indicating that the turnover of PaDHPAO is relatively fast compared to other DHPAOs. The pH-rate profile of the PaDHPAO reaction shows a bell-shaped plot that exhibits a maximum activity at pH 7.5 with two pKa values of 6.5 ± 0.1 and 8.9 ± 0.1. Study of the effect of temperature on PaDHPAO activity indicated that the enzyme activity increases as temperature increases up to 55°C. The Arrhenius plot of ln(k’cat) versus the reciprocal of the absolute temperature shows two correlations with a transition temperature at 35°C. Two activation energy values (Ea) above and below the transition temperature were calculated as 42 and 14 kJ/mol, respectively. The data imply that the rate determining steps of the PaDHPAO reaction at temperatures above and below 35°C may be different. Sequence similarity network analysis indicated that PaDHPAO belongs to the enzyme clusters that are largely unexplored. As PaDHPAO has a high turnover number compared to most of the enzymes previously reported, understanding its biochemical and biophysical properties should be useful for future applications in biotechnology. PMID:28158217

Substrate binding interferes with active site conformational dynamics in endoglucanase Cel5A from Thermobifida fusca.

PubMed

Jiang, Xukai; Wang, Yuying; Xu, Limei; Chen, Guanjun; Wang, Lushan

2017-09-09

The role of protein dynamics in enzyme catalysis is one of the most active areas in current enzymological research. Here, using endoglucanase Cel5A from Thermobifida fusca (TfCel5A) as a model, we applied molecular dynamics simulations to explore the dynamic behavior of the enzyme upon substrate binding. The collective motions of the active site revealed that the mechanism of TfCel5A substrate binding can likely be described by the conformational-selection model; however, we observed that the conformations of active site residues changed differently along with substrate binding. Although most active site residues retained their native conformational ensemble, some (Tyr163 and Glu355) generated newly induced conformations, whereas others (Phe162 and Tyr189) exhibited shifts in the equilibration of their conformational distributions. These results showed that TfCel5A substrate binding relied on a hybrid mechanism involving induced fit and conformational selection. Interestingly, we found that TfCel5A active site could only partly rebalance its conformational dynamics upon substrate dissociation within the same simulation time, which implies that the conformational rebalance upon substrate dissociation is likely more difficult than the conformational selection upon substrate binding at least in the view of the time required. Our findings offer new insight into enzyme catalysis and potential applications for future protein engineering. Copyright © 2017 Elsevier Inc. All rights reserved.

Neural ECM proteases in learning and synaptic plasticity.

PubMed

Tsilibary, Effie; Tzinia, Athina; Radenovic, Lidija; Stamenkovic, Vera; Lebitko, Tomasz; Mucha, Mariusz; Pawlak, Robert; Frischknecht, Renato; Kaczmarek, Leszek

2014-01-01

Recent studies implicate extracellular proteases in synaptic plasticity, learning, and memory. The data are especially strong for such serine proteases as thrombin, tissue plasminogen activator, neurotrypsin, and neuropsin as well as matrix metalloproteinases, MMP-9 in particular. The role of those enzymes in the aforementioned phenomena is supported by the experimental results on the expression patterns (at the gene expression and protein and enzymatic activity levels) and functional studies, including knockout mice, specific inhibitors, etc. Counterintuitively, the studies have shown that the extracellular proteolysis is not responsible mainly for an overall degradation of the extracellular matrix (ECM) and loosening perisynaptic structures, but rather allows for releasing signaling molecules from the ECM, transsynaptic proteins, and latent form of growth factors. Notably, there are also indications implying those enzymes in the major neuropsychiatric disorders, probably by contributing to synaptic aberrations underlying such diseases as schizophrenia, bipolar, autism spectrum disorders, and drug addiction.

Aroma Release in Wine Using Co-Immobilized Enzyme Aggregates.

PubMed

Ahumada, Katherine; Martínez-Gil, Ana; Moreno-Simunovic, Yerko; Illanes, Andrés; Wilson, Lorena

2016-11-08

Aroma is a remarkable factor of quality and consumer preference in wine, representing a distinctive feature of the product. Most aromatic compounds in varietals are in the form of glycosidic precursors, which are constituted by a volatile aglycone moiety linked to a glucose residue by an O -glycosidic bond; glucose is often linked to another sugar (arabinose, rhamnose or apiose). The use of soluble β-glycosidases for aroma liberation implies the addition of a precipitating agent to remove it from the product and precludes its reuse after one batch. An attractive option from a technological perspective that will aid in removing such constraints is the use of immobilized glycosidases. Immobilization by aggregation and crosslinking is a simple strategy producing enzyme catalysts of very high specific activity, being an attractive option to conventional immobilization to solid inert supports. The purpose of this work was the evaluation of co-immobilized β-glycosidases crosslinked aggregates produced from the commercial preparation AR2000, which contains the enzymes involved in the release of aromatic terpenes in Muscat wine (α-l-arabinofuranosidase and β-d-glucopyranosidase). To do so, experiments were conducted with co-immobilized crosslinked enzyme aggregates (combi-CLEAs), and with the soluble enzymes, using an experiment without enzyme addition as control. Stability of the enzymes at the conditions of winemaking was assessed and the volatiles composition of wine was determined by SPE-GC-MS. Stability of enzymes in combi-CLEAs was much higher than in soluble form, 80% of the initial activity remaining after 60 days in contact with the wine; at the same conditions, the soluble enzymes had lost 80% of their initial activities after 20 days. Such higher stabilities will allow prolonged use of the enzyme catalyst reducing its impact in the cost of winemaking. Wine treated with combi-CLEAs was the one exhibiting the highest concentration of total terpenes (18% higher than the control) and the highest concentrations of linalool (20% higher), nerol (20% higher) and geraniol (100% higher), which are the most important terpenes in determining Muscat typicity. Co-immobilized enzymes were highly stable at winemaking conditions, so their reutilization is possible and technologically attractive by reducing the impact of enzyme cost on winemaking cost.

Short-term incorporation of organic manures and biofertilizers influences biochemical and microbial characteristics of soils under an annual crop [Turmeric (Curcuma longa L.)].

PubMed

Dinesh, R; Srinivasan, V; Hamza, S; Manjusha, A

2010-06-01

The study was conducted to determine whether short-term incorporation of organic manures and biofertilizers influence biochemical and microbial variables reflecting soil quality. For the study, soils were collected from a field experiment conducted on turmeric (Curcuma longa L.) involving organic nutrient management (ONM), chemical nutrient management (CNM) and integrated nutrient management (INM). The findings revealed that application of organic manures and biofertilizers (ONM and INM) positively influenced microbial biomass C, N mineralization, soil respiration and enzymes activities. Contrarily, greater metabolic quotient levels in CNM indicated a stressed soil microbial community. Principal component analysis indicated the strong relationship between microbial activity and the availability of labile and easily mineralizable organic matter. The findings imply that even short-term incorporation of organic manures and biofertilizers promoted soil microbial and enzyme activities and these parameters are sensitive enough to detect changes in soil quality due to short-term incorporation of biological fertilizers. (c) 2010 Elsevier Ltd. All rights reserved.

Postnatal growth hormone deficiency in growing rats causes marked decline in the activity of spinal cord acetylcholinesterase but not butyrylcholinesterase.

PubMed

Koohestani, Faezeh; Brown, Chester M; Meisami, Esmail

2012-11-01

The effects of growth hormone (GH) deficiency on the developmental changes in the abundance and activity of cholinesterase enzymes were studied in the developing spinal cord (SC) of postnatal rats by measuring the specific activity of acetylcholinesterase (AChE), a marker for cholinergic neurons and their synaptic compartments, and butyrylcholinesterase (BuChE), a marker for glial cells and neurovascular cells. Specific activities of these two enzymes were measured in SC tissue of 21- and 90 day-old (P21, weaning age; P90, young adulthood) GH deficient spontaneous dwarf (SpDwf) mutant rats which lack anterior pituitary and circulating plasma GH, and were compared with SC tissue of normal age-matched control animals. Assays were carried out for AChE and BuChE activity in the presence of their specific chemical inhibitors, BW284C51 and iso-OMPA, respectively. Results revealed that mean AChE activity was markedly and significantly reduced [28% at P21, 49% at P90, (p<0.01)] in the SC of GH deficient rats compared to age-matched controls. GH deficiency had a higher and more significant effect on AChE activity of the older (P90) rats than the younger ones (P21) ones. In contrast, BuChE activity in SC showed no significant changes in GH deficient rats at either of the two ages studied. Results imply that, in the absence of pituitary GH, the postnatal proliferation of cholinergic synapses in the rat SC, a CNS structure, where AChE activity is abundant, is markedly reduced during both the pre- and postweaning periods; more so in the postweaning than preweaning ages. In contrast, the absence of any effects on BuChE activity implies that GH does not affect the development of non-neuronal elements, e.g., glia, as much as the neuronal and synaptic compartments of the developing rat SC. Copyright © 2012 ISDN. Published by Elsevier Ltd. All rights reserved.

Close relationship of a novel Flavobacteriaceae α-amylase with archaeal α-amylases and good potentials for industrial applications

PubMed Central

2014-01-01

Background Bioethanol production from various starchy materials has received much attention in recent years. α-Amylases are key enzymes in the bioconversion process of starchy biomass to biofuels, food or other products. The properties of thermostability, pH stability, and Ca-independency are important in the development of such fermentation process. Results A novel Flavobacteriaceae Sinomicrobium α-amylase (FSA) was identified and characterized from genomic analysis of a novel Flavobacteriaceae species. It is closely related with archaeal α-amylases in the GH13_7 subfamily, but is evolutionary distant with other bacterial α-amylases. Based on the conserved sequence alignment and homology modeling, with minor variation, the Zn2+- and Ca2+-binding sites of FSA were predicated to be the same as those of the archaeal thermophilic α-amylases. The recombinant α-amylase was highly expressed and biochemically characterized. It showed optimum activity at pH 6.0, high enzyme stability at pH 6.0 to 11.0, but weak thermostability. A disulfide bond was introduced by site-directed mutagenesis in domain C and resulted in the apparent improvement of the enzyme activity at high temperature and broad pH range. Moreover, about 50% of the enzyme activity was detected under 100°C condition, whereas no activity was observed for the wild type enzyme. Its thermostability was also enhanced to some extent, with the half-life time increasing from 25 to 55 minutes at 50°C. In addition, after the introduction of the disulfide bond, the protein became a Ca-independent enzyme. Conclusions The improved stability of FSA suggested that the domain C contributes to the overall stability of the enzyme under extreme conditions. In addition, successfully directed modification and special evolutionary status of FSA imply its directional reconstruction potentials for bioethanol production, as well as for other industrial applications. PMID:24485248

Close relationship of a novel Flavobacteriaceae α-amylase with archaeal α-amylases and good potentials for industrial applications.

PubMed

Li, Chunfang; Du, Miaofen; Cheng, Bin; Wang, Lushan; Liu, Xinqiang; Ma, Cuiqing; Yang, Chunyu; Xu, Ping

2014-01-31

Bioethanol production from various starchy materials has received much attention in recent years. α-Amylases are key enzymes in the bioconversion process of starchy biomass to biofuels, food or other products. The properties of thermostability, pH stability, and Ca-independency are important in the development of such fermentation process. A novel Flavobacteriaceae Sinomicrobium α-amylase (FSA) was identified and characterized from genomic analysis of a novel Flavobacteriaceae species. It is closely related with archaeal α-amylases in the GH13_7 subfamily, but is evolutionary distant with other bacterial α-amylases. Based on the conserved sequence alignment and homology modeling, with minor variation, the Zn2+- and Ca2+-binding sites of FSA were predicated to be the same as those of the archaeal thermophilic α-amylases. The recombinant α-amylase was highly expressed and biochemically characterized. It showed optimum activity at pH 6.0, high enzyme stability at pH 6.0 to 11.0, but weak thermostability. A disulfide bond was introduced by site-directed mutagenesis in domain C and resulted in the apparent improvement of the enzyme activity at high temperature and broad pH range. Moreover, about 50% of the enzyme activity was detected under 100°C condition, whereas no activity was observed for the wild type enzyme. Its thermostability was also enhanced to some extent, with the half-life time increasing from 25 to 55 minutes at 50°C. In addition, after the introduction of the disulfide bond, the protein became a Ca-independent enzyme. The improved stability of FSA suggested that the domain C contributes to the overall stability of the enzyme under extreme conditions. In addition, successfully directed modification and special evolutionary status of FSA imply its directional reconstruction potentials for bioethanol production, as well as for other industrial applications.

Structure and catalytic activation of the TRIM23 RING E3 ubiquitin ligase: DAWIDZIAK et al.

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

Dawidziak, Daria M.; Sanchez, Jacint G.; Wagner, Jonathan M.

Tripartite motif (TRIM) proteins comprise a large family of RING-type ubiquitin E3 ligases that regulate important biological processes. An emerging general model is that TRIMs form elongated antiparallel coiled-coil dimers that prevent interaction of the two attendant RING domains. The RING domains themselves bind E2 conjugating enzymes as dimers, implying that an active TRIM ligase requires higher-order oligomerization of the basal coiled-coil dimers. Here, we report crystal structures of the TRIM23 RING domain in isolation and in complex with an E2–ubiquitin conjugate. Our results indicate that TRIM23 enzymatic activity requires RING dimerization, consistent with the general model of TRIM activation.

Kinetic studies and molecular modelling attribute a crucial role in the specificity and stereoselectivity of penicillin acylase to the pair ArgA145-ArgB263.

PubMed

Guncheva, Maya; Ivanov, Ivaylo; Galunsky, Boris; Stambolieva, Nicolina; Kaneti, Jose

2004-06-01

Kinetic experiments with a substrate series of phenylacetyl-arylamides reveal that at least one polar group in the amine moiety is required for the proper orientation of the substrate in the large nucleophile-binding subsite of penicillin acylase of Escherichia coli. Quantum mechanical molecular modelling of enzyme-substrate interactions in the enzyme active site shows that in the case of substrates lacking local symmetry, the productive binding implies two nonsymmetrical arrangements with respect to the two positively charged guanidinium residues of ArgA145 and ArgB263. This indicates a crucial role of the specified arginine pair in the substrate- and stereoselectivity of penicillin acylase.

Insights into the activity change of spore photoproduct lyase induced by mutations at a peripheral glycine residue

NASA Astrophysics Data System (ADS)

Yang, Linlin; Li, Lei

2017-03-01

UV radiation triggers the formation of 5-thyminyl-5,6-dihydrothymine, i.e. the spore photoproduct (SP), in the genomic DNA of bacterial endospores. These SPs, if not repaired in time, may lead to genome instability and cell death. SP is mainly repaired by spore photoproduct lyase (SPL) during spore outgrowth via an unprecedented protein-harbored radical transfer pathway that is composed of at least a cysteine and two tyrosine residues. This mechanism is consistent with the recently solved SPL structure that shows all three residues are located in proximity and thus able to participate in the radical transfer process during the enzyme catalysis. In contrast, an earlier in vivo mutational study identified a glycine to arginine mutation at the position 168 on the B. subtilis SPL that was later found to be > 15 Å away from the enzyme active site. This mutation appears to abolish the enzyme activity because endospores carrying this mutant were sensitive to UV light. To understand the molecular basis for this rendered enzyme activity, we constructed two SPL mutations G168A and G168R, examined their repair of dinucleotide SP TpT, and found that both mutants exhibit reduced enzyme activity. Comparing with the wildtype (WT) SPL enzyme, the G168A mutant slows down the SP TpT repair by 3 4 fold while the G168R mutant by 80 fold. Both mutants exhibit a smaller apparent (DV) kinetic isotope effect (KIE) but a bigger competitive (DV/K) KIE than that by the WT SPL. Moreover, the G168R mutant also produces a large portion of the abortive repair product TpT-SO2-; the formation of which indicates that cysteine 141 is no longer well positioned as the H-donor to the thymine allylic radical intermediate. All these data imply that the mutation at the remote glycine 168 residue alters the enzyme 3D structure, subsequently reducing the SPL activity by changing the positions of the essential amino acids involved in the radical transfer process.

Citrate Accumulation-Related Gene Expression and/or Enzyme Activity Analysis Combined With Metabolomics Provide a Novel Insight for an Orange Mutant

PubMed Central

Guo, Ling-Xia; Shi, Cai-Yun; Liu, Xiao; Ning, Dong-Yuan; Jing, Long-Fei; Yang, Huan; Liu, Yong-Zhong

2016-01-01

‘Hong Anliu’ (HAL, Citrus sinensis cv. Hong Anliu) is a bud mutant of ‘Anliu’ (AL), characterized by a comprehensive metabolite alteration, such as lower accumulation of citrate, high accumulation of lycopene and soluble sugars in fruit juice sacs. Due to carboxylic acid metabolism connects other metabolite biosynthesis and/or catabolism networks, we therefore focused analyzing citrate accumulation-related gene expression profiles and/or enzyme activities, along with metabolic fingerprinting between ‘HAL’ and ‘AL’. Compared with ‘AL’, the transcript levels of citrate biosynthesis- and utilization-related genes and/or the activities of their respective enzymes such as citrate synthase, cytosol aconitase and ATP-citrate lyase were significantly higher in ‘HAL’. Nevertheless, the mitochondrial aconitase activity, the gene transcript levels of proton pumps, including vacuolar H+-ATPase, vacuolar H+-PPase, and the juice sac-predominant p-type proton pump gene (CsPH8) were significantly lower in ‘HAL’. These results implied that ‘HAL’ has higher abilities for citrate biosynthesis and utilization, but lower ability for the citrate uptake into vacuole compared with ‘AL’. Combined with the metabolites-analyzing results, a model was then established and suggested that the reduction in proton pump activity is the key factor for the low citrate accumulation and the comprehensive metabolite alterations as well in ‘HAL’. PMID:27385485

A single mutation in the castor Delta9-18:0-desaturase changes reaction partitioning from desaturation to oxidase chemistry.

PubMed

Guy, Jodie E; Abreu, Isabel A; Moche, Martin; Lindqvist, Ylva; Whittle, Edward; Shanklin, John

2006-11-14

Sequence analysis of the diiron cluster-containing soluble desaturases suggests they are unrelated to other diiron enzymes; however, structural alignment of the core four-helix bundle of desaturases to other diiron enzymes reveals a conserved iron binding motif with similar spacing in all enzymes of this structural class, implying a common evolutionary ancestry. Detailed structural comparison of the castor desaturase with that of a peroxidase, rubrerythrin, shows remarkable conservation of both identity and geometry of residues surrounding the diiron center, with the exception of residue 199. Position 199 is occupied by a threonine in the castor desaturase, but the equivalent position in rubrerythrin contains a glutamic acid. We previously hypothesized that a carboxylate in this location facilitates oxidase chemistry in rubrerythrin by the close apposition of a residue capable of facilitating proton transfer to the activated oxygen (in a hydrophobic cavity adjacent to the diiron center based on the crystal structure of the oxygen-binding mimic azide). Here we report that desaturase mutant T199D binds substrate but its desaturase activity decreases by approximately 2 x 10(3)-fold. However, it shows a >31-fold increase in peroxide-dependent oxidase activity with respect to WT desaturase, as monitored by single-turnover stopped-flow spectrometry. A 2.65-A crystal structure of T199D reveals active-site geometry remarkably similar to that of rubrerythrin, consistent with its enhanced function as an oxidase enzyme. That a single amino acid substitution can switch reactivity from desaturation to oxidation provides experimental support for the hypothesis that the desaturase evolved from an ancestral oxidase enzyme.

A single mutation in the castor Δ9-18:0-desaturase changes reaction partitioning from desaturation to oxidase chemistry

PubMed Central

Guy, Jodie E.; Abreu, Isabel A.; Moche, Martin; Lindqvist, Ylva; Whittle, Edward; Shanklin, John

2006-01-01

Sequence analysis of the diiron cluster-containing soluble desaturases suggests they are unrelated to other diiron enzymes; however, structural alignment of the core four-helix bundle of desaturases to other diiron enzymes reveals a conserved iron binding motif with similar spacing in all enzymes of this structural class, implying a common evolutionary ancestry. Detailed structural comparison of the castor desaturase with that of a peroxidase, rubrerythrin, shows remarkable conservation of both identity and geometry of residues surrounding the diiron center, with the exception of residue 199. Position 199 is occupied by a threonine in the castor desaturase, but the equivalent position in rubrerythrin contains a glutamic acid. We previously hypothesized that a carboxylate in this location facilitates oxidase chemistry in rubrerythrin by the close apposition of a residue capable of facilitating proton transfer to the activated oxygen (in a hydrophobic cavity adjacent to the diiron center based on the crystal structure of the oxygen-binding mimic azide). Here we report that desaturase mutant T199D binds substrate but its desaturase activity decreases by ≈2 × 103-fold. However, it shows a >31-fold increase in peroxide-dependent oxidase activity with respect to WT desaturase, as monitored by single-turnover stopped-flow spectrometry. A 2.65-Å crystal structure of T199D reveals active-site geometry remarkably similar to that of rubrerythrin, consistent with its enhanced function as an oxidase enzyme. That a single amino acid substitution can switch reactivity from desaturation to oxidation provides experimental support for the hypothesis that the desaturase evolved from an ancestral oxidase enzyme. PMID:17088542

The release of alginate lyase from growing Pseudomonas syringae pathovar phaseolicola

NASA Technical Reports Server (NTRS)

Ott, C. M.; Day, D. F.; Koenig, D. W.; Pierson, D. L.

2001-01-01

Pseudomonas syringae pathovar phaseolicola, which produces alginate during stationary growth phase, displayed elevated extracellular alginate lyase activity during both mid-exponential and late-stationary growth phases of batch growth. Intracellular activity remained below 22% of the total activity during exponential growth, suggesting that alginate lyase has an extracellular function for this organism. Extracellular enzyme activity in continuous cultures, grown in either nutrient broth or glucose-simple salts medium, peaked at 60% of the washout rate, although nutrient broth-grown cultures displayed more than twice the activity per gram of cell mass. These results imply that growth rate, nutritional composition, or both initiate a release of alginate lyase from viable P. syringae pv. phaseolicola, which could modify its entrapping biofilm.

Small-Angle X-Ray Scattering Analysis of the Bifunctional Antibiotic Resistance Enzyme Aminoglycoside (6′) Acetyltransferase-Ie/Aminoglycoside (2″) Phosphotransferase-Ia Reveals a Rigid Solution Structure

PubMed Central

Caldwell, Shane J.

2012-01-01

Aminoglycoside (6′) acetyltransferase-Ie/aminoglycoside (2″) phosphotransferase-Ia [AAC(6′)-Ie/APH(2″)-Ia] is one of the most problematic aminoglycoside resistance factors in clinical pathogens, conferring resistance to almost every aminoglycoside antibiotic available to modern medicine. Despite 3 decades of research, our understanding of the structure of this bifunctional enzyme remains limited. We used small-angle X-ray scattering (SAXS) to model the structure of this bifunctional enzyme in solution and to study the impact of substrate binding on the enzyme. It was observed that the enzyme adopts a rigid conformation in solution, where the N-terminal AAC domain is fixed to the C-terminal APH domain and not loosely tethered. The addition of acetyl-coenzyme A, coenzyme A, GDP, guanosine 5′-[β,γ-imido]triphosphate (GMPPNP), and combinations thereof to the protein resulted in only modest changes to the radius of gyration (RG) of the enzyme, which were not consistent with any large changes in enzyme structure upon binding. These results imply some selective advantage to the bifunctional enzyme beyond coexpression as a single polypeptide, likely linked to an improvement in enzymatic properties. We propose that the rigid structure contributes to improved electrostatic steering of aminoglycoside substrates toward the two active sites, which may provide such an advantage. PMID:22290965

Biochemical characterization in Norway spruce (Picea abies) of SABATH methyltransferases that methylate phytohormones.

PubMed

Chaiprasongsuk, Minta; Zhang, Chi; Qian, Ping; Chen, Xinlu; Li, Guanglin; Trigiano, Robert N; Guo, Hong; Chen, Feng

2018-05-01

Indole-3-acetic acid (IAA), gibberellins (GAs), salicylic acid (SA) and jasmonic acid (JA) exist in methyl ester forms in plants in addition to their free acid forms. The enzymes that catalyze methylation of these carboxylic acid phytohormones belong to a same protein family, the SABATH methyltransferases. While the genes encoding these enzymes have been isolated from a small number of flowering plants, little is known about their occurrence and evolution in non-flowering plants. Here, we report the systematic characterization of the SABATH family from Norway spruce (Picea abies), a gymnosperm. The Norway spruce genome contains ten SABATH genes (PaSABATH1-10). Full-length cDNA for each of the ten PaSABATH genes was cloned and expressed in Escherichia coli. Recombinant PaSABATHs were tested for activity with IAA, GA, SA, and JA. Among the ten PaSABATHs, five had activity with one or more of the four substrates. PaSABATH1 and PaSABATH2 had the highest activities with IAA and SA, respectively. PaSABATH4, PaSABATH5 and PaSABATH10 all had JA as a preferred substrate but with notable differences in biochemical properties. The structural basis of PaSABATHs in discriminating various phytohormone substrates was inferred based on structural models of the enzyme-substrate complexes. The phylogeny of PaSABATHs with selected SABATHs from other plants implies that the enzymes methylating IAA are conserved in seed plants whereas the enzymes methylating JA and SA have independent evolution in gymnosperms and angiosperms. Copyright © 2018 Elsevier Ltd. All rights reserved.

Elucidation of the biosynthesis of the di-C-glycosylflavone isoschaftoside, an allelopathic component from Desmodium spp. that inhibits Striga spp. development.

PubMed

Hamilton, Mary L; Kuate, Serge P; Brazier-Hicks, Melissa; Caulfield, John C; Rose, Ruth; Edwards, Robert; Torto, Baldwyn; Pickett, John A; Hooper, Antony M

2012-12-01

Isoschaftoside, an allelopathic di-C-glycosylflavone from Desmodium spp. root exudates, is biosynthesised through sequential glucosylation and arabinosylation of 2-hydroxynaringenin with UDP-glucose and UDP-arabinose. Complete conversion to the flavone requires chemical dehydration implying a dehydratase enzyme has a role in vivo to complete the biosynthesis. The C-glucosyltransferase has been partially characterised and its activity demonstrated in highly purified fractions. Copyright © 2012 Elsevier Ltd. All rights reserved.

Novel Method of Preparation and Activity Research on Arctigenin from Fructus Arctii.

PubMed

Cai, Enbo; Han, Jiahong; Yang, Limin; Zhang, Weiyuan; Zhao, Yan; Chen, Qiulian; Guo, Meng; He, Xinhong

2018-01-01

Arctigenin has many pharmacological activities with clinical significance and is derived from Arctium lappa L. However, the present extraction method is inefficient and does not have meaningful industrial production. A new method to directly prepare arctigenin was established by combining enzyme-assisted extraction and central composite design. Arctigenin's further pharmacological activity was also surveyed in vitro . β-D-Glucosidase, a food-grade enzyme, was added directly to the fruits of A. lappa L. to hydrolyze the arctiin to arctigenin, and the obtained samples were subsequently subjected to ethanol (30%, v/v) extraction. The pharmacological activity of the extraction and arctigenin was determined by inhibiting acetylcholinesterase (AChE) and scavenging nitrite. The factors investigated include the enzyme concentration (0.5%-2.5%), ultrasound time (10 min -3 0 min), and extraction temperature (30°C-50°C). From the analysis of the results by Design-Expert (V8.0.6), the optimal extraction conditions were obtained: enzyme concentration (1.4%), ultrasound time (25 min), and extraction temperature (45°C). The highest yield of arctigenin, obtained under the optimal conditions was 6.39%, representing an increase of 28.15% compared to the reference extraction without enzyme processing. The IC 50 values of the extraction and arctigenin, respectively, for inhibiting AChE were 0.572 mg/ml and 0.462 mg/ml, and those for nitrite-scavenging were 34.571 mg/ml and 17.49 mg/ml. The results demonstrate that using an enzyme directly in the production is an effective means for extracting arctigenin from Fructus arctii. The extraction has the activities of inhibiting AChE and scavenging nitrite, probably because there has arctigenin in it. It is implied that the extraction and arctigenin could contribute to human health in clinical applications. The new method of adding enzyme directly to the preparation of arctigenin was carried out instead of preparing arctigenin by two-step methodThree factors affecting the efficiency of preparation were analyzed and discussed include the enzyme concentration, ultrasound time, and extraction temperature by central composite designThis new method of preparing arctigenin improved the yield significantly than other methodsArctigenin has remarkable pharmacological activities of inhibiting acetylcholinesterase and scavenging nitrite. Abbreviations used: AChE: Acetylcholinesterase, CCD: Central composite design, TCM: Traditional Chinese medicines, AD.

Novel Method of Preparation and Activity Research on Arctigenin from Fructus Arctii

PubMed Central

Cai, Enbo; Han, Jiahong; Yang, Limin; Zhang, Weiyuan; Zhao, Yan; Chen, Qiulian; Guo, Meng; He, Xinhong

2018-01-01

Background: Arctigenin has many pharmacological activities with clinical significance and is derived from Arctium lappa L. However, the present extraction method is inefficient and does not have meaningful industrial production. Objective: A new method to directly prepare arctigenin was established by combining enzyme-assisted extraction and central composite design. Arctigenin's further pharmacological activity was also surveyed in vitro. Materials and Methods: β-D-Glucosidase, a food-grade enzyme, was added directly to the fruits of A. lappa L. to hydrolyze the arctiin to arctigenin, and the obtained samples were subsequently subjected to ethanol (30%, v/v) extraction. The pharmacological activity of the extraction and arctigenin was determined by inhibiting acetylcholinesterase (AChE) and scavenging nitrite. Results: The factors investigated include the enzyme concentration (0.5%–2.5%), ultrasound time (10 min−3 0 min), and extraction temperature (30°C–50°C). From the analysis of the results by Design-Expert (V8.0.6), the optimal extraction conditions were obtained: enzyme concentration (1.4%), ultrasound time (25 min), and extraction temperature (45°C). The highest yield of arctigenin, obtained under the optimal conditions was 6.39%, representing an increase of 28.15% compared to the reference extraction without enzyme processing. The IC50 values of the extraction and arctigenin, respectively, for inhibiting AChE were 0.572 mg/ml and 0.462 mg/ml, and those for nitrite-scavenging were 34.571 mg/ml and 17.49 mg/ml. Conclusions: The results demonstrate that using an enzyme directly in the production is an effective means for extracting arctigenin from Fructus arctii. The extraction has the activities of inhibiting AChE and scavenging nitrite, probably because there has arctigenin in it. It is implied that the extraction and arctigenin could contribute to human health in clinical applications. SUMMARY The new method of adding enzyme directly to the preparation of arctigenin was carried out instead of preparing arctigenin by two-step methodThree factors affecting the efficiency of preparation were analyzed and discussed include the enzyme concentration, ultrasound time, and extraction temperature by central composite designThis new method of preparing arctigenin improved the yield significantly than other methodsArctigenin has remarkable pharmacological activities of inhibiting acetylcholinesterase and scavenging nitrite. Abbreviations used: AChE: Acetylcholinesterase, CCD: Central composite design, TCM: Traditional Chinese medicines, AD: PMID:29576707

A route from darkness to light: emergence and evolution of luciferase activity in AMP-CoA-ligases inferred from a mealworm luciferase-like enzyme.

PubMed

Viviani, V R; Prado, R A; Neves, D R; Kato, D; Barbosa, J A

2013-06-11

The origin of luciferases and of bioluminescence is enigmatic. In beetles, luciferases seem to have evolved from AMP-CoA-ligases. How the new oxygenase luminogenic function originated from AMP-ligases leading to luciferases is one of the most challenging mysteries of bioluminescence. Comparison of the cloned luciferase-like enzyme from the nonluminescent Zophobas morio mealworm and beetle luciferases showed that the oxygenase activity may have emerged as a stereoselective oxidative drift with d-luciferin, a substrate that cannot be easily thioesterified to CoA as in the case of the l-isomer. While the overall kcat displayed by beetle luciferases is orders of magnitude greater than that of the luciferase-like enzyme, the respective oxidation rates and quantum yields of bioluminescence are roughly similar, suggesting that the rate constant of the AMP-ligase activity exerted on the new d-luciferin substrate in beetle protoluciferases was the main enzymatic property that suffered optimization during the evolution of luciferases. The luciferase-like enzyme and luciferases boost the rate of luciferyl-adenylate chemiluminescent oxidation by factors of 10(6) and 10(7), respectively, as compared to the substrate spontaneous oxidation in buffer. A similar enhancement of luciferyl-adenylate chemiluminescence is provided by nucleophilic aprotic solvents, implying that the peptide bonds in the luciferin binding site of beetle luciferase could provide a similar catalytically favorable environment. These data suggest that the luciferase-like enzyme and other similar AMP-ligases are potential alternative oxygenases. Site-directed mutagenesis studies of the luciferase-like enzyme and the red light-producing luciferase of Phrixotrix hirtus railroadworm confirm here a critical role for T/S345 in luciferase function. Mutations such as I327T/S in the luciferase-like enzyme, which simultaneously increases luciferase activity and promotes blue shifts in the emission spectrum, could have been critical for evolving functional bioluminescence from red-emitting protoluciferases. Through the combination of I327T/S mutations and N-terminal fusion, the luminescence activity of this enzyme was increased to visible levels, with the development of a totally new orange-emitting luciferase. These results open the possibility of engineering luciferase activity in a set of AMP-CoA-ligases.

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.

Soil microbial carbon utilization, enzyme activities and nutrient availability responses to Bidens pilosa and a non-invasive congener under different irradiances.

PubMed

Wei, Hui; Yan, Wenbin; Quan, Guoming; Zhang, Jiaen; Liang, Kaiming

2017-09-12

Two Bidens species (Bidens pilosa and B. bipinnata) that originate from America have been introduced widely in pan-tropics, with the former regarded as a noxious invasive weed whereas the latter naturalized as a plant resource. Whether the two species exhibit different effects on the belowground system remains rarely studied. This study was conducted to investigate soil microbial carbon (C) utilization, enzyme activities and available nitrogen, phosphorus and potassium contents under the two species in a subtropical garden soil of southern China under different levels of light intensity. Results showed that the microbial C utilization and enzyme activities were not significantly different under the two species, implying that the strong invasiveness of B. pilosa could not be due to the plant-soil microbe interactions, at least plant-induced alterations of microbial community function to utilize C substrates. Alternatively, available soil nitrogen and potassium contents were significantly higher under B. pilosa than under B. bipinnata in full sun, indicating that the strong invasiveness of B. pilosa could result from rapid nutrient mobilizations by B. pilosa. However, the differences turned non-significant as light intensity decreased, suggesting that light availability could substantially alter the plant effects on soil nutrient mobilizations.

Interactive response of photosynthetic characteristics in Haloxylon ammodendron and Hedysarum scoparium exposed to soil water and air vapor pressure deficits.

PubMed

Gong, Chunmei; Wang, Jiajia; Hu, Congxia; Wang, Junhui; Ning, Pengbo; Bai, Juan

2015-08-01

C4 plants possess better drought tolerance than C3 plants. However, Hedysarum scoparium, a C3 species, is dominant and widely distributed in the desert areas of northwestern China due to its strong drought tolerance. This study compared it with Haloxylon ammodendron, a C4 species, regarding the interactive effects of drought stress and different leaf-air vapor pressure deficits. Variables of interest included gas exchange, the activity levels of key C4 photosynthetic enzymes, and cellular anatomy. In both species, gas exchange parameters were more sensitive to high vapor pressure deficit than to strong water stress, and the net CO2 assimilation rate (An) was enhanced as vapor pressure deficits increased. A close relationship between An and stomatal conductance (gs) suggested that the species shared a similar response mechanism. In H. ammodendron, the activity levels of key C4 enzymes were higher, including those of phosphoenolpyruvate carboxylase (PEPC) and nicotinamide adenine dinucleotide phosphate-malate enzyme (NADP-ME), whereas in H. scoparium, the activity level of nicotinamide adenine dinucleotide-malate enzyme (NAD-ME) was higher. Meanwhile, H. scoparium utilized adaptive structural features, including a larger relative vessel area and a shorter distance from vein to stomata, which facilitated the movement of water. These findings implied that some C4 biochemical pathways were present in H. scoparium to respond to environmental challenges. Copyright © 2015. Published by Elsevier B.V.

Indigofera suffruticosa Mill extracts up-regulate the expression of the π class of glutathione S-transferase and NAD(P)H: quinone oxidoreductase 1 in rat Clone 9 liver cells.

PubMed

Chen, Chun-Chieh; Liu, Chin-San; Li, Chien-Chun; Tsai, Chia-Wen; Yao, Hsien-Tsung; Liu, Te-Chung; Chen, Haw-Wen; Chen, Pei-Yin; Wu, Yu-Ling; Lii, Chong-Kuei; Liu, Kai-Li

2013-09-01

Because induction of phase II detoxification enzyme is important for chemoprevention, we study the effects of Indigofera suffruticosa Mill, a medicinal herb, on the expression of π class of glutathione S-transferase (GSTP) and NAD(P)H: quinone oxidoreductase 1 (NQO1) in rat Clone 9 liver cells. Both water and ethanolic extracts of I. suffruticosa significantly increased the expression and enzyme activities of GSTP and NQO1. I. suffruticosa extracts up-regulated GSTP promoter activity and the binding affinity of nuclear factor erythroid 2-related factor 2 (Nrf2) with the GSTP enhancer I oligonucleotide. Moreover, I. suffruticosa extracts increased nuclear Nrf2 accumulation as well as ARE transcriptional activity. The level of phospho-ERK was augmented by I. suffruticosa extracts, and the ERK inhibitor PD98059 abolished the I. suffruticosa extract-induced ERK activation and GSTP and NQO-1 expression. Moreover, I. suffruticosa extracts, especially the ethanolic extract increased the glutathione level in mouse liver and red blood cells as well as Clone 9 liver cells. The efficacy of I. suffruticosa extracts in induction of phase II detoxification enzymes and glutathione content implies that I. suffruticosa could be considered as a potential chemopreventive agent. Copyright © 2013 Elsevier Ltd. All rights reserved.

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.

From molecular engineering to process engineering: development of high-throughput screening methods in enzyme directed evolution.

PubMed

Ye, Lidan; Yang, Chengcheng; Yu, Hongwei

2018-01-01

With increasing concerns in sustainable development, biocatalysis has been recognized as a competitive alternative to traditional chemical routes in the past decades. As nature's biocatalysts, enzymes are able to catalyze a broad range of chemical transformations, not only with mild reaction conditions but also with high activity and selectivity. However, the insufficient activity or enantioselectivity of natural enzymes toward non-natural substrates limits their industrial application, while directed evolution provides a potent solution to this problem, thanks to its independence on detailed knowledge about the relationship between sequence, structure, and mechanism/function of the enzymes. A proper high-throughput screening (HTS) method is the key to successful and efficient directed evolution. In recent years, huge varieties of HTS methods have been developed for rapid evaluation of mutant libraries, ranging from in vitro screening to in vivo selection, from indicator addition to multi-enzyme system construction, and from plate screening to computation- or machine-assisted screening. Recently, there is a tendency to integrate directed evolution with metabolic engineering in biosynthesis, using metabolites as HTS indicators, which implies that directed evolution has transformed from molecular engineering to process engineering. This paper aims to provide an overview of HTS methods categorized based on the reaction principles or types by summarizing related studies published in recent years including the work from our group, to discuss assay design strategies and typical examples of HTS methods, and to share our understanding on HTS method development for directed evolution of enzymes involved in specific catalytic reactions or metabolic pathways.

Conservation and Role of Electrostatics in Thymidylate Synthase.

PubMed

Garg, Divita; Skouloubris, Stephane; Briffotaux, Julien; Myllykallio, Hannu; Wade, Rebecca C

2015-11-27

Conservation of function across families of orthologous enzymes is generally accompanied by conservation of their active site electrostatic potentials. To study the electrostatic conservation in the highly conserved essential enzyme, thymidylate synthase (TS), we conducted a systematic species-based comparison of the electrostatic potential in the vicinity of its active site. Whereas the electrostatics of the active site of TS are generally well conserved, the TSs from minimal organisms do not conform to the overall trend. Since the genomes of minimal organisms have a high thymidine content compared to other organisms, the observation of non-conserved electrostatics was surprising. Analysis of the symbiotic relationship between minimal organisms and their hosts, and the genetic completeness of the thymidine synthesis pathway suggested that TS from the minimal organism Wigglesworthia glossinidia (W.g.b.) must be active. Four residues in the vicinity of the active site of Escherichia coli TS were mutated individually and simultaneously to mimic the electrostatics of W.g.b TS. The measured activities of the E. coli TS mutants imply that conservation of electrostatics in the region of the active site is important for the activity of TS, and suggest that the W.g.b. TS has the minimal activity necessary to support replication of its reduced genome.

Widening and Elaboration of Consecutive Research into Therapeutic Antioxidant Enzyme Derivatives

PubMed Central

2016-01-01

Undiminishing actuality of enzyme modification for therapeutic purposes has been confirmed by application of modified enzymes in clinical practice and numerous research data on them. Intravenous injection of the superoxide dismutase-chondroitin sulfate-catalase (SOD-CHS-CAT) conjugate in preventive and medicative regimes in rats with endotoxin shock induced with a lipopolysaccharide bolus has demonstrated that antioxidant agents not only effectively prevent damage caused by oxidative stress (as believed previously) but also can be used for antioxidative stress therapy. The results obtained emphasize the importance of investigation into the pathogenesis of vascular damage and the role of oxidative stress in it. The effects of intravenous medicative injection of SOD-CHS-CAT in a rat model of endotoxin shock have demonstrated a variety in the activity of this conjugate in addition to prevention of NO conversion in peroxynitrite upon interaction with O2 ∙− superoxide radical. Together with the literature data, these findings offer a prospect for the study of NO-independent therapeutic effects of SOD-CHS-CAT, implying the importance of a better insight into the mechanisms of the conjugate activity in modeled cardiovascular damage involving vasoactive agents other than NO. PMID:27148430

Parvoviruses Cause Nuclear Envelope Breakdown by Activating Key Enzymes of Mitosis

PubMed Central

Porwal, Manvi; Cohen, Sarah; Snoussi, Kenza; Popa-Wagner, Ruth; Anderson, Fenja; Dugot-Senant, Nathalie; Wodrich, Harald; Dinsart, Christiane; Kleinschmidt, Jürgen A.; Panté, Nelly; Kann, Michael

2013-01-01

Disassembly of the nuclear lamina is essential in mitosis and apoptosis requiring multiple coordinated enzymatic activities in nucleus and cytoplasm. Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases. Here we used the ability of parvoviruses to induce nuclear membrane breakdown to understand the triggers of key mitotic enzymes. Nuclear envelope disintegration was shown upon infection, microinjection but also upon their application to permeabilized cells. The latter technique also showed that nuclear envelope disintegration was independent upon soluble cytoplasmic factors. Using time-lapse microscopy, we observed that nuclear disassembly exhibited mitosis-like kinetics and occurred suddenly, implying a catastrophic event irrespective of cell- or type of parvovirus used. Analyzing the order of the processes allowed us to propose a model starting with direct binding of parvoviruses to distinct proteins of the nuclear pore causing structural rearrangement of the parvoviruses. The resulting exposure of domains comprising amphipathic helices was required for nuclear envelope disintegration, which comprised disruption of inner and outer nuclear membrane as shown by electron microscopy. Consistent with Ca++ efflux from the lumen between inner and outer nuclear membrane we found that Ca++ was essential for nuclear disassembly by activating PKC. PKC activation then triggered activation of cdk-2, which became further activated by caspase-3. Collectively our study shows a unique interaction of a virus with the nuclear envelope, provides evidence that a nuclear pool of executing enzymes is sufficient for nuclear disassembly in quiescent cells, and demonstrates that nuclear disassembly can be uncoupled from initial phases of mitosis. PMID:24204256

Inhibition of Actinobacillus actinomycetemcomitans leukotoxicity by bacteria from the subgingival flora.

PubMed

Johansson, A; Hänström, L; Kalfas, S

2000-08-01

Actinobacillus actinomycetemcomitans produces a pore-forming leukotoxin that lyses human polymorphonuclear leukocytes and monocytes. Certain proteolytic bacteria may coexist with A. actinomycetemcomitans in periodontal pockets. We aimed therefore to examine whether oral bacteria can modify the leukotoxicity of A. actinomycetemcomitans. A total of 55 strains representing 45 bacterial species of the subgingival flora were tested. Each strain was incubated with the highly toxic strain of A. actinomycetemcomitans HK 1519 and the leukotoxic activity of the suspension against human polymorphonuclear leukocytes was determined from the activity of the lactate dehydrogenase released upon lysis of the leukocytes. Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Prevotella melaninogenica and Prevotella loeschii inhibited the leukotoxicity of A. actinomycetemcomitans cells as well as the activity of leukotoxin purified from the same strain. The bacterial strains without the ability to block leukotoxic activity also failed to destroy pure leukotoxin even after 5 h of incubation. The proteolytic degradation of leukotoxin by P. gingivalis was mainly dependent on the activity of the enzymes R- and K-gingipains. P. intermedia and P. nigrescens also degraded the leukotoxin by enzymes. The results imply a role of the periodontal microflora in modifying the virulence of A. actinomycetemcomitans by destroying its leukotoxin.

Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity

PubMed Central

Yang, Guang; Yao, Hua; Mozzicafreddo, Matteo; Ballarini, Patrizia; Pucciarelli, Sandra

2017-01-01

ABSTRACT The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii (EfAmy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, EfAmy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active EfAmy with improved thermostability and catalytic efficiency at low temperatures. We engineered two EfAmy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of EfAmy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model. IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the psychrophilic Antarctic ciliate Euplotes focardii (named EfAmy) is a cold-adapted enzyme with optimal catalytic activity in an alkaline environment. These unique features distinguish it from most α-amylases characterized so far. In this work, we engineered a novel EfAmy with improved thermostability, substrate binding affinity, and catalytic efficiency to various extents, without impacting its pH preference. These characteristics can be considered important properties for use in the food, detergent, and textile industries and in other industrial applications. The enzyme engineering strategy developed in this study may also provide useful knowledge for future optimization of molecules to be used in particular industrial applications. PMID:28455329

Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity.

PubMed

Yang, Guang; Yao, Hua; Mozzicafreddo, Matteo; Ballarini, Patrizia; Pucciarelli, Sandra; Miceli, Cristina

2017-07-01

The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii ( Ef Amy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, Ef Amy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active Ef Amy with improved thermostability and catalytic efficiency at low temperatures. We engineered two Ef Amy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of Ef Amy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model. IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the psychrophilic Antarctic ciliate Euplotes focardii (named Ef Amy) is a cold-adapted enzyme with optimal catalytic activity in an alkaline environment. These unique features distinguish it from most α-amylases characterized so far. In this work, we engineered a novel Ef Amy with improved thermostability, substrate binding affinity, and catalytic efficiency to various extents, without impacting its pH preference. These characteristics can be considered important properties for use in the food, detergent, and textile industries and in other industrial applications. The enzyme engineering strategy developed in this study may also provide useful knowledge for future optimization of molecules to be used in particular industrial applications. Copyright © 2017 Yang et al.

Short communication: Assessing antihypertensive activity in native and model Queso Fresco cheeses.

PubMed

Paul, M; Van Hekken, D L

2011-05-01

Hispanic-style cheeses are one of the fastest growing varieties in the United States, making up approximately 2% of the total cheese production in this country. Queso Fresco is one of most popular Hispanic-style cheeses. Protein extracts from several varieties of Mexican Queso Fresco and model Queso Fresco were analyzed for potential antihypertensive activity. Many Quesos Frescos obtained from Mexico are made from raw milk and therefore the native microflora is included in the cheese-making process. Model Queso Fresco samples were made from pasteurized milk and did not utilize starter cultures. Water-soluble protein extracts from 6 Mexican Quesos Frescos and 12 model cheeses were obtained and assayed for their ability to inhibit angiotensin-converting enzyme, implying potential as foods that can help to lower blood pressure. All model cheeses displayed antihypertensive activity, but mainly after 8 wk of aging when they were no longer consumable, whereas the Mexican samples did display some angiotensin-converting enzyme inhibitory action after minimal aging. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Characterization of a novel debranching enzyme from Nostoc punctiforme possessing a high specificity for long branched chains

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

Choi, Ji-Hye; Lee, Heeseob; Kim, Young-Wan

2009-01-09

A novel debranching enzyme from Nostoc punctiforme PCC73102 (NPDE) exhibits hydrolysis activity toward both {alpha}-(1,6)- and {alpha}-(1,4)-glucosidic linkages. The action patterns of NPDE revealed that branched chains are released first, and the resulting maltooligosaccharides are then hydrolyzed. Analysis of the reaction with maltooligosaccharide substrates labeled with {sup 14}C-glucose at the reducing end shows that NPDE specifically liberates glucose from the reducing end. Kinetic analyses showed that the hydrolytic activity of NPDE is greatly affected by the length of the substrate. The catalytic efficiency of NPDE increased considerably upon using substrates that can occupy at least eight glycone subsites such asmore » maltononaose and maltooctaosyl-{alpha}-(1,6)-{beta}-cyclodextrin. These results imply that NPDE has a unique subsite structure consisting of -8 to +1 subsites. Given its unique subsite structure, side chains shorter than maltooctaose in amylopectin were resistant to hydrolysis by NPDE, and the population of longer side chains was reduced.« less

Bovine brain ribonuclease is the functional homolog of human ribonuclease 1.

PubMed

Eller, Chelcie H; Lomax, Jo E; Raines, Ronald T

2014-09-19

Mounting evidence suggests that human pancreatic ribonuclease (RNase 1) plays important roles in vivo, ranging from regulating blood clotting and inflammation to directly counteracting tumorigenic cells. Understanding these putative roles has been pursued with continual comparisons of human RNase 1 to bovine RNase A, an enzyme that appears to function primarily in the ruminant gut. Our results imply a different physiology for human RNase 1. We demonstrate distinct functional differences between human RNase 1 and bovine RNase A. Moreover, we characterize another RNase 1 homolog, bovine brain ribonuclease, and find pronounced similarities between that enzyme and human RNase 1. We report that human RNase 1 and bovine brain ribonuclease share high catalytic activity against double-stranded RNA substrates, a rare quality among ribonucleases. Both human RNase 1 and bovine brain RNase are readily endocytosed by mammalian cells, aided by tight interactions with cell surface glycans. Finally, we show that both human RNase 1 and bovine brain RNase are secreted from endothelial cells in a regulated manner, implying a potential role in vascular homeostasis. Our results suggest that brain ribonuclease, not RNase A, is the true bovine homolog of human RNase 1, and provide fundamental insight into the ancestral roles and functional adaptations of RNase 1 in mammals. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

AMP-activated protein kinase is physiologically regulated by inositol polyphosphate multikinase

PubMed Central

Bang, Sookhee; Kim, Seyun; Dailey, Megan J.; Chen, Yong; Moran, Timothy H.; Snyder, Solomon H.; Kim, Sangwon F.

2012-01-01

The AMP-activated kinase (AMPK) senses the energy status of cells and regulates fuel availability, whereas hypothalamic AMPK regulates food intake. We report that inositol polyphosphate multikinase (IPMK) regulates glucose signaling to AMPK in a pathway whereby glucose activates phosphorylation of IPMK at tyrosine 174 enabling the enzyme to bind to AMPK and regulate its activation. Thus, refeeding fasted mice rapidly and markedly stimulates transcriptional enhancement of IPMK expression while down-regulating AMPK. Also, AMPK is up-regulated in mice with genetic depletion of hypothalamic IPMK. IPMK physiologically binds AMPK, with binding enhanced by glucose treatment. Regulation by glucose of phospho-AMPK in hypothalamic cell lines is prevented by blocking AMPK-IPMK binding. These findings imply that IPMK inhibitors will be beneficial in treating obesity and diabetes. PMID:22203993

Clinical and biochemical studies support smokeless tobacco’s carcinogenic potential in the human oral cavity

PubMed Central

Mallery, Susan R.; Tong, Meng; Michaels, Gregory C.; Kiyani, Amber R.; Hecht, Stephen S.

2014-01-01

In 2007, International Agency for Cancer Research presented compelling evidence that linked smokeless tobacco use to the development of human oral cancer. While these findings imply vigorous local carcinogen metabolism, little is known regarding levels and distribution of Phase I, II and drug egress enzymes in human oral mucosa. In the study presented here, we integrated clinical data, imaging and histopathologic analyses of an oral squamous cell carcinoma that arose at the site of smokeless tobacco quid placement in a patient. Immunoblot and immunohistochemical (IHC) analyses were employed to identify tumor and normal human oral mucosal smokeless tobacco-associated metabolic activation and detoxification enzymes. Human oral epithelium contains every known Phase I enzyme associated with nitrosamine oxidative bioactivation with ~2 fold inter-donor differences in protein levels. Previous studies have confirmed ~3.5 fold inter-donor variations in intraepithelial Phase II enzymes. Unlike the superficially located enzymes in non-replicating esophageal surface epithelium, IHC studies confirmed oral mucosal nitrosamine metabolizing enzymes reside in the basilar and suprabasilar region which notably is the site of ongoing keratinocyte DNA replication. Clearly, variations in product composition, nitrosamine metabolism and exposure duration will modulate clinical outcomes. The data presented here form a coherent picture consistent with the abundant experimental data that links tobacco-specific nitrosamines to human oral cancer. PMID:24265177

Adenine phosphoribosyltransferase from Sulfolobus solfataricus is an enzyme with unusual kinetic properties and a crystal structure that suggests it evolved from a 6-oxopurine phosphoribosyltransferase.

PubMed

Jensen, Kaj Frank; Hansen, Michael Riis; Jensen, Kristine Steen; Christoffersen, Stig; Poulsen, Jens-Christian Navarro; Mølgaard, Anne; Kadziola, Anders

2015-04-14

The adenine phosphoribosyltransferase (APRTase) encoded by the open reading frame SSO2342 of Sulfolobus solfataricus P2 was subjected to crystallographic, kinetic, and ligand binding analyses. The enzyme forms dimers in solution and in the crystals, and binds one molecule of the reactants 5-phosphoribosyl-α-1-pyrophosphate (PRPP) and adenine or the product adenosine monophosphate (AMP) or the inhibitor adenosine diphosphate (ADP) in each active site. The individual subunit adopts an overall structure that resembles a 6-oxopurine phosphoribosyltransferase (PRTase) more than known APRTases implying that APRT functionality in Crenarchaeotae has its evolutionary origin in this family of PRTases. Only the N-terminal two-thirds of the polypeptide chain folds as a traditional type I PRTase with a five-stranded β-sheet surrounded by helices. The C-terminal third adopts an unusual three-helix bundle structure that together with the nucleobase-binding loop undergoes a conformational change upon binding of adenine and phosphate resulting in a slight contraction of the active site. The inhibitor ADP binds like the product AMP with both the α- and β-phosphates occupying the 5'-phosphoribosyl binding site. The enzyme shows activity over a wide pH range, and the kinetic and ligand binding properties depend on both pH and the presence/absence of phosphate in the buffers. A slow hydrolysis of PRPP to ribose 5-phosphate and pyrophosphate, catalyzed by the enzyme, may be facilitated by elements in the C-terminal three-helix bundle part of the protein.

Litter type affects the activity of aerobic decomposers in a boreal peatland more than site nutrient and water table regimes

NASA Astrophysics Data System (ADS)

Straková, P.; Niemi, R. M.; Freeman, C.; Peltoniemi, K.; Toberman, H.; Heiskanen, I.; Fritze, H.; Laiho, R.

2011-09-01

Peatlands are carbon (C) storage ecosystems sustained by a high water table (WT). High WT creates anoxic conditions that suppress the activity of aerobic decomposers and provide conditions for peat accumulation. Peatland function can be dramatically affected by WT drawdown caused by climate and/or land-use change. Aerobic decomposers are directly affected by WT drawdown through environmental factors such as increased oxygenation and nutrient availability. Additionally, they are indirectly affected via changes in plant community composition and litter quality. We studied the relative importance of direct and indirect effects of WT drawdown on aerobic decomposer activity in plant litter at two stages of decomposition (incubated in the field for 1 or 2 years). We did this by profiling 11 extracellular enzymes involved in the mineralization of organic C, nitrogen (N), phosphorus (P) and sulphur. Our study sites represented a three-stage chronosequence from pristine to short-term (years) and long-term (decades) WT drawdown conditions under two nutrient regimes (bog and fen). The litter types included reflected the prevalent vegetation: Sphagnum mosses, graminoids, shrubs and trees. Litter type was the main factor shaping microbial activity patterns and explained about 30 % of the variation in enzyme activities and activity allocation. Overall, enzyme activities were higher in vascular plant litters compared to Sphagnum litters, and the allocation of enzyme activities towards C or nutrient acquisition was related to the initial litter quality (chemical composition). Direct effects of WT regime, site nutrient regime and litter decomposition stage (length of incubation period) summed to only about 40 % of the litter type effect. WT regime alone explained about 5 % of the variation in enzyme activities and activity allocation. Generally, enzyme activity increased following the long-term WT drawdown and the activity allocation turned from P and N acquisition towards C acquisition. This caused an increase in the rate of litter decomposition. The effects of the short-term WT drawdown were minor compared to those of the long-term WT drawdown: e.g., the increase in the activity of C-acquiring enzymes was up to 120 % (bog) or 320 % (fen) higher after the long-term WT drawdown compared to the short-term WT drawdown. In general, the patterns of microbial activity as well as their responses to WT drawdown depended on peatland type: e.g., the shift in activity allocation to C-acquisition was up to 100 % stronger at the fen compared to the bog. Our results imply that changes in plant community composition in response to persistent WT drawdown will strongly affect the C dynamics of peatlands. The predictions of decomposer activity under changing climate and/or land-use thus cannot be based on the direct effects of the changed environment only, but need to consider the indirect effects of environmental changes: the changes in plant community composition, their dependence on peatland type, and their time scale.

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

Strategies for Determining Correct Cytochrome P450 Contributions in Hepatic Clearance Predictions: In Vitro-In Vivo Extrapolation as Modelling Approach and Tramadol as Proof-of Concept Compound.

PubMed

T'jollyn, Huybrecht; Snoeys, Jan; Van Bocxlaer, Jan; De Bock, Lies; Annaert, Pieter; Van Peer, Achiel; Allegaert, Karel; Mannens, Geert; Vermeulen, An; Boussery, Koen

2017-06-01

Although the measurement of cytochrome P450 (CYP) contributions in metabolism assays is straightforward, determination of actual in vivo contributions might be challenging. How representative are in vitro for in vivo CYP contributions? This article proposes an improved strategy for the determination of in vivo CYP enzyme-specific metabolic contributions, based on in vitro data, using an in vitro-in vivo extrapolation (IVIVE) approach. Approaches are exemplified using tramadol as model compound, and CYP2D6 and CYP3A4 as involved enzymes. Metabolism data for tramadol and for the probe substrates midazolam (CYP3A4) and dextromethorphan (CYP2D6) were gathered in human liver microsomes (HLM) and recombinant human enzyme systems (rhCYP). From these probe substrates, an activity-adjustment factor (AAF) was calculated per CYP enzyme, for the determination of correct hepatic clearance contributions. As a reference, tramadol CYP contributions were scaled-back from in vivo data (retrograde approach) and were compared with the ones derived in vitro. In this view, the AAF is an enzyme-specific factor, calculated from reference probe activity measurements in vitro and in vivo, that allows appropriate scaling of a test drug's in vitro activity to the 'healthy volunteer' population level. Calculation of an AAF, thus accounts for any 'experimental' or 'batch-specific' activity difference between in vitro HLM and in vivo derived activity. In this specific HLM batch, for CYP3A4 and CYP2D6, an AAF of 0.91 and 1.97 was calculated, respectively. This implies that, in this batch, the in vitro CYP3A4 activity is 1.10-fold higher and the CYP2D6 activity 1.97-fold lower, compared to in vivo derived CYP activities. This study shows that, in cases where the HLM pool does not represent the typical mean population CYP activities, AAF correction of in vitro metabolism data, optimizes CYP contributions in the prediction of hepatic clearance. Therefore, in vitro parameters for any test compound, obtained in a particular batch, should be corrected with the AAF for the respective enzymes. In the current study, especially the CYP2D6 contribution was found, to better reflect the average in vivo situation. It is recommended that this novel approach is further evaluated using a broader range of compounds.

Comparative effects of dietary flavanols on antioxidant defences and their response to oxidant-induced stress on Caco2 cells.

PubMed

Rodríguez-Ramiro, Ildefonso; Martín, María Angeles; Ramos, Sonia; Bravo, Laura; Goya, Luis

2011-08-01

Flavanols are an important fraction of our diet both for their antioxidant capacity and because they are constituents of greatly accepted foodstuffs such as tea, wine and cocoa. In addition to their antioxidant activity by directly scavenging intracellular reactive oxygen species (ROS), flavanols have been recently shown to enhance protective enzymes. The objective was to evaluate the antioxidant response of colon-derived Caco2 cells to dietary flavanols. Four representative flavanols were selected: epicatechin (EC), epicatechin-3-gallate (ECG), epigallocatechin-3-gallate (EGCG) and procyanidin B2 (PB2). Cell viability, concentration of ROS and reduced glutathione (GSH), and activity of antioxidant/detoxification enzymes and caspase 3 were determined. Treatment of Caco2 cells with flavanols decreased ROS production but did not affect GSH content. ECG induced glutathione peroxidase (GPx), whereas PB2 evoked a dose-dependent increase in GPx, glutathione reductase and glutathione-S-transferase. Enhancement of the antioxidant defences implies an improved cell response to an oxidative challenge. Hence, Caco2 cells treated 20 h with the flavanols, especially PB2, and then submitted to an oxidative stress induced by a pro-oxidant, tert-butyl-hydroperoxide, showed a reduced ROS production, restricted activation of caspase 3 and higher viability than cells plainly submitted to the stressor. Flavanols protect Caco2 cells against an induced oxidative stress and subsequent cellular death by reducing ROS production and preventing caspase-3 activation. In particular, PB2 increases the activity of antioxidant/detoxification enzymes and thus protects Caco2 cells by directly counteracting free radicals and also by activating the antioxidant defence system.

A synthetic urinary probe–coated nanoparticles sensitive to fibroblast activation protein α for solid tumor diagnosis

PubMed Central

Feng, Xinwei; Wang, Qifan; Liao, Yuehua; Zhou, Xie; Wang, Yidan; Liu, Wanli; Zhang, Ge

2017-01-01

We developed fibroblast activation protein α (FAPα)-sensitive magnetic iron oxide nanoparticles (MNPs) by conjugating a substrate-reporter tandem peptide as a synthetic biomarker to the surface of MNPs (marker-MNPs). In vitro, the marker-MNPs showed stability when treated with serum or urine and exhibited high susceptibility and specificity for FAPα enzyme and 3T3/FAPα cell line. Furthermore, the marker-MNPs were administered to esophageal squamous cell carcinoma xenograft tumor mice; they reached the tumor tissues in the mice, where they were cleaved effectively by the local overexpressed FAPα to release the reporter peptide and filter it into the urine. The tumor targeting and biodistribution of marker-MNPs were verified by in vivo imaging. The cleaved reporter peptides in urine detected by enzyme-linked immunosorbent assay have high diagnostic accuracy for esophageal squamous cell carcinoma (area under the receiver-operating characteristic curve =1.0). Our study implies a promising strategy of utilizing the low-cost and noninvasive synthetic urinary probe–coated nanoparticles for the diagnosis of FAPα-positive solid tumors, except for in renal cancer. PMID:28794628

Effects of porcine pancreatic enzymes on the pancreas of hamsters. Part 1: basic studies.

PubMed

Saruc, Murat; Nozawa, Fumiaki; Yalniz, Mehmet; Itami, Atsushi; Pour, Parviz M

2012-09-10

Porcine pancreatic enzymes (PPE) extracted from glandular stomach has been used for the treatment of pancreatic cancer patients. Unfortunately, no information is available on the in vitro and in vivo effect on the pancreas and other tissues. We used Syrian Golden hamsters, a unique pancreatic cancer model, to obtain basic information on PPE for its eventual use for the treatment of pancreatic cancer. PPE was used in different concentrations in vitro and in vivo. The stability of the enzyme in the water solution was investigated. It was given to the hamsters by gavage in concentrations of 1g/kg and 400 mg/kg for short periods and in aqueous solution for 65 days. Plasma enzyme and insulin, the size of islets and the number of the insulin cells per islet were examined. The enzyme activity of PPE was maintained in water solution for at least 24 hours. Due to its content of calcium chloride it showed a high toxicity to normal and malignant hamster pancreatic cancer cells and human pancreatic cancer cell lines in vitro. PPE did not alter the plasma pancreatic enzyme levels regardless of the dose, duration and application route. On the contrary, PPE reduced their levels significantly. Remarkably, it also reduced the level of insulin, the size of the islets and the number of insulin cells in the islets significantly. The results imply that PPE does not enter the blood circulation but it appears to slow down the function of both the exocrine and endocrine pancreas.

Degradation of 2,4-D in soils by Fe₃O₄ nanoparticles combined with stimulating indigenous microbes.

PubMed

Fang, Guodong; Si, Youbin; Tian, Chao; Zhang, Gangya; Zhou, Dongmei

2012-03-01

Degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) in soils by Fe₃O₄ nanoparticles combined with soil indigenous microbes was investigated, and the effects of Fe₃O₄ nanoparticles on soil microbial populations and enzyme activities were also studied. The soils contaminated with 2,4-D were treated with Fe₃O₄ nanoparticles. The microbial populations and enzyme activities were analyzed by dilution plate method and chemical assay, respectively, and the concentration of 2,4-D in soil was determined by high-performance liquid chromatography (HPLC). The results indicated that Fe₃O₄ nanoparticles combined with soil indigenous microbes led to a higher degradation efficiency of 2,4-D than the treatments with Fe₃O₄ nanoparticles or indigenous microbes alone. The degradation of 2,4-D in soils followed the pseudo first-order kinetic. The half-lives of 2,4-D degradation (DT₅₀) of the combined treatments were 0.9, 1.9 and 3.1 days in a Red soil, Vertisol and Alfisol, respectively, which implied that the DT₅₀ of the combination treatments were significantly shorter than that of the treatments Fe₃O₄ nanoparticles or indigenous microbes alone. The effects of Fe₃O₄ nanoparticles on soil microbial populations and enzyme activities were also investigated and compared with the α-Fe₂O₃ nanoparticles. The results suggested that the α-Fe₂O₃ nanoparticles had only comparatively small effects on degradation of 2,4-D in soils, while the Fe₃O₄ nanoparticles not only degraded 2,4-D in soils but also increased the soil microbial populations and enzyme activities; the maximum increase in enzyme activities were 67.8% (amylase), 53.8% (acid phosphatase), 26.5% (catalase) and 38.0% (urease), compared with the untreated soil. Moreover, the introduction of Fe₃O₄ nanoparticles at the different dosage resulted in a variable degradation efficiency of 2,4-D in soil. The method of combining Fe₃O₄ nanoparticles with indigenous soil microbes may offer great benefits for the application of nanotechnology in remediation of herbicide contaminated soil.

Influence of UV radiation on chlorophyll, and antioxidant enzymes of wetland plants in different types of constructed wetland.

PubMed

Xu, Defu; Wu, Yinjuan; Li, Yingxue; Howard, Alan; Jiang, Xiaodong; Guan, Yidong; Gao, Yongxia

2014-09-01

A surface- and vertical subsurface-flow-constructed wetland were designed to study the response of chlorophyll and antioxidant enzymes to elevated UV radiation in three types of wetland plants (Canna indica, Phragmites austrail, and Typha augustifolia). Results showed that (1) chlorophyll content of C. indica, P. austrail, and T. augustifolia in the constructed wetland was significantly lower where UV radiation was increased by 10 and 20 % above ambient solar level than in treatment with ambient solar UV radiation (p < 0.05). (2) The malondialdehyde (MDA) content, guaiacol peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activities of wetland plants increased with elevated UV radiation intensity. (3) The increased rate of MDA, SOD, POD, and CAT activities of C. indica, P. australis, and T. angustifolia by elevated UV radiation of 10 % was higher in vertical subsurface-flow-constructed wetland than in surface-flow-constructed wetland. The sensitivity of MDA, SOD, POD, and CAT activities of C. indica, P. austrail, and T. augustifolia to the elevated UV radiation was lower in surface-flow-constructed wetland than in the vertical subsurface-flow-constructed wetland, which was related to a reduction in UV radiation intensity through the dissolved organic carbon and suspended matter in the water. C. indica had the highest SOD and POD activities, which implied it is more sensitive to enhanced UV radiation. Therefore, different wetland plants had different antioxidant enzymes by elevated UV radiation, which were more sensitive in vertical subsurface-flow-constructed wetland than in surface-flow-constructed wetland.

Site-directed decapsulation of bolaamphiphilic vesicles with enzymatic cleavable surface groups.

PubMed

Popov, Mary; Grinberg, Sarina; Linder, Charles; Waner, Tal; Levi-Hevroni, Bosmat; Deckelbaum, Richard J; Heldman, Eliahu

2012-06-10

Stable nano-sized vesicles with a monolayer encapsulating membrane were prepared from novel bolaamphiphiles with choline ester head groups. The head groups were covalently bound to the alkyl chain of the bolaamphiphiles either via the nitrogen atom of the choline moiety, or via the choline ester's methyl group. Both types of bolaamphiphiles competed with acetylthiocholine for binding to acetylcholine esterase (AChE), yet, only the choline ester head groups bound to the alkyl chain via the nitrogen atom of the choline moiety were hydrolyzed by the enzyme. Likewise, only vesicles composed of bolaamphiphiles with head groups that were hydrolyzed by AChE released their encapsulated material upon exposure to the enzyme. Injection of carboxyfluorescein (CF)-loaded vesicles with cleavable choline ester head groups into mice resulted in the accumulation of CF in tissues that express high AChE activity, including the brain. By comparison, when vesicles with choline ester head groups that are not hydrolyzed by AChE were injected into mice, there was no accumulation of CF in tissues that highly express the enzyme. These results imply that bolaamphiphilic vesicles with surface groups that are substrates to enzymes which are highly expressed in target organs may potentially be used as a drug delivery system with controlled site-directed drug release. Copyright © 2011 Elsevier B.V. All rights reserved.

Differential Contributions of Ubiquitin-Modified APOBEC3G Lysine Residues to HIV-1 Vif-Induced Degradation.

PubMed

Turner, Tiffany; Shao, Qiujia; Wang, Weiran; Wang, Yudi; Wang, Chenliang; Kinlock, Ballington; Liu, Bindong

2016-08-28

Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (A3G) is a host restriction factor that impedes HIV-1 replication. Viral integrity is salvaged by HIV-1 virion infectivity factor (Vif), which mediates A3G polyubiquitination and subsequent cellular depletion. Previous studies have implied that A3G polyubiquitination is essential for Vif-induced degradation. However, the contribution of polyubiquitination to the rate of A3G degradation remains unclear. Here, we show that A3G polyubiquitination is essential for degradation. Inhibition of ubiquitin-activating enzyme E1 by PYR-41 or blocking the formation of ubiquitin chains by over-expressing the lysine to arginine mutation of ubiquitin K48 (K48R) inhibited A3G degradation. Our A3G mutagenesis study showed that lysine residues 297, 301, 303, and 334 were not sufficient to render lysine-free A3G sensitive to Vif-mediated degradation. Our data also confirm that Vif could induce ubiquitin chain formation on lysine residues interspersed throughout A3G. Notably, A3G degradation relied on the lysine residues involved in polyubiquitination. Although A3G and the A3G C-terminal mutant interacted with Vif and were modified by ubiquitin chains, the latter remained more resistant to Vif-induced degradation. Furthermore, the A3G C-terminal mutant, but not the N-terminal mutant, maintained potent antiviral activity in the presence of Vif. Taken together, our results suggest that the location of A3G ubiquitin modification is a determinant for Vif-mediated degradation, implying that in addition to polyubiquitination, other factors may play a key role in the rate of A3G degradation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Regulation of 2-carboxyarabinitol 1-phosphatase.

PubMed

Holbrook, G P; Galasinski, S C; Salvucci, M E

1991-11-01

The regulation of 2-carboxyarabinitol 1-phosphatase (CA 1-Pase) by phosphorylated effectors was studied with enzyme purified from tobacco (Nicotiana tabacum) leaves. CA 1-Pase activity was most stimulated by fructose 1,6-bisphosphate, exhibiting an A(0.5) value of 1.9 millimolar and a 10-fold enhancement of catalysis. With ribulose-1,5-bisphosphate, the A(0.5) was 0.6 millimolar, and maximal stimulation of activity was 5.3-fold. Among the monophosphates, 3-phosphoglycerate and phosphoglycolate were more potent positive effectors than glyceraldehyde 3-phosphate, glucose 1-phosphate, glucose 6-phosphate, and dihydroxyacetone phosphate. Stimulation of CA 1-Pase by ribulose-1,5-bisphosphate and fructose 1,6-bisphosphate increased V(max) but did not appreciably alter K(m) (2-carboxyarabinitol 1-phosphate) values. Inorganic phosphate appeared to inhibit CA 1-Pase noncompetitively with respect to 2-carboxyarabinitol 1-phosphate, exhibiting a K(i) of 0.3 millimolar. The results suggest that these positive and negative effectors bind to a regulatory site on CA 1-Pase and may have a physiologial role in the light regulation of this enzyme. Related experiments with CA 1-Pase inactivated by dialysis in the absence of dithiothreitol show that partial reactivation can be achieved in the presence of a range of reducing reagents, including dithiothreitol, cysteine, and reduced glutathione. This could imply an ancillary involvement of sulfhydryl reduction during light activation of CA 1-Pase in vivo. The enzyme was thermally stable up to 35 degrees C, in contrast to ribulose-1,5-bisphosphate carboxylase/oxygenase activase which lost activity above 30 degrees C. The activation energy for CA 1-Pase was calculated to be 56.14 kilojoules per mole.

Regulation of 2-Carboxyarabinitol 1-Phosphatase 1

PubMed Central

Holbrook, Gabriel P.; Galasinski, Scott C.; Salvucci, Michael E.

1991-01-01

The regulation of 2-carboxyarabinitol 1-phosphatase (CA 1-Pase) by phosphorylated effectors was studied with enzyme purified from tobacco (Nicotiana tabacum) leaves. CA 1-Pase activity was most stimulated by fructose 1,6-bisphosphate, exhibiting an A0.5 value of 1.9 millimolar and a 10-fold enhancement of catalysis. With ribulose-1,5-bisphosphate, the A0.5 was 0.6 millimolar, and maximal stimulation of activity was 5.3-fold. Among the monophosphates, 3-phosphoglycerate and phosphoglycolate were more potent positive effectors than glyceraldehyde 3-phosphate, glucose 1-phosphate, glucose 6-phosphate, and dihydroxyacetone phosphate. Stimulation of CA 1-Pase by ribulose-1,5-bisphosphate and fructose 1,6-bisphosphate increased Vmax but did not appreciably alter Km (2-carboxyarabinitol 1-phosphate) values. Inorganic phosphate appeared to inhibit CA 1-Pase noncompetitively with respect to 2-carboxyarabinitol 1-phosphate, exhibiting a Ki of 0.3 millimolar. The results suggest that these positive and negative effectors bind to a regulatory site on CA 1-Pase and may have a physiologial role in the light regulation of this enzyme. Related experiments with CA 1-Pase inactivated by dialysis in the absence of dithiothreitol show that partial reactivation can be achieved in the presence of a range of reducing reagents, including dithiothreitol, cysteine, and reduced glutathione. This could imply an ancillary involvement of sulfhydryl reduction during light activation of CA 1-Pase in vivo. The enzyme was thermally stable up to 35°C, in contrast to ribulose-1,5-bisphosphate carboxylase/oxygenase activase which lost activity above 30°C. The activation energy for CA 1-Pase was calculated to be 56.14 kilojoules per mole. PMID:16668528

Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA

PubMed Central

Kandiah, Eaazhisai; Carriel, Diego; Perard, Julien; Malet, Hélène; Bacia, Maria; Liu, Kaiyin; Chan, Sze W. S.; Houry, Walid A.; Ollagnier de Choudens, Sandrine; Elsen, Sylvie; Gutsche, Irina

2016-01-01

The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation. Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding. Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions. PMID:27080013

Disaggregation of adenylate cyclase during polyacrylamide-gel electrophoresis in mixtures of ionic and non-ionic detergents.

PubMed

Newby, A C; Chrambach, A

1979-02-01

1. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] solubilized from the rat liver plasma membrane with 1% Lubrol PX and partially purified by gel filtration in buffer containing 0.01% Lubrol PX was physically characterized by polyacrylamide-gel electrophoresis. 2. The molecular radius determined for the partially purified enzyme was 4.9nm, compared with the value of 3.9nm obtained for the enzyme before gel filtration. 3. This difference, representing an approximate doubling of the molecular volume of the enzyme, implied that aggregation with itself or other proteins had occurred during partial purification. 4. Aggregation was not reversed by electrophoresis in the presence of high Lubrol concentrations. 5. Substitution of deoxycholate or N-dodecylsarcosinate for Lubrol PX either for solubilization or during electrophoresis led to poorer resolution of membrane proteins at concentrations giving greater than 70% loss of enzyme activity. 6. Partially purified adenylate cyclase was electrophoresed in the presence of mixed micelles of Lubrol PX and deoxycholate or Lubrol PX and N-dodecylsarcosinate. Different mixtures were examined simultaneously in a suitable apparatus. 7. Electrophoresis in the presence of 0.1% Lubrol plus 0.03% deoxycholate decreased the molecular radius of the cyclase to 4.0nm, with greater than 90% recovery of enzymic activity. The net charge of the enzyme was also increased, indicating ionic detergent binding. 8. With 0.1% Lubrol plus 0.03% N-dodecylsarcosinate the molecular radius was 4.3nm, recovery approx. 50% and net charge similar to that seen in Lubrol plus deoxycholate. 9. The resolution of cyclase from bulk protein, on an analytical scale, was improved in the presence of detergent mixtures, as compared with resolution in Lubrol alone. 10. The results demonstrate the usefulness of polyacrylamide-gel electrophoresis to detect and overcome aggregation problems with membrane proteins and suggest that detergent mixtures in specific ratios may be useful in the purification of adenylate cyclase and other intrinsic membrane proteins.

Evolutionary patterns of proteinase activity in attine ant fungus gardens

PubMed Central

2011-01-01

Background Attine ants live in symbiosis with a basidiomycetous fungus that they rear on a substrate of plant material. This indirect herbivory implies that the symbiosis is likely to be nitrogen deprived, so that specific mechanisms may have evolved to enhance protein availability. We therefore hypothesized that fungal proteinase activity may have been under selection for efficiency and that different classes of proteinases might be involved. Results We determined proteinase activity profiles across a wide pH range for fungus gardens of 14 Panamanian species of fungus-growing ants, representing eight genera. We mapped these activity profiles on an independently obtained molecular phylogeny of the symbionts and show that total proteinase activity in lower attine symbionts peaks at ca. pH 6. The higher attine symbionts that have no known free-living relatives had much higher proteinase activities than the lower attine symbionts. Their total in vitro proteinase activity peaked at pH values around 5, which is close to the pH that the ants maintain in their fungus gardens, suggesting that the pH optimum of fungal proteinases may have changed after the irreversible domestication of evolutionary more derived fungal symbionts. This notion is also supported by buffering capacities of fungus gardens at pH 5.2 being remarkably high, and suggests that the fungal symbiont actively helps to maintain garden acidity at this specific level. Metalloproteinases dominated the activity profiles of lower attine gardens and may thus represent the ancestral type of proteinase production, whereas serine proteinase activity dominated the activity profiles of the higher attine gardens reared by Trachymyrmex and Sericomyrmex, suggesting that there may be trade-offs in the production of these enzyme classes. Remarkably, the single symbiont that is shared by species of the crown group of Atta and Acromyrmex leaf-cutting ants mostly showed metalloproteinase activity, suggesting that recurrent changes in enzyme production may have occurred throughout the domestication history of fungus-garden symbionts. Conclusions Proteinase pH optima and buffering capacities of fungal symbionts appear to have evolved remarkable adaptations to living in obligate symbiosis with farming ants. Although the functional roles of serine and metalloproteinases in fungus gardens are unknown, the differential production of these classes of proteolytic enzymes suggest that substrate specificity may be important and that trade-offs may prevent the simultaneous upregulation of both classes of enzymes. PMID:21247468

Interaction of methotrexate with trypsin analyzed by spectroscopic and molecular modeling methods

NASA Astrophysics Data System (ADS)

Wang, Yanqing; Zhang, Hongmei; Cao, Jian; Zhou, Qiuhua

2013-11-01

Trypsin is one of important digestive enzymes that have intimate correlation with human health and illness. In this work, the interaction of trypsin with methotrexate was investigated by spectroscopic and molecular modeling methods. The results revealed that methotrexate could interact with trypsin with about one binding site. Methotrexate molecule could enter into the primary substrate-binding pocket, resulting in inhibition of trypsin activity. Furthermore, the thermodynamic analysis implied that electrostatic force, hydrogen bonding, van der Waals and hydrophobic interactions were the main interactions for stabilizing the trypsin-methotrexate system, which agreed well with the results from the molecular modeling study.

The activity of detoxifying enzymes in the infective juveniles of Heterorhabditis bacteriophora strains: Purification and characterization of two acetylcholinesterases.

PubMed

Mohamed, Magda A; Mahdy, El-Sayed M E; Ghazy, Abd-El-Hady M; Ibrahim, Nihal M; El-Mezayen, Hatem A; Ghanem, Manal M E

2016-02-01

The infectivity and detoxifying enzyme activities including glutathione-S-transferase (GST), acetylcholinesterase (AChE) and carboxylesterase (CaE) are investigated in the infective juveniles (IJs) of six different strains of Heterorhabditis bacteriophora as a biocontrol agent against insect pests. The specific activities ranged from 10.8-29.8 and 50-220units/mg protein for GST and AChE, respectively; and from 24.7-129 and 22.6-77.3units/mg protein for CaE as estimated by P-nitrophenyl and α-naphthyl acetates, respectively. H. bacteriophora EM2 strain has the highest infectivity and the highest enzymatic activities as well. AChE is the predominant detoxifying enzyme that might imply its major role in the detoxification of insecticide(s). The isoenzyme pattern demonstrated two major slow-moving isoforms in all EPN strains examined. Purification of two AChE isoforms, AChEAII and AChEBI, from H. bacteriophora EM2 strain is performed by ammonium sulfate precipitation, gel filtration on Sephacryl S-200 and chromatography on DEAE-Sepharose. AChEAII and AChEBII have specific activities of 1207 and 1560unit/mg protein, native molecular weights of 180 and 68kDa, and are found in dimeric and monomeric forms, respectively. Both isoforms showed optimum activity at pH8.5 and 35°C. AChEBI exhibited higher thermal stability and higher activation energy than AChEAII. The enzymatic activities of purified AChEs are completely inhibited by Hg(+2) and Ni(+2) and greatly enhanced by Mn(+2). The substrate specificity, the relative efficiency of substrates hydrolysis, substrate inhibition and inhibition by BW284C51, but not by iso-OMPA, clearly indicated that they are true AChEs; their properties are compared with those recorded for insects as target hosts for H. bacteriophora EM2. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparison of human plasma low- and high-density lipoproteins as substrates for lecithin: cholesterol acyltransferase.

PubMed

Barter, P J; Hopkins, G J; Gorjatschko, L

1984-01-17

A recent observation that lecithin: cholesterol acyltransferase (EC 2.3.1.43) interacts with both low-density lipoproteins (LDL) and high-density lipoproteins (HDL) in human plasma is in apparent conflict with an earlier finding that the purified enzyme, while highly reactive with isolated HDL, was only minimally reactive with LDL. There is evidence, however, that lecithin: cholesterol acyltransferase may exist physiologically as a component of a complex with other proteins and that studies with the isolated enzyme may therefore provide misleading results. Consequently, interactions of the enzyme with isolated human lipoproteins have been re-examined in incubations containing lecithin: cholesterol acyltransferase as a component of human lipoprotein-free plasma in which a physiologically active complex of the enzyme with other proteins may have been preserved. In this system there was a ready esterification of the free cholesterol associated with both LDL and HDL-subfraction 3 (HDL3) in reactions that obeyed typical enzyme-saturation kinetics. For a given preparation of lipoprotein-free plasma the Vmax values with LDL and with HDL3 were virtually identical. The apparent Km for free cholesterol associated with HDL3 was 5.6 X 10(-5) M, while for that associated with LDL it was 4.1 X 10(-4) M. This implied that, in terms of free cholesterol concentration, the affinity of HDL3 for lecithin: cholesterol acyltransferase was about 7-times greater than that of LDL. When expressed in terms of lipoprotein particle concentration, however, it was apparent that the affinity of LDL for the enzyme was considerably greater than that of HDL3. When the lipoprotein fractions were equated in terms of lipoprotein surface area, the apparent affinities of the two fractions for the enzyme were found to be comparable.

Insights into Enzyme Catalysis and Thyroid Hormone Regulation of Cerebral Ketimine Reductase/μ-Crystallin Under Physiological Conditions.

PubMed

Hallen, André; Cooper, Arthur J L; Jamie, Joanne F; Karuso, Peter

2015-06-01

Mammalian ketimine reductase is identical to μ-crystallin (CRYM)-a protein that is also an important thyroid hormone binding protein. This dual functionality implies a role for thyroid hormones in ketimine reductase regulation and also a reciprocal role for enzyme catalysis in thyroid hormone bioavailability. In this research we demonstrate potent sub-nanomolar inhibition of enzyme catalysis at neutral pH by the thyroid hormones L-thyroxine and 3,5,3'-triiodothyronine, whereas other thyroid hormone analogues were shown to be far weaker inhibitors. We also investigated (a) enzyme inhibition by the substrate analogues pyrrole-2-carboxylate, 4,5-dibromopyrrole-2-carboxylate and picolinate, and (b) enzyme catalysis at neutral pH of the cyclic ketimines S-(2-aminoethyl)-L-cysteine ketimine (owing to the complex nomenclature trivial names are used for the sulfur-containing cyclic ketimines as per the original authors' descriptions) (AECK), Δ(1)-piperideine-2-carboxylate (P2C), Δ(1)-pyrroline-2-carboxylate (Pyr2C) and Δ(2)-thiazoline-2-carboxylate. Kinetic data obtained at neutral pH suggests that ketimine reductase/CRYM plays a major role as a P2C/Pyr2C reductase and that AECK is not a major substrate at this pH. Thus, ketimine reductase is a key enzyme in the pipecolate pathway, which is the main lysine degradation pathway in the brain. In silico docking of various ligands into the active site of the X-ray structure of the enzyme suggests an unusual catalytic mechanism involving an arginine residue as a proton donor. Given the critical importance of thyroid hormones in brain function this research further expands on our knowledge of the connection between amino acid metabolism and regulation of thyroid hormone levels.

Cystathionine β-Synthase Inhibition Is a Potential Therapeutic Approach to Treatment of Ischemic Injury

PubMed Central

Chan, Su Jing; Chai, Chou; Lim, Tze Wei; Yamamoto, Mie; Lo, Eng H; Lai, Mitchell Kim Peng

2015-01-01

Hydrogen sulfide (H2S) has been reported to exacerbate stroke outcome in experimental models. Cystathionine β-synthase (CBS) has been implicated as the predominant H2S-producing enzyme in central nervous system. When SH-SY5Y cells were transfected to overexpress CBS, these cells were able to synthesize H2S when exposed to high levels of enzyme substrates but not substrate concentrations that may reflect normal physiological conditions. At the same time, these cells demonstrated exacerbated cell death when subjected to oxygen and glucose deprivation (OGD) together with high substrate concentrations, indicating that H2S production has a detrimental effect on cell survival. This effect could be abolished by CBS inhibition. The same effect was observed with primary astrocytes exposed to OGD and high substrates or sodium hydrosulfide. In addition, CBS was upregulated and activated by truncation in primary astrocytes subjected to OGD. When rats were subjected to permanent middle cerebral artery occlusion, CBS activation was also observed. These results imply that in acute ischemic conditions, CBS is upregulated and activated by truncation causing an increased production of H2S, which exacerbate the ischemic injuries. Therefore, CBS inhibition may be a viable approach to stroke treatment. PMID:25873304

Role of the C-terminal residue of the DNA polymerase of bacteriophage T7.

PubMed

Kumar, J K; Tabor, S; Richardson, C C

2001-09-14

The crystal structure of the DNA polymerase encoded by gene 5 of bacteriophage T7, in a complex with its processivity factor, Escherichia coli thioredoxin, a primer-template, and an incoming deoxynucleoside triphosphate reveals a putative hydrogen bond between the C-terminal residue, histidine 704 of gene 5 protein, and an oxygen atom on the penultimate phosphate diester of the primer strand. Elimination of this electrostatic interaction by replacing His(704) with alanine renders the phage nonviable, and no DNA synthesis is observed in vivo. Polymerase activity of the genetically altered enzyme on primed M13 DNA is only 12% of the wild-type enzyme, and its processivity is drastically reduced. Kinetic parameters for binding a primer-template (K(D)(app)), nucleotide binding (K(m)), and k(off) for dissociation of the altered polymerase from a primer-template are not significantly different from that of wild-type T7 DNA polymerase. However, the decrease in polymerase activity is concomitant with increased hydrolytic activity, judging from the turnover of nucleoside triphosphate into the corresponding nucleoside monophosphate (percentage of turnover, 65%) during DNA synthesis. Biochemical data along with structural observations imply that the terminal amino acid residue of T7 DNA polymerase plays a critical role in partitioning DNA between the polymerase and exonuclease sites.

Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas.

PubMed

Sneeringer, Christopher J; Scott, Margaret Porter; Kuntz, Kevin W; Knutson, Sarah K; Pollock, Roy M; Richon, Victoria M; Copeland, Robert A

2010-12-07

EZH2, the catalytic subunit of the PRC2 complex, catalyzes the mono- through trimethylation of lysine 27 on histone H3 (H3K27). Histone H3K27 trimethylation is a mechanism for suppressing transcription of specific genes that are proximal to the site of histone modification. Point mutations of the EZH2 gene (Tyr641) have been reported to be linked to subsets of human B-cell lymphoma. The mutant allele is always found associated with a wild-type allele (heterozygous) in disease cells, and the mutations were reported to ablate the enzymatic activity of the PRC2 complex for methylating an unmodified peptide substrate. Here we demonstrate that the WT enzyme displays greatest catalytic efficiency (k(cat)/K) for the zero to monomethylation reaction of H3K27 and diminished efficiency for subsequent (mono- to di- and di- to trimethylation) reactions. In stark contrast, the disease-associated Y641 mutations display very limited ability to perform the first methylation reaction, but have enhanced catalytic efficiency for the subsequent reactions, relative to the WT enzyme. These results imply that the malignant phenotype of disease requires the combined activities of a H3K27 monomethylating enzyme (PRC2 containing WT EZH2 or EZH1) together with the mutant PRC2s for augmented conversion of H3K27 to the trimethylated form. To our knowledge, this is the first example of a human disease that is dependent on the coordinated activities of normal and disease-associated mutant enzymatic function.

Paraoxonase (PON1) polymorphism and activity as the determinants of sensitivity to organophosphates in human subjects.

PubMed

Sirivarasai, Jintana; Kaojarern, Sming; Yoovathaworn, Krongtong; Sura, Thanyachai

2007-07-20

Paraoxonase (PON1) plays an important role in mechanism of organophosphorus compound (OP) toxicity, as seen both in vitro and in vivo studies. Polymorphisms of PON1 gene at coding and promoter regions have also been to affect on the hydrolytic activity and PON1 level. The objectives of this study were to determine PON1 polymorphism and activity in an OP-exposed population and the effects on inhibition of cholinesterase activity. The studied population consisted of control (n=30) and exposed groups (n=90). All enzyme activities (AChE, BuChE, paraoxonase, arylesterase and diazonase) were measured once for control group and two periods of exposure for exposed group. Three polymorphisms of PON1 (Q192R, L55M and T-108C) were identified only in the exposed subjects. The results demonstrated that AChE activity in both high (345.5 microkat/gHb) and low exposure periods (496.9 microkat/gHb) of the exposed group were significantly different from control group (649.7 microkat/gHb, p<0.01). For BuChE activity, the exposed group also showed the statistically lower level in both periods (high exposure period: 62.17 microkat/L and low exposure period: 81.84 microkat/L) than those in the control group (93.35 microkat/L). Serum paraoxonase activity was significantly different among individual genotypes, RR>QR>RR, LL>LM and -108CC>-108CT>-108TT, but this was not found for those of arylesterase and diazonase activities. Q192R and L55M as well as Q192R and T-108C also presented substantial linkage disequilibrium. Further analysis was performed with haplotypes and various enzyme activities. AChE activity was not affected by haplotypes. Individuals with "211" haplotype showed significantly higher paraoxonase activity and BuChE activity than other haplotypes but not in diazonase activity. In conclusion, PON1 gene exhibited a wide variation in enzyme activities both within and between genotypes which implied insights of a potentially difference in sensitivity to OP toxicity.

Purification of α-glucosidase from mouse intestine by countercurrent chromatography coupled with a reverse micelle solvent system.

PubMed

He, Kai; Zou, Zongyao; Hu, Yinran; Yang, Yong; Xiao, Yubo; Gao, Pincao; Li, Xuegang; Ye, Xiaoli

2016-02-01

Countercurrent chromatography coupled with a reverse micelle solvent was applied to separate α-glucosidase, which is stable at pH 6.0-8.8, 15-50°C. The separation conditions are as follows: stationary phase: pH 4.0 Tris-HCl buffer phase containing 50 mM Tris-HCl and 50 mM KCl; mobile phase A: isooctane containing 50 mM anionic surfactant sodium di(2-ethylhexyl)sulfosuccinate; mobile phase B: 50 mM Tris-HCl buffer containing 500 mM KCl (pH 8.0); In total, 25 mL (23.9 mg) crude enzyme was injected through the injection valve, the enzymatic reaction and sodium dodecylsulfate polyacrylamide gel electrophoresis results imply that the activity of purified α-glucosidase is 6.63-fold higher than that of the crude enzyme. Therefore, countercurrent chromatography coupled with a reverse micelle solvent is capable for protein separation and enrichment. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Catalase-only nanoparticles prepared by shear alone: Characteristics, activity and stability evaluation.

PubMed

Huang, Xiao-Nan; Du, Xin-Ying; Xing, Jin-Feng; Ge, Zhi-Qiang

2016-09-01

Catalase is a promising therapeutic enzyme; however, it carries risks of inactivation and rapid degradation when it is used in practical bioprocess, such as delivery in vivo. To overcome the issue, we made catalase-only nanoparticles using shear stress alone at a moderate shear rate of 217s(-1) in a coaxial cylinder flow cell. Properties of nanoparticles, including particle size, polydispersity index and zeta potential, were characterized. The conformational changes of pre- and post-sheared catalase were determined using spectroscopy techniques. The results indicated that the conformational changes of catalase and reduction in α-helical content caused by shear alone were less significant than that by desolvation method. Catalase-only nanoparticles prepared by single shear retained over 90% of its initial activity when compared with the native catalase. Catalase nanoparticles lost only 20% of the activity when stored in phosphate buffer solution for 72h at 4°C, whereas native catalase lost 53% under the same condition. Especially, the activity of nanogranulated catalase was decreased only slightly in the simulated intestinal fluid containing α-chymotrypsin during 4h incubation at 37°C, implying that the catalase nanoparticle was more resistant to the degradation of proteases than native catalase molecules. Overall, catalase-only nanoparticles offered a great potential to stabilize enzymes for various pharmaceutical applications. Copyright © 2015 Elsevier B.V. All rights reserved.

Adenosylcobinamide methyl phosphate as a pseudocoenzyme for diol dehydrase.

PubMed

Ishida, A; Toraya, T

1993-02-16

Adenosylcobinamide methyl phosphate, a novel analog of adenosylcobalamin lacking the nucleotide loop moiety, was synthesized. It did not show detectable coenzymic activity but behaved as a strong competitive inhibitor against AdoCbl with relatively high affinity (Ki = 2.5 microM). When apoenzyme was incubated at 37 degrees C with this analog in the presence of substrate, the Co-C bond of the analog was almost completely and irreversibly cleaved within 10 min, forming an enzyme-bound Co(II)-containing species. The cleavage was not observed in the absence of substrate. The Co-C bond cleavage in the presence of substrate was not catalytic but stoichiometric, implying that the Co-C bond of the analog undergoes activation when the analog binds to the active site of the enzyme. 5'-Deoxyadenosine was the only product derived from the adenosyl group of the analog upon the Co-C bond cleavage. Apoenzyme did not undergo modification during this process. Therefore, it seems likely that adenosylcobinamide methyl phosphate acts as a pseudocoenzyme or a potent suicide coenzyme. Since adenosylcobinamide neither functions as coenzyme nor binds tightly to apoenzyme, it can be concluded that the phosphodiester moiety of the nucleotide loop of adenosylcobalamin is essential for tight binding to apoenzyme and therefore for subsequent activation of the Co-C bond and catalysis. It is also evident that the nucleotide loop is obligatory for the normal progress of catalytic cycle.

Targeted metabolomics connects thioredoxin-interacting protein (TXNIP) to mitochondrial fuel selection and regulation of specific oxidoreductase enzymes in skeletal muscle.

PubMed

DeBalsi, Karen L; Wong, Kari E; Koves, Timothy R; Slentz, Dorothy H; Seiler, Sarah E; Wittmann, April H; Ilkayeva, Olga R; Stevens, Robert D; Perry, Christopher G R; Lark, Daniel S; Hui, Simon T; Szweda, Luke; Neufer, P Darrell; Muoio, Deborah M

2014-03-21

Thioredoxin-interacting protein (TXNIP) is an α-arrestin family member involved in redox sensing and metabolic control. Growing evidence links TXNIP to mitochondrial function, but the molecular nature of this relationship has remained poorly defined. Herein, we employed targeted metabolomics and comprehensive bioenergetic analyses to evaluate oxidative metabolism and respiratory kinetics in mouse models of total body (TKO) and skeletal muscle-specific (TXNIP(SKM-/-)) Txnip deficiency. Compared with littermate controls, both TKO and TXNIP(SKM-/-) mice had reduced exercise tolerance in association with muscle-specific impairments in substrate oxidation. Oxidative insufficiencies in TXNIP null muscles were not due to perturbations in mitochondrial mass, the electron transport chain, or emission of reactive oxygen species. Instead, metabolic profiling analyses led to the discovery that TXNIP deficiency causes marked deficits in enzymes required for catabolism of branched chain amino acids, ketones, and lactate, along with more modest reductions in enzymes of β-oxidation and the tricarboxylic acid cycle. The decrements in enzyme activity were accompanied by comparable deficits in protein abundance without changes in mRNA expression, implying dysregulation of protein synthesis or stability. Considering that TXNIP expression increases in response to starvation, diabetes, and exercise, these findings point to a novel role for TXNIP in coordinating mitochondrial fuel switching in response to nutrient availability.

Targeted Metabolomics Connects Thioredoxin-interacting Protein (TXNIP) to Mitochondrial Fuel Selection and Regulation of Specific Oxidoreductase Enzymes in Skeletal Muscle*

PubMed Central

DeBalsi, Karen L.; Wong, Kari E.; Koves, Timothy R.; Slentz, Dorothy H.; Seiler, Sarah E.; Wittmann, April H.; Ilkayeva, Olga R.; Stevens, Robert D.; Perry, Christopher G. R.; Lark, Daniel S.; Hui, Simon T.; Szweda, Luke; Neufer, P. Darrell; Muoio, Deborah M.

2014-01-01

Thioredoxin-interacting protein (TXNIP) is an α-arrestin family member involved in redox sensing and metabolic control. Growing evidence links TXNIP to mitochondrial function, but the molecular nature of this relationship has remained poorly defined. Herein, we employed targeted metabolomics and comprehensive bioenergetic analyses to evaluate oxidative metabolism and respiratory kinetics in mouse models of total body (TKO) and skeletal muscle-specific (TXNIPSKM−/−) Txnip deficiency. Compared with littermate controls, both TKO and TXNIPSKM−/− mice had reduced exercise tolerance in association with muscle-specific impairments in substrate oxidation. Oxidative insufficiencies in TXNIP null muscles were not due to perturbations in mitochondrial mass, the electron transport chain, or emission of reactive oxygen species. Instead, metabolic profiling analyses led to the discovery that TXNIP deficiency causes marked deficits in enzymes required for catabolism of branched chain amino acids, ketones, and lactate, along with more modest reductions in enzymes of β-oxidation and the tricarboxylic acid cycle. The decrements in enzyme activity were accompanied by comparable deficits in protein abundance without changes in mRNA expression, implying dysregulation of protein synthesis or stability. Considering that TXNIP expression increases in response to starvation, diabetes, and exercise, these findings point to a novel role for TXNIP in coordinating mitochondrial fuel switching in response to nutrient availability. PMID:24482226

Estimating the magnitude of near-membrane PDE4 activity in living cells.

PubMed

Xin, Wenkuan; Feinstein, Wei P; Britain, Andrea L; Ochoa, Cristhiaan D; Zhu, Bing; Richter, Wito; Leavesley, Silas J; Rich, Thomas C

2015-09-15

Recent studies have demonstrated that functionally discrete pools of phosphodiesterase (PDE) activity regulate distinct cellular functions. While the importance of localized pools of enzyme activity has become apparent, few studies have estimated enzyme activity within discrete subcellular compartments. Here we present an approach to estimate near-membrane PDE activity. First, total PDE activity is measured using traditional PDE activity assays. Second, known cAMP concentrations are dialyzed into single cells and the spatial spread of cAMP is monitored using cyclic nucleotide-gated channels. Third, mathematical models are used to estimate the spatial distribution of PDE activity within cells. Using this three-tiered approach, we observed two pharmacologically distinct pools of PDE activity, a rolipram-sensitive pool and an 8-methoxymethyl IBMX (8MM-IBMX)-sensitive pool. We observed that the rolipram-sensitive PDE (PDE4) was primarily responsible for cAMP hydrolysis near the plasma membrane. Finally, we observed that PDE4 was capable of blunting cAMP levels near the plasma membrane even when 100 μM cAMP were introduced into the cell via a patch pipette. Two compartment models predict that PDE activity near the plasma membrane, near cyclic nucleotide-gated channels, was significantly lower than total cellular PDE activity and that a slow spatial spread of cAMP allowed PDE activity to effectively hydrolyze near-membrane cAMP. These results imply that cAMP levels near the plasma membrane are distinct from those in other subcellular compartments; PDE activity is not uniform within cells; and localized pools of AC and PDE activities are responsible for controlling cAMP levels within distinct subcellular compartments. Copyright © 2015 the American Physiological Society.

Estimating the magnitude of near-membrane PDE4 activity in living cells

PubMed Central

Xin, Wenkuan; Feinstein, Wei P.; Britain, Andrea L.; Ochoa, Cristhiaan D.; Zhu, Bing; Richter, Wito; Leavesley, Silas J.

2015-01-01

Recent studies have demonstrated that functionally discrete pools of phosphodiesterase (PDE) activity regulate distinct cellular functions. While the importance of localized pools of enzyme activity has become apparent, few studies have estimated enzyme activity within discrete subcellular compartments. Here we present an approach to estimate near-membrane PDE activity. First, total PDE activity is measured using traditional PDE activity assays. Second, known cAMP concentrations are dialyzed into single cells and the spatial spread of cAMP is monitored using cyclic nucleotide-gated channels. Third, mathematical models are used to estimate the spatial distribution of PDE activity within cells. Using this three-tiered approach, we observed two pharmacologically distinct pools of PDE activity, a rolipram-sensitive pool and an 8-methoxymethyl IBMX (8MM-IBMX)-sensitive pool. We observed that the rolipram-sensitive PDE (PDE4) was primarily responsible for cAMP hydrolysis near the plasma membrane. Finally, we observed that PDE4 was capable of blunting cAMP levels near the plasma membrane even when 100 μM cAMP were introduced into the cell via a patch pipette. Two compartment models predict that PDE activity near the plasma membrane, near cyclic nucleotide-gated channels, was significantly lower than total cellular PDE activity and that a slow spatial spread of cAMP allowed PDE activity to effectively hydrolyze near-membrane cAMP. These results imply that cAMP levels near the plasma membrane are distinct from those in other subcellular compartments; PDE activity is not uniform within cells; and localized pools of AC and PDE activities are responsible for controlling cAMP levels within distinct subcellular compartments. PMID:26201952

Distinct expression of alkaline phosphatase activity in epilimnetic bacteria: Implication for persistent DOC consumption in a P-limited reservoir

NASA Astrophysics Data System (ADS)

Tseng, Y.; Kao, S.; Shiah, F.

2013-12-01

In a P-deficient system, P availability usually controls the microbial activity and thus the ecosystem function. Thingstad et al. (1997) first addressed a 'Malfunctioning Microbial-loop' theory, which stated that low bacterial production (BP) caused by insufficient nutrient supply would result in DOC accumulation in an oligotrophic ecosystem. In this study we re-examined the theory by conducting seasonal patterns and correlations among soluble reactive phosphate (SRP) and DOC, microbial abundances (picocyanobacteria, bacteria, and heterotrophic nanoflagellate; HNF) and activities (primary production, bacterial production, and alkaline phosphatase activity; APA) coupled with enzyme-labeled fluorescence (ELF) assays on bacterioplankton in a subtropical reservoir sharing the common features, nitrate-replete and P-deficient, with most natural freshwater system during Oct 2007-Oct 2008. Persistently high APA was recorded during most of time, implying that the system was P-deficient. Size fractionated APA and ELF assay revealed that bacteria were the major APA contributor. However, significantly low epilimnion DOC was recorded during the stratified summer season accompanying with high BP and APA as well as high PP, implying that heterotrophic bacteria can well sustain in P-deficient system by utilizing DOP to rapidly lower down DOC under relatively high PP. Such findings oppose the 'Malfunctioning Microbial-loop' theory. On the other hand, strong epilimnetic DOC accumulation occurred in Oct 2007 under low light and low PP condition accompanying with high abundance of HNF, implying that HNF grazing may contribute to a certain degree of DOC accumulation. Correlation matrix supported our suggestions. This study testified the DOC dynamics in P-deficient ecosystem are tightly coupled with the source (PP and grazing) and sink (BP). We also suggested that in SRP-limited freshwater systems bacteria are capable of breaking down autochthonous DOC to reduce the chance of DOC accumulation during transport even dam construction continues to increase.

Litter type affects the activity of aerobic decomposers in a boreal peatland more than site nutrient and water level regimes

NASA Astrophysics Data System (ADS)

Straková, P.; Niemi, R. M.; Freeman, C.; Peltoniemi, K.; Toberman, H.; Heiskanen, I.; Fritze, H.; Laiho, R.

2011-02-01

Peatlands are carbon (C) storage ecosystems sustained by a high water level (WL). High WL creates anoxic conditions that suppress the activity of aerobic decomposers and provide conditions for peat accumulation. Peatland function can be dramatically affected by WL drawdown caused by land-use and/or climate change. Aerobic decomposers are directly affected by WL drawdown through environmental factors such as increased oxygenation and nutrient availability. Additionally, they are indirectly affected via changes in plant community composition and litter quality. We studied the relative importance of direct and indirect effects of WL drawdown on aerobic decomposer activity in plant litter. We did this by profiling 11 extracellular enzymes involved in the mineralization of organic C, nitrogen, phosphorus and sulphur. Our study sites represented a three-stage chronosequence from pristine (undrained) to short-term (years) and long-term (decades) WL drawdown conditions under two nutrient regimes. The litter types included reflected the prevalent vegetation, i.e., Sphagnum mosses, graminoids, shrubs and trees. WL drawdown had a direct and positive effect on microbial activity. Enzyme allocation shifted towards C acquisition, which caused an increase in the rate of decomposition. However, litter type overruled the direct effects of WL drawdown and was the main factor shaping microbial activity patterns. Our results imply that changes in plant community composition in response to persistent WL drawdown will strongly affect the C dynamics of peatlands.

Viral evolution in response to the broad-based retroviral protease inhibitor TL-3.

PubMed

Bühler, B; Lin, Y C; Morris, G; Olson, A J; Wong, C H; Richman, D D; Elder, J H; Torbett, B E

2001-10-01

TL-3 is a protease inhibitor developed using the feline immunodeficiency virus protease as a model. It has been shown to efficiently inhibit replication of human, simian, and feline immunodeficiency viruses and therefore has broad-based activity. We now demonstrate that TL-3 efficiently inhibits the replication of 6 of 12 isolates with confirmed resistance mutations to known protease inhibitors. To dissect the spectrum of molecular changes in protease and viral properties associated with resistance to TL-3, a panel of chronological in vitro escape variants was generated. We have virologically and biochemically characterized mutants with one (V82A), three (M46I/F53L/V82A), or six (L24I/M46I/F53L/L63P/V77I/V82A) changes in the protease and structurally modeled the protease mutant containing six changes. Virus containing six changes was found to be 17-fold more resistant to TL-3 in cell culture than was wild-type virus but maintained similar in vitro replication kinetics compared to the wild-type virus. Analyses of enzyme activity of protease variants with one, three, and six changes indicated that these enzymes, compared to wild-type protease, retained 40, 47, and 61% activity, respectively. These results suggest that deficient protease enzymatic activity is sufficient for function, and the observed protease restoration might imply a selective advantage, at least in vitro, for increased protease activity.

Viral Evolution in Response to the Broad-Based Retroviral Protease Inhibitor TL-3†

PubMed Central

Bühler, Bernd; Lin, Ying-Chuan; Morris, Garrett; Olson, Arthur J.; Wong, Chi-Huey; Richman, Douglas D.; Elder, John H.; Torbett, Bruce E.

2001-01-01

TL-3 is a protease inhibitor developed using the feline immunodeficiency virus protease as a model. It has been shown to efficiently inhibit replication of human, simian, and feline immunodeficiency viruses and therefore has broad-based activity. We now demonstrate that TL-3 efficiently inhibits the replication of 6 of 12 isolates with confirmed resistance mutations to known protease inhibitors. To dissect the spectrum of molecular changes in protease and viral properties associated with resistance to TL-3, a panel of chronological in vitro escape variants was generated. We have virologically and biochemically characterized mutants with one (V82A), three (M46I/F53L/V82A), or six (L24I/M46I/F53L/L63P/V77I/V82A) changes in the protease and structurally modeled the protease mutant containing six changes. Virus containing six changes was found to be 17-fold more resistant to TL-3 in cell culture than was wild-type virus but maintained similar in vitro replication kinetics compared to the wild-type virus. Analyses of enzyme activity of protease variants with one, three, and six changes indicated that these enzymes, compared to wild-type protease, retained 40, 47, and 61% activity, respectively. These results suggest that deficient protease enzymatic activity is sufficient for function, and the observed protease restoration might imply a selective advantage, at least in vitro, for increased protease activity. PMID:11533212

Structure of the prolyl-tRNA synthetase from the eukaryotic pathogen Giardia lamblia

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

Larson, Eric T.; Kim, Jessica E.; Napuli, Alberto J.

2012-09-01

The structure of Giardia prolyl-tRNA synthetase cocrystallized with proline and ATP shows evidence for half-of-the-sites activity, leading to a corresponding mixture of reaction substrates and product (prolyl-AMP) in the two active sites of the dimer. The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolutionmore » crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine.« less

Multiple complexes of nitrogen assimilatory enzymes in spinach chloroplasts: possible mechanisms for the regulation of enzyme function.

PubMed

Kimata-Ariga, Yoko; Hase, Toshiharu

2014-01-01

Assimilation of nitrogen is an essential biological process for plant growth and productivity. Here we show that three chloroplast enzymes involved in nitrogen assimilation, glutamate synthase (GOGAT), nitrite reductase (NiR) and glutamine synthetase (GS), separately assemble into distinct protein complexes in spinach chloroplasts, as analyzed by western blots under blue native electrophoresis (BN-PAGE). GOGAT and NiR were present not only as monomers, but also as novel complexes with a discrete size (730 kDa) and multiple sizes (>120 kDa), respectively, in the stromal fraction of chloroplasts. These complexes showed the same mobility as each monomer on two-dimensional (2D) SDS-PAGE after BN-PAGE. The 730 kDa complex containing GOGAT dissociated into monomers, and multiple complexes of NiR reversibly converted into monomers, in response to the changes in the pH of the stromal solvent. On the other hand, the bands detected by anti-GS antibody were present not only in stroma as a conventional decameric holoenzyme complex of 420 kDa, but also in thylakoids as a novel complex of 560 kDa. The polypeptide in the 560 kDa complex showed slower mobility than that of the 420 kDa complex on the 2D SDS-PAGE, implying the assembly of distinct GS isoforms or a post-translational modification of the same GS protein. The function of these multiple complexes was evaluated by in-gel GS activity under native conditions and by the binding ability of NiR and GOGAT with their physiological electron donor, ferredoxin. The results indicate that these multiplicities in size and localization of the three nitrogen assimilatory enzymes may be involved in the physiological regulation of their enzyme function, in a similar way as recently described cases of carbon assimilatory enzymes.

Endocytosis of a maltose permease is induced when amylolytic enzyme production is repressed in Aspergillus oryzae.

PubMed

Hiramoto, Tetsuya; Tanaka, Mizuki; Ichikawa, Takanori; Matsuura, Yuka; Hasegawa-Shiro, Sachiko; Shintani, Takahiro; Gomi, Katsuya

2015-09-01

In the filamentous fungus Aspergillus oryzae, amylolytic enzyme production is induced by the presence of maltose. Previously, we identified a putative maltose permease (MalP) gene in the maltose-utilizing cluster of A. oryzae. malP disruption causes a significant decrease in α-amylase activity and maltose consumption, indicating that MalP is a maltose transporter required for amylolytic enzyme production in A. oryzae. Although the expression of amylase genes and malP is repressed by the presence of glucose, the effect of glucose on the abundance of functional MalP is unknown. In this study, we examined the effect of glucose and other carbon sources on the subcellular localization of green fluorescence protein (GFP)-tagged MalP. After glucose addition, GFP-MalP at the plasma membrane was internalized and delivered to the vacuole. This glucose-induced internalization of GFP-MalP was inhibited by treatment with latrunculin B, an inhibitor of actin polymerization. Furthermore, GFP-MalP internalization was inhibited by repressing the HECT ubiquitin ligase HulA (ortholog of yeast Rsp5). These results suggest that MalP is transported to the vacuole by endocytosis in the presence of glucose. Besides glucose, mannose and 2-deoxyglucose also induced the endocytosis of GFP-MalP and amylolytic enzyme production was inhibited by the addition of these sugars. However, neither the subcellular localization of GFP-MalP nor amylolytic enzyme production was influenced by the addition of xylose or 3-O-methylglucose. These results imply that MalP endocytosis is induced when amylolytic enzyme production is repressed. Copyright © 2015 Elsevier Inc. All rights reserved.

Crystal Structures of Wild-type and Mutant Methicillin-resistant Staphylococcus aureus Dihydrofolate Reductase Reveal an Alternative Conformation of NADPH that may be Linked to Trimethoprim Resistance

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

Frey, K.; Liu, J; Lombardo, M

2009-01-01

Both hospital- and community-acquired Staphylococcus aureus infections have become major health concerns in terms of morbidity, suffering and cost. Trimethoprim-sulfamethoxazole (TMP-SMZ) is an alternative treatment for methicillin-resistant S. aureus (MRSA) infections. However, TMP-resistant strains have arisen with point mutations in dihydrofolate reductase (DHFR), the target for TMP. A single point mutation, F98Y, has been shown biochemically to confer the majority of this resistance to TMP. Using a structure-based approach, we have designed a series of novel propargyl-linked DHFR inhibitors that are active against several trimethoprim-resistant enzymes. We screened this series against wild-type and mutant (F98Y) S. aureus DHFR and foundmore » that several are active against both enzymes and specifically that the meta-biphenyl class of these inhibitors is the most potent. In order to understand the structural basis of this potency, we determined eight high-resolution crystal structures: four each of the wild-type and mutant DHFR enzymes bound to various propargyl-linked DHFR inhibitors. In addition to explaining the structure-activity relationships, several of the structures reveal a novel conformation for the cofactor, NADPH. In this new conformation that is predominantly associated with the mutant enzyme, the nicotinamide ring is displaced from its conserved location and three water molecules complete a network of hydrogen bonds between the nicotinamide ring and the protein. In this new position, NADPH has reduced interactions with the inhibitor. An equilibrium between the two conformations of NADPH, implied by their occupancies in the eight crystal structures, is influenced both by the ligand and the F98Y mutation. The mutation induced equilibrium between two NADPH-binding conformations may contribute to decrease TMP binding and thus may be responsible for TMP resistance.« less

Ionization of isocitrate bound to pig hear NADP/sup +/-dependent isocitrate dehydrogenase: /sup 13/C NMR study of substrate binding

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

Ehrlich, R.S.; Colman, R.F.

1987-06-16

Isocitrate and ..cap alpha..-ketoglutarate have been synthesized with carbon-13 enrichment at specific positions. The /sup 13/C NMR spectra of these derivatives were measured as a function of pH. The magnitudes of the changes in chemical shifts with pH for free isocitrate and the magnesium-isocitrate complex suggest that the primary site of ionization at the ..beta..-carboxyl. In the presence of the enzyme NADP/sup +/-dependent isocitrate dehydrogenase and the activating metal magnesium, the carbon-13 resonances of all three carboxyls remain constant from pH 5.5 to pH 7.5. Thus, the carboxyls remain in the ionized form in the enzyme-isocitrate complex. The ..cap alpha..-hydroxylmore » carbon resonance could not be located in the enzyme-isocitrate complex, suggesting immobilization of this group. Magnesium produces a 2 ppm downfield shift of the ..beta..-carboxyl but does not change the resonances of the ..cap alpha..- and ..gamma..-carboxyls. This result is consistent with metal activation of both the dehydrogenation and decarboxylation reactions. The /sup 13/C NMR spectrum of ..cap alpha..-ketoglutarate remains unchanged in the presence of isocitrate dehydrogenase, implying the absence of alterations in geometry in the enzyme-bound form. Formation of the quaternary complex with Mg/sup 2 +/ and NADPH leads to loss of the ..cap alpha..-ketoglutarate resonances and the appearance of new resonances characteristic of ..cap alpha..-hydroxyglutarate. In addition, a broad peak ascribed to the enol form of ..cap alpha..-ketoglutarate is observed. The substantial change in the shift of the ..beta..-carboxyl of isocitrate and the lack of significant shifts in the other carboxyls of isocitrate or ..cap alpha..-ketoglutarate suggest that interaction of the ..beta..-carboxyl with the enzyme contributes to the tighter binding of isocitrate and may be significant for the oxidative decarboxylation function of isocitrate dehydrogenase.« less

Hypogravity-induced atrophy of rat soleus and extensor digitorum longus muscles

NASA Technical Reports Server (NTRS)

Riley, D. A.; Ellis, S.; Slocum, G. R.; Satyanarayana, T.; Bain, J. L.; Sedlak, F. R.

1987-01-01

Prolonged exposure of humans to hypogravity causes weakening of their skeletal muscles. This problem was studied in rats exposed to hypogravity for 7 days aboard Spacelab 3. Hindlimb muscles were harvested 12-16 hours postflight for histochemical, biochemical, and ultrastructural analyses. The majority of the soleus and extensor digitorum longus fibers exhibited simple cell shrinkage. However, approximately 1% of the fibers in flight soleus muscles appeared necrotic. Flight muscle fibers showed increased glycogen, lower subsarcolemmal staining for mitochondrial enzymes, and fewer subsarcolemmal mitochondria. During atrophy, myofibrils were eroded by multiple focal losses of myofilaments; lysosomal autophagy was not evident. Tripeptidylaminopeptidase and calcium-activated protease activities of flight soleus fibers were significantly increased, implying a role in myofibril breakdown. Simple fiber atrophy appears to account for muscle weakening during spaceflight, but fiber necrosis is also a contributing factor.

A first principle study on the interaction between acetylcholinesterase and acetylcholine, and also rivastigmine in alzheimer's disease case

NASA Astrophysics Data System (ADS)

Khoirunisa, V.; Rusydi, F.; Kasai, H.; Gandaryus, A. G.; Dipojono, H. K.

2016-08-01

The catalytic activity of acetylcholinesterase enzyme (AChE) relates to the symptom progress in Alzheimer's disease. Interaction of AChE with rivastigmine (from the medicine) can reduce its catalytic activity toward acetylcholine to decelerate the progression of Alzheimer's disease. This research attempts to study the interaction between AChE and rivastigmine, and also acetylcholine (without the presence of rivastigmine) using density functional theory by simplifying the reaction occurs in the active site, which is assumed to be C2H5OH, C3N2H3(Ch3), and CH3COO-. The results suggest that AChE interacts easier with acetylcholine than with rivastigmine, which implies that the medicine does not effectively reduce the catalytic activity of AChE. At this stage, no experimental data is available to be compared with the calculation results. Nonetheless, this study has shown a good prospect to understand the AChE-substrate interaction using a first-principles calculation.

Extending double modulation: combinatorial rules for identifying the modulations necessary for determining elasticities in metabolic pathways.

PubMed

Giersch, C; Cornish-Bowden, A

1996-10-07

The double modulation method for determining the elasticities of pathway enzymes, originally devised by Kacser & Burns (Biochem. Soc. Trans. 7, 1149-1160, 1979), is extended to pathways of complex topological structure, including branching and feedback loops. An explicit system of linear equations for the unknown elasticities is derived. The constraints imposed on this linear system imply that modulations of more than one enzyme are not necessarily independent. Simple combinatorial rules are described for identifying without using any algebra the set of independent modulations that allow the determination of the elasticities of any enzyme. By repeated application, the minimum numbers of modulations required to determine the elasticities of all enzymes of a given pathway can be determined. The procedure is illustrated with numerous examples.

Insecticide susceptibility status and major detoxifying enzymes' activity in Aedes albopictus (Skuse), vector of dengue and chikungunya in Northern part of West Bengal, India.

PubMed

Bharati, Minu; Saha, Dhiraj

2017-06-01

Mosquitoes belonging to Aedes genus, Aedes aegypti and Aedes albopictus transmit many globally important arboviruses including Dengue (DENV) and Chikungunya (CHIKV). Vector control with the use of insecticide remains the suitable method of choice to stop the transmission of these diseases. However, vector control throughout the world is failing to achieve its target results because of the worldwide development of insecticide resistance in mosquitoes. To assess the insecticide susceptibility status of Aedes albopictus from northern part of West Bengal, the susceptibility of eight different Aedes albopictus populations were tested against a commonly used larvicide (temephos) and some adulticides (malathion, deltamethrin and lambda cyhalothrin) along with the major insecticide detoxifying enzymes' activity in them. Through this study, it was revealed that most of the populations were found susceptible to temephos except Nagrakata (NGK) and Siliguri (SLG), which showed both a higher resistance ratio (RR 99 ) and a lower susceptibility, thereby reflecting the development of resistance against temephos in them. However, all tested adulticides caused 100% mortality in all the population implying their potency in control of this mosquito in this region of India. Through the study of carboxylesterase activity, it was revealed that the NGK population showed a 9.6 fold higher level of activity than susceptible population. The same population also showed a lower level of susceptibility and a higher resistance ratio (RR 99 ), indicating a clear correlation between susceptibility to temephos and carboxylesterase enzymes' activity in this population. This preliminary data reflects that the NGK population is showing a trend towards resistance development and with time, there is possibility that this resistance phenomenon will spread to other populations. With the recurrence of dengue and chikungunya, this data on insecticide susceptibility status of Aedes albopictus could help the authorities engaged in vector control programmes to formulate effective measures against this mosquito in this region. Copyright © 2017 Elsevier B.V. All rights reserved.

Cigarette smoke–induced induction of antioxidant enzyme activities in airway leukocytes is absent in active smokers with COPD

PubMed Central

Dove, Rosamund E.; Leong-Smith, Pheneatia; Roos-Engstrand, Ester; Pourazar, Jamshid; Shah, Mittal; Behndig, Annelie F.; Mudway, Ian S.; Blomberg, Anders

2015-01-01

Background Oxidative injury to the airway has been proposed as an important underlying mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD). As the extent of oxidant-mediated damage is dependent on the endogenous antioxidant defences within the airways, we examined whether COPD was associated with deficiencies in the antioxidant network within the respiratory tract lining fluids (RTLFs) and resident airway leukocytes. We hypothesised that COPD would be associated with both basal depression of antioxidant defences and impaired adaptive antioxidant responses to cigarette smoke. Methods Low molecular weight and enzymatic antioxidants together with metal-handling proteins were quantified in bronchoalveolar lavage fluid and airway leukocytes, derived from current (n=9) and ex-smoking COPD patients (n=15), as well as from smokers with normal lung function (n=16) and healthy never smokers (n=13). Results Current cigarette smoking was associated with an increase in ascorbate and glutathione within peripheral RTLFs in both smokers with normal lung function compared with healthy never smokers and in COPD smokers compared with COPD ex-smokers. In contrast, intra-cellular antioxidant enzyme activities (glutathione peroxidase, glutathione reductase, and catalase) were only up-regulated in smokers with normal lung function compared with healthy never smokers and not in actively smoking COPD patients relative to COPD ex-smokers. Conclusions We found no evidence of impaired basal antioxidant defences, within either the RTLFs or airway leukocytes in stable ex-smoking COPD patients compared with healthy never smoking controls. Current cigarette smoking induced an up-regulation of low molecular weight antioxidants in the RTLFs of both control subjects with normal lung function and patients with COPD. Importantly, the present data demonstrated a cigarette smoke–induced increase in intra-cellular antioxidant enzyme activities only within the smokers with normal lung function, implying that patients with COPD who continue to smoke will experience enhanced oxidative stress, prompting disease progression. PMID:26557249

Gene knockout and overexpression analysis revealed the role of N-acetylmuramidase in autolysis of Lactobacillus delbrueckii subsp. bulgaricus ljj-6.

PubMed

Pang, Xiao-Yang; Cui, Wen-Ming; Liu, Lu; Zhang, Shu-Wen; Lv, Jia-Ping

2014-01-01

Autolysis of lactic acid bacteria (LAB) plays a vital role in dairy processing. During cheese making, autolysis of LAB affects cheese flavor development through release of intracellular enzymes and restricts the proliferation of cells in yogurt fermentation and probiotics production. In order to explore the mechanism of autolysis, the gene for the autolytic enzymes of L. bulgaricus, N-acetylmuramidase (mur), was cloned and sequenced (GenBank accession number: KF157911). Mur gene overexpression and gene knockout vectors were constructed based on pMG76e and pUC19 vectors. Recombinant plasmids were transformed into L. bulgaricus ljj-6 by electroporation, then three engineered strains with pMG76e-mur vector and fifteen engineered strains with pUC19-mur::EryBII were screened. The autolysis of the mur knockout strain was significantly lower and autolysis of the mur overexpressed strain was significantly higher compared with that of the wild type strain ljj-6. This result suggested that the mur gene played an important role in autolysis of L. bulgaricus. On the other hand, autolytic activity in a low degree was still observed in the mur knockout strain, which implied that other enzymes but autolysin encoded by mur were also involved in autolysis of L. bulgaricus.

Role of AMACR (α-methylacyl-CoA racemase) and MFE-1 (peroxisomal multifunctional enzyme-1) in bile acid synthesis in mice.

PubMed

Autio, Kaija J; Schmitz, Werner; Nair, Remya R; Selkälä, Eija M; Sormunen, Raija T; Miinalainen, Ilkka J; Crick, Peter J; Wang, Yuqin; Griffiths, William J; Reddy, Janardan K; Baes, Myriam; Hiltunen, J Kalervo

2014-07-01

Cholesterol is catabolized to bile acids by peroxisomal β-oxidation in which the side chain of C27-bile acid intermediates is shortened by three carbon atoms to form mature C24-bile acids. Knockout mouse models deficient in AMACR (α-methylacyl-CoA racemase) or MFE-2 (peroxisomal multifunctional enzyme type 2), in which this β-oxidation pathway is prevented, display a residual C24-bile acid pool which, although greatly reduced, implies the existence of alternative pathways of bile acid synthesis. One alternative pathway could involve Mfe-1 (peroxisomal multifunctional enzyme type 1) either with or without Amacr. To test this hypothesis, we generated a double knockout mouse model lacking both Amacr and Mfe-1 activities and studied the bile acid profiles in wild-type, Mfe-1 and Amacr single knockout mouse line and Mfe-1 and Amacr double knockout mouse lines. The total bile acid pool was decreased in Mfe-1-/- mice compared with wild-type and the levels of mature C24-bile acids were reduced in the double knockout mice when compared with Amacr-deficient mice. These results indicate that Mfe-1 can contribute to the synthesis of mature bile acids in both Amacr-dependent and Amacr-independent pathways.

Presence of a plant-like proton-pumping pyrophosphatase in acidocalcisomes of Trypanosoma cruzi.

PubMed

Scott, D A; de Souza, W; Benchimol, M; Zhong, L; Lu, H G; Moreno, S N; Docampo, R

1998-08-21

The vacuolar-type proton-translocating pyrophosphatase (V-H+-PPase) is an enzyme previously described in detail only in plants. This paper demonstrates its presence in the trypanosomatid Trypanosoma cruzi. Pyrophosphate promoted organellar acidification in permeabilized amastigotes, epimastigotes, and trypomastigotes of T. cruzi. This activity was stimulated by K+ ions and was inhibited by Na+ ions and pyrophosphate analogs, as is the plant activity. Separation of epimastigote extracts on Percoll gradients yielded a dense fraction that contained H+-PPase activity measured both by proton uptake and phosphate release but lacked markers for mitochondria, lysosomes, glycosomes, cytosol, and plasma membrane. Antiserum raised against specific sequences of the plant V-H+-PPase cross-reacted with a T. cruzi protein, which was also detectable in the dense Percoll fraction. The organelles in this fraction appeared by electron microscopy to consist mainly of acidocalcisomes (acidic calcium storage organelles). This identification was confirmed by x-ray microanalysis. Immunofluorescence and immunoelectron microscopy indicated that the V-H+-PPase was located in the plasma membrane and acidocalcisomes of the three different forms of the parasite. Pyrophosphate was able to drive calcium uptake in permeabilized T. cruzi. This uptake depended upon a proton gradient and was reversed by a specific V-H+-PPase inhibitor. Our results imply that the phylogenetic distribution of V-H+-PPases is much wider than previously perceived but that the enzyme has a unique subcellular location in trypanosomes.

Disaggregation of adenylate cyclase during polyacrylamide-gel electrophoresis in mixtures of ionic and non-ionic detergents

PubMed Central

Newby, Andrew C.; Chrambach, Andreas

1979-01-01

1. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] solubilized from the rat liver plasma membrane with 1% Lubrol PX and partially purified by gel filtration in buffer containing 0.01% Lubrol PX was physically characterized by polyacrylamide-gel electrophoresis. 2. The molecular radius determined for the partially purified enzyme was 4.9nm, compared with the value of 3.9nm obtained for the enzyme before gel filtration. 3. This difference, representing an approximate doubling of the molecular volume of the enzyme, implied that aggregation with itself or other proteins had occurred during partial purification. 4. Aggregation was not reversed by electrophoresis in the presence of high Lubrol concentrations. 5. Substitution of deoxycholate or N-dodecylsarcosinate for Lubrol PX either for solubilization or during electrophoresis led to poorer resolution of membrane proteins at concentrations giving greater than 70% loss of enzyme activity. 6. Partially purified adenylate cyclase was electrophoresed in the presence of mixed micelles of Lubrol PX and deoxycholate or Lubrol PX and N-dodecylsarcosinate. Different mixtures were examined simultaneously in a suitable apparatus. 7. Electrophoresis in the presence of 0.1% Lubrol plus 0.03% deoxycholate decreased the molecular radius of the cyclase to 4.0nm, with greater than 90% recovery of enzymic activity. The net charge of the enzyme was also increased, indicating ionic detergent binding. 8. With 0.1% Lubrol plus 0.03% N-dodecylsarcosinate the molecular radius was 4.3nm, recovery approx. 50% and net charge similar to that seen in Lubrol plus deoxycholate. 9. The resolution of cyclase from bulk protein, on an analytical scale, was improved in the presence of detergent mixtures, as compared with resolution in Lubrol alone. 10. The results demonstrate the usefulness of polyacrylamide-gel electrophoresis to detect and overcome aggregation problems with membrane proteins and suggest that detergent mixtures in specific ratios may be useful in the purification of adenylate cyclase and other intrinsic membrane proteins. ImagesFig. 3. PMID:435255

Dynamic Reorganization of Metabolic Enzymes into Intracellular Bodies

PubMed Central

O’Connell, Jeremy D.; Zhao, Alice; Ellington, Andrew D.; Marcotte, Edward M.

2013-01-01

Both focused and large-scale cell biological and biochemical studies have revealed that hundreds of metabolic enzymes across diverse organisms form large intracellular bodies. These proteinaceous bodies range in form from fibers and intracellular foci—such as those formed by enzymes of nitrogen and carbon utilization and of nucleotide biosynthesis—to high-density packings inside bacterial microcompartments and eukaryotic microbodies. Although many enzymes clearly form functional mega-assemblies, it is not yet clear for many recently discovered cases whether they represent functional entities, storage bodies, or aggregates. In this article, we survey intracellular protein bodies formed by metabolic enzymes, asking when and why such bodies form and what their formation implies for the functionality—and dysfunctionality—of the enzymes that comprise them. The panoply of intracellular protein bodies also raises interesting questions regarding their evolution and maintenance within cells. We speculate on models for how such structures form in the first place and why they may be inevitable. PMID:23057741

Molecular Basis for Antioxidant Enzymes in Mediating Copper Detoxification in the Nematode Caenorhabditis elegans

PubMed Central

Song, Shaojuan; Zhang, Xueyao; Wu, Haihua; Han, Yan; Zhang, Jianzhen; Ma, Enbo; Guo, Yaping

2014-01-01

Antioxidant enzymes play a major role in defending against oxidative damage by copper. However, few studies have been performed to determine which antioxidant enzymes respond to and are necessary for copper detoxification. In this study, we examined both the activities and mRNA levels of SOD, CAT, and GPX under excessive copper stress in Caenorhabditis elegans, which is a powerful model for toxicity studies. Then, taking advantage of the genetics of this model, we assessed the lethal concentration (LC50) values of copper for related mutant strains. The results showed that the SOD, CAT, and GPX activities were significantly greater in treated groups than in controls. The mRNA levels of sod-3, sod-5, ctl-1, ctl-2, and almost all gpx genes were also significantly greater in treated groups than in controls. Among tested mutants, the sod-5, ctl-1, gpx-3, gpx-4, and gpx-6 variants exhibited hypersensitivity to copper. The strains with SOD or CAT over expression were reduced sensitive to copper. Mutations in daf-2 and age-1, which are involved in the insulin/insulin-like growth factor-1 signaling pathway, result in reduced sensitivity to stress. Here, we showed that LC50 values for copper in daf-2 and age-1 mutants were significantly greater than in N2 worms. However, the LC50 values in daf-16;daf-2 and daf-16;age-1 mutants were significantly reduced than in daf-2 and age-1 mutants, implying that reduced copper sensitivity is influenced by DAF-16-related functioning. SOD, CAT, and GPX activities and the mRNA levels of the associated copper responsive genes were significantly increased in daf-2 and age-1 mutants compared to N2. Additionally, the activities of SOD, CAT, and GPX were greater in these mutants than in N2 when treated with copper. Our results not only support the theory that antioxidant enzymes play an important role in copper detoxification but also identify the response and the genes involved in these processes. PMID:25243607

Function of isoamylase-type starch debranching enzymes ISA1 and ISA2 in the Zea mays leaf.

PubMed

Lin, Qiaohui; Facon, Maud; Putaux, Jean-Luc; Dinges, Jason R; Wattebled, Fabrice; D'Hulst, Christophe; Hennen-Bierwagen, Tracie A; Myers, Alan M

2013-12-01

Conserved isoamylase-type starch debranching enzymes (ISAs), including the catalytic ISA1 and noncatalytic ISA2, are major starch biosynthesis determinants. Arabidopsis thaliana leaves require ISA1 and ISA2 for physiological function, whereas endosperm starch is near normal with only ISA1. ISA functions were characterized in maize (Zea mays) leaves to determine whether species-specific distinctions in ISA1 primary structure, or metabolic differences in tissues, are responsible for the differing ISA2 requirement. Genetic methods provided lines lacking ISA1 or ISA2. Biochemical analyses characterized ISA activities in mutant tissues. Starch content, granule morphology, and amylopectin fine structure were determined. Three ISA activity forms were observed in leaves, two ISA1/ISA2 heteromultimers and one ISA1 homomultimer. ISA1 homomultimer activity existed in mutants lacking ISA2. Mutants without ISA2 differed in leaf starch content, granule morphology, and amylopectin structure compared with nonmutants or lines lacking both ISA1 and ISA2. The data imply that both the ISA1 homomultimer and ISA1/ISA2 heteromultimer function in the maize leaf. The ISA1 homomultimer is present and functions in the maize leaf. Evolutionary divergence between monocots and dicots probably explains the ability of ISA1 to function as a homomultimer in maize leaves, in contrast to other species where the ISA1/ISA2 heteromultimer is the only active form. No claim to original US goverment works. New Phytologist © 2013 New Phytologist Trust.

New lives for old: evolution of pseudoenzyme function illustrated by iRhoms.

PubMed

Adrain, Colin; Freeman, Matthew

2012-07-11

Large-scale sequencing of genomes has revealed that most enzyme families include inactive homologues. These pseudoenzymes are often well conserved, implying a selective pressure to retain them during evolution, and therefore that they have significant function. Mechanistic insights and evolutionary lessons are now emerging from the study of a broad range of such 'dead' enzymes. The recently discovered iRhoms - inactive homologues of rhomboid proteases - have joined derlins and other members of the rhomboid-like clan in regulating the fate of proteins as they pass through the secretory pathway. There is a strong case that dead enzymes, which have been rather overlooked, may be a rich source of biological regulators.

A combination of He-Ne laser irradiation and exogenous NO application efficiently protect wheat seedling from oxidative stress caused by elevated UV-B stress.

PubMed

Li, Yongfeng; Gao, Limei; Han, Rong

2016-12-01

The elevated ultraviolet-B (UV-B) stress induces the accumulation of a variety of intracellular reactive oxygen species (ROS), which seems to cause oxidative stress for plants. To date, very little work has been done to evaluate the biological effects of a combined treatment with He-Ne laser irradiation and exogenous nitric oxide (NO) application on oxidative stress resulting from UV-B radiation. Thus, our study investigated the effects of a combination with He-Ne laser irradiation and exogenous NO treatment on oxidative damages in wheat seedlings under elevated UV-B stress. Our data showed that the reductions in ROS levels, membrane damage parameters, while the increments in antioxidant contents and antioxidant enzyme activity caused by a combination with He-Ne laser and exogenous NO treatment were greater than those of each individual treatment. Furthermore, these treatments had a similar effect on transcriptional activities of plant antioxidant enzymes. This implied that the protective effects of a combination with He-Ne laser irradiation and exogenous NO treatment on oxidative stress resulting from UV-B radiation was more efficient than each individual treatment with He-Ne laser or NO molecule. Our findings might provide beneficial theoretical references for identifying some effective new pathways for plant UV-B protection.

coq7/clk-1 regulates mitochondrial respiration and the generation of reactive oxygen species via coenzyme Q.

PubMed

Nakai, Daisuke; Shimizu, Takahiko; Nojiri, Hidetoshi; Uchiyama, Satoshi; Koike, Hideo; Takahashi, Mayumi; Hirokawa, Katsuiku; Shirasawa, Takuji

2004-10-01

coq7/clk-1 was isolated from a long-lived mutant of Caenorhabditis elegans, and shows sluggish behaviours and an extended lifespan. In C. elegans and Saccharomyces cerevisiae, coq7/clk-1 is required for the biosynthesis of coenzyme Q (CoQ), an essential co-factor in mitochondrial respiration. The clk-1 mutant contains dietary CoQ(8) from Escherichia coli and demethoxyubiquinone 9 (DMQ9) instead of CoQ(9). In a previous study, we generated COQ7-deficient mice by targeted disruption of the coq7 gene and reported that mouse coq7/clk-1 is also essential for CoQ synthesis, maintenance of mitochondrial integrity and neurogenesis. In the present study, we rescued COQ7-deficient mice from embryonic lethality and established a mouse model with decreased CoQ level by transgene expression of COQ7/CLK-1. A biochemical analysis showed a concomitant decrease in CoQ(9), mitochondrial respiratory enzyme activity and the generation of reactive oxygen species (ROS) in the mitochondria of CoQ-insufficient mice. This implied that the depressed activity of respiratory enzymes and the depressed production of ROS may play a physiological role in the control of lifespan in mammalian species and of C. elegans.

Investigating Information Dynamics in Living Systems through the Structure and Function of Enzymes.

PubMed

Gatenby, Robert; Frieden, B Roy

2016-01-01

Enzymes are proteins that accelerate intracellular chemical reactions often by factors of 105-1012s-1. We propose the structure and function of enzymes represent the thermodynamic expression of heritable information encoded in DNA with post-translational modifications that reflect intra- and extra-cellular environmental inputs. The 3 dimensional shape of the protein, determined by the genetically-specified amino acid sequence and post translational modifications, permits geometric interactions with substrate molecules traditionally described by the key-lock best fit model. Here we apply Kullback-Leibler (K-L) divergence as metric of this geometric "fit" and the information content of the interactions. When the K-L 'distance' between interspersed substrate pn and enzyme rn positions is minimized, the information state, reaction probability, and reaction rate are maximized. The latter obeys the Arrhenius equation, which we show can be derived from the geometrical principle of minimum K-L distance. The derivation is first limited to optimum substrate positions for fixed sets of enzyme positions. However, maximally improving the key/lock fit, called 'induced fit,' requires both sets of positions to be varied optimally. We demonstrate this permits and is maximally efficient if the key and lock particles pn, rn are quantum entangled because the level of entanglement obeys the same minimized value of the Kullback-Leibler distance that occurs when all pn ≈ rn. This implies interchanges pn ⇄ brn randomly taking place during a reaction successively improves key/lock fits, reducing the activation energy Ea and increasing the reaction rate k. Our results demonstrate the summation of heritable and environmental information that determines the enzyme spatial configuration, by decreasing the K-L divergence, is converted to thermodynamic work by reducing Ea and increasing k of intracellular reactions. Macroscopically, enzyme information increases the order in living systems, similar to the Maxwell demon gedanken, by selectively accelerating specific reaction thus generating both spatial and temporal concentration gradients.

Micro-CT X-ray imaging exposes structured diffusion barriers within biofilms.

PubMed

Keren-Paz, Alona; Brumfeld, Vlad; Oppenheimer-Shaanan, Yaara; Kolodkin-Gal, Ilana

2018-01-01

In nature, bacteria predominantly exist as highly structured biofilms, which are held together by extracellular polymeric substance and protect their residents from environmental insults, such as antibiotics. The mechanisms supporting this phenotypic resistance are poorly understood. Recently, we identified a new mechanism maintaining biofilms - an active production of calcite minerals. In this work, a high-resolution and robust µCT technique is used to study the mineralized areas within intact bacterial biofilms. µCT is a vital tool for visualizing bacterial communities that can provide insights into the relationship between bacterial biofilm structure and function. Our results imply that dense and structured calcium carbonate lamina forms a diffusion barrier sheltering the inner cell mass of the biofilm colony. Therefore, µCT can be employed in clinical settings to predict the permeability of the biofilms. It is demonstrated that chemical interference with urease, a key enzyme in biomineralization, inhibits the assembly of complex bacterial structures, prevents the formation of mineral diffusion barriers and increases biofilm permeability. Therefore, biomineralization enzymes emerge as novel therapeutic targets for highly resistant infections.

Systematic revelation of the protective effect and mechanism of Cordycep sinensis on diethylnitrosamine-induced rat hepatocellular carcinoma with proteomics.

PubMed

Wang, Pei-Wen; Hung, Yu-Chiang; Li, Wen-Tai; Yeh, Chau-Ting; Pan, Tai-Long

2016-09-13

Cordyceps sinensis (C. sinensis) has been reported to treat liver diseases. Here, we investigated the inhibitory effect of C. sinensis on hepatocarcinoma in a diethylnitrosamine (DEN)-induced rat model with functional proteome tools.In the DEN-exposed group, levels of serum alanine aminotransferase and aspartate aminotransferase were increased while C. sinensis application remarkably inhibited the activities of these enzymes. Histopathological analysis also indicated that C. sinensis could substantially restore hypertrophic hepatocytes caused by DEN, suggesting that C. sinensis is effective in preventing DEN-induced hepatocarcinogenesis.We therefore comprehensively delineated the global protein alterations using a proteome platform. The most meaningful changes were found among proteins involved in oxidative stress and detoxification. Meanwhile, C. sinensis application could attenuate the carbonylation level of several enzymes as well as chaperone proteins. Network analysis implied that C. sinensis could obviously alleviate hepatocarcinoma via modulating redox imbalance, protein ubiquitination and tumor growth-associated transcription factors.Our findings provide new insight into the potential effects of C. sinensis in preventing carcinogenesis and might help in developing novel therapeutic strategies against chemical-induced hepatocarcinoma.

Systematic revelation of the protective effect and mechanism of Cordycep sinensis on diethylnitrosamine-induced rat hepatocellular carcinoma with proteomics

PubMed Central

Wang, Pei-Wen; Hung, Yu-Chiang; Li, Wen-Tai; Yeh, Chau-Ting; Pan, Tai-Long

2016-01-01

Cordyceps sinensis (C. sinensis) has been reported to treat liver diseases. Here, we investigated the inhibitory effect of C. sinensis on hepatocarcinoma in a diethylnitrosamine (DEN)-induced rat model with functional proteome tools. In the DEN-exposed group, levels of serum alanine aminotransferase and aspartate aminotransferase were increased while C. sinensis application remarkably inhibited the activities of these enzymes. Histopathological analysis also indicated that C. sinensis could substantially restore hypertrophic hepatocytes caused by DEN, suggesting that C. sinensis is effective in preventing DEN-induced hepatocarcinogenesis. We therefore comprehensively delineated the global protein alterations using a proteome platform. The most meaningful changes were found among proteins involved in oxidative stress and detoxification. Meanwhile, C. sinensis application could attenuate the carbonylation level of several enzymes as well as chaperone proteins. Network analysis implied that C. sinensis could obviously alleviate hepatocarcinoma via modulating redox imbalance, protein ubiquitination and tumor growth–associated transcription factors. Our findings provide new insight into the potential effects of C. sinensis in preventing carcinogenesis and might help in developing novel therapeutic strategies against chemical-induced hepatocarcinoma. PMID:27531890

Association of A(313)G glutathione S-transferase P1 germline polymorphism with susceptibility to de novo myelodysplastic syndrome.

PubMed

Zachaki, Sophia; Stavropoulou, Chrysa; Kalomoiraki, Marina; Koromila, Theodora; Daraki, Aggeliki; Manola, Kalliopi N; Mavrou, Ariadni; Kanavakis, Emmanuel; Pantelias, Gabriel E; Sambani, Constantina

2013-08-01

Models for the pathogenesis of myelodysplastic syndrome (MDS) imply the role of individual genetic variations in genes involved in detoxification mechanisms. GSTP1 enzyme plays a key role in the biotransformation of a variety of carcinogens. The corresponding gene is subject to a single nucleotide polymorphism (A(313)G) leading to abolished enzyme activity. In order to evaluate whether the GSTP1 polymorphism influences MDS susceptibility, we conducted a case-control study comprising 310 de novo patients and 370 healthy controls using a real-time polymerase chain reaction (PCR) genotyping method. The GSTP1 gene status was also evaluated in relation to patients' characteristics and chromosomal abnormalities. A significantly higher incidence of the GSTP1 variant genotypes was observed in patients with MDS compared to controls (p < 0.0001). The results revealed increased frequencies of heterozygotes in patients younger than 60 years old and of homozygotes G/G in older patients (p = 0.007). Our results provide evidence for a pathogenetic role of the GSTP1 polymorphism in MDS risk, probably in an age-dependent manner.

A central role for S-nitrosothiols in plant disease resistance

PubMed Central

Feechan, Angela; Kwon, Eunjung; Yun, Byung-Wook; Wang, Yiqin; Pallas, Jacqueline A.; Loake, Gary J.

2005-01-01

Animal S-nitrosoglutathione reductase (GSNOR) governs the extent of cellular S-nitrosylation, a key redox-based posttranslational modification. Mutations in AtGSNOR1, an Arabidopsis thaliana GSNOR, modulate the extent of cellular S-nitrosothiol (SNO) formation in this model plant species. Loss of AtGSNOR1 function increased SNO levels, disabling plant defense responses conferred by distinct resistance (R) gene subclasses. Furthermore, in the absence of AtGSNOR1, both basal and nonhost disease resistance are also compromised. Conversely, increased AtGSNOR1 activity reduced SNO formation, enhancing protection against ordinarily virulent microbial pathogens. Here we demonstrate that AtGSNOR1 positively regulates the signaling network controlled by the plant immune system activator, salicylic acid. This contrasts with the function of this enzyme in mice during endotoxic shock, where GSNOR antagonizes inflammatory responses. Our data imply SNO formation and turnover regulate multiple modes of plant disease resistance. PMID:15911759

Homology modelling of Drosophila cytochrome P450 enzymes associated with insecticide resistance.

PubMed

Jones, Robert T; Bakker, Saskia E; Stone, Deborah; Shuttleworth, Sally N; Boundy, Sam; McCart, Caroline; Daborn, Phillip J; ffrench-Constant, Richard H; van den Elsen, Jean M H

2010-10-01

Overexpression of the cytochrome P450 gene Cyp6g1 confers resistance against DDT and a broad range of other insecticides in Drosophila melanogaster Meig. In the absence of crystal structures of CYP6G1 or complexes with its substrates, structural studies rely on homology modelling and ligand docking to understand P450-substrate interactions. Homology models are presented for CYP6G1, a P450 associated with resistance to DDT and neonicotinoids, and two other enzymes associated with insecticide resistance in D. melanogaster, CYP12D1 and CYP6A2. The models are based on a template of the X-ray structure of the phylogenetically related human CYP3A4, which is known for its broad substrate specificity. The model of CYP6G1 has a much smaller active site cavity than the template. The cavity is also 'V'-shaped and is lined with hydrophobic residues, showing high shape and chemical complementarity with the molecular characteristics of DDT. Comparison of the DDT-CYP6G1 complex and a non-resistant CYP6A2 homology model implies that tight-fit recognition of this insecticide is important in CYP6G1. The active site can accommodate differently shaped substrates ranging from imidacloprid to malathion but not the pyrethroids permethrin and cyfluthrin. The CYP6G1, CYP12D1 and CYP6A2 homology models can provide a structural insight into insecticide resistance in flies overexpressing P450 enzymes with broad substrate specificities.

Heavy metals induce oxidative stress and trigger oxidative stress-mediated heat shock protein (hsp) modulation in the intertidal copepod Tigriopus japonicus.

PubMed

Kim, Bo-Mi; Rhee, Jae-Sung; Jeong, Chang-Bum; Seo, Jung Soo; Park, Gyung Soo; Lee, Young-Mi; Lee, Jae-Seong

2014-11-01

Heat shock proteins (hsps) are induced by a wide range of environmental stressors including heavy metals in aquatic organisms. However, the effect of heavy metals on zooplankton at the molecular level remains still unclear. In this study, we measured the intracellular reactive oxygen species (ROS) level and the antioxidant enzyme activities for 96 h after exposure to five heavy metals: arsenic (As), cadmium (Cd), copper (Cu), silver (Ag), and zinc (Zn) in the intertidal copepod Tigriopus japonicus. Activities of the antioxidant enzymes were highly elevated in metal-exposed copepods, indicating that heavy metals can induce oxidative stress by generating ROS, and stimulate the involvement of antioxidant enzymes as cellular defense mechanisms. Subsequently, transcriptional changes in hsp gene families were further investigated in the metal-exposed groups for 96 h. The ROS level and glutathione (GSH) content were significantly increased in Ag-, As-, and Cu-exposed copepods, while they were only slightly elevated in Cd- and Zn-exposed groups. Based on the numbers of significantly modulated hsp genes and their expression levels for 96 h, we measured the effect of heavy metals to stress genes of T. japonicus in the following order: Cu > Zn > Ag > As > Cd, implying that Cu acts as a stronger oxidative stress inducer than other heavy metals. Of them, the expression of hsp20 and hsp70 genes was substantially modulated by exposure to heavy metals, indicating that these genes would provide a sensitive molecular biomarker for aquatic monitoring of heavy metal pollution. Copyright © 2014 Elsevier Inc. All rights reserved.

Seasonal changes in testicular steroidogenesis in the toad Bufo arenarum H.

PubMed

Canosa, L F; Ceballos, N R

2002-02-15

The biosynthesis of androgens in Bufo arenarum takes place through the 5-ene pathway that includes 5-androstane-3beta,17beta-diol as intermediate in testosterone biosynthesis. Besides testosterone and 5alpha-dihydrotestosterone, testes are able to synthesize 5alpha-pregnan-3,20-dione and several 3alpha- and 20alpha-reduced derivatives. Steroid biosynthesis changes during the breeding period (spring and early summer), turning from androgen to C21 steroid production. During the reproductive season, the production of progesterone, 5alpha-pregnan-3alpha,20alpha-diol, 3alpha-hydroxy-5alpha-pregnan-20-one, and 5alpha-pregnan-3,20-dione increases significantly. The function of most of these steroids in amphibians remains unknown. However, 5alpha-androstan-3alpha,17beta-diol and 3alpha-hydroxy-5alpha-pregnan-20-one were shown to be neuroactive in mammals, modulating sexual behavior. Thus, 5alpha/3alpha-reduced steroids could be involved in the regulation of the reproductive behavior in B. arenarum, a species with a dissociated reproductive pattern. Percentage contribution of each enzymes to the total metabolism reveals that neither 3beta-hydroxysteroid dehydrogenase/isomerase nor 5alpha-reductase change throughout the reproductive cycle. However, a strong reduction in 17-hydroxylase-C(17-20) lyase activity occurs in the reproductive season, suggesting that this enzyme could represent a key enzyme in the regulation of the seasonal change of steroidogenesis. Also, 3alpha-hydroxysteroid dehydrogenase and 20-hydroxysteroid dehydrogenase activities increase during the reproductive period, implying that steroid metabolism is clearly focused on C21-reduced steroids. (C)2002 Elsevier Science (USA).

Moonlighting Glutamate Formiminotransferases Can Functionally Replace 5-Formyltetrahydrofolate Cycloligase*

PubMed Central

Jeanguenin, Linda; Lara-Núñez, Aurora; Pribat, Anne; Mageroy, Melissa Hamner; Gregory, Jesse F.; Rice, Kelly C.; de Crécy-Lagard, Valérie; Hanson, Andrew D.

2010-01-01

5-Formyltetrahydrofolate (5-CHO-THF) is formed by a side reaction of serine hydroxymethyltransferase. Unlike other folates, it is not a one-carbon donor but a potent inhibitor of folate enzymes and must therefore be metabolized. Only 5-CHO-THF cycloligase (5-FCL) is generally considered to do this. However, comparative genomic analysis indicated (i) that certain prokaryotes lack 5-FCL, implying that they have an alternative 5-CHO-THF-metabolizing enzyme, and (ii) that the histidine breakdown enzyme glutamate formiminotransferase (FT) might moonlight in this role. A functional complementation assay for 5-CHO-THF metabolism was developed in Escherichia coli, based on deleting the gene encoding 5-FCL (ygfA). The deletion mutant accumulated 5-CHO-THF and, with glycine as sole nitrogen source, showed a growth defect; both phenotypes were complemented by bacterial or archaeal genes encoding FT. Furthermore, utilization of supplied 5-CHO-THF by Streptococcus pyogenes was shown to require expression of the native FT. Recombinant bacterial and archaeal FTs catalyzed formyl transfer from 5-CHO-THF to glutamate, with kcat values of 0.1–1.2 min−1 and Km values for 5-CHO-THF and glutamate of 0.4–5 μm and 0.03–1 mm, respectively. Although the formyltransferase activities of these proteins were far lower than their formiminotransferase activities, the Km values for both substrates relative to their intracellular levels in prokaryotes are consistent with significant in vivo flux through the formyltransferase reaction. Collectively, these data indicate that FTs functionally replace 5-FCL in certain prokaryotes. PMID:20952389

Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase[OPEN

PubMed Central

Niehaus, Thomas D.; Hasnain, Ghulam; Gidda, Satinder K.; Nguyen, Thuy N.D.; Anderson, Erin M.; Brown, Greg; Yakunin, Alexander F.; de Crécy-Lagard, Valérie; Gregory, Jesse F.

2016-01-01

To synthesize the cofactor thiamin diphosphate (ThDP), plants must first hydrolyze thiamin monophosphate (ThMP) to thiamin, but dedicated enzymes for this hydrolysis step were unknown and widely doubted to exist. The classical thiamin-requiring th2-1 mutation in Arabidopsis thaliana was shown to reduce ThDP levels by half and to increase ThMP levels 5-fold, implying that the THIAMIN REQUIRING2 (TH2) gene product could be a dedicated ThMP phosphatase. Genomic and transcriptomic data indicated that TH2 corresponds to At5g32470, encoding a HAD (haloacid dehalogenase) family phosphatase fused to a TenA (thiamin salvage) family protein. Like the th2-1 mutant, an insertional mutant of At5g32470 accumulated ThMP, and the thiamin requirement of the th2-1 mutant was complemented by wild-type At5g32470. Complementation tests in Escherichia coli and enzyme assays with recombinant proteins confirmed that At5g32470 and its maize (Zea mays) orthologs GRMZM2G148896 and GRMZM2G078283 are ThMP-selective phosphatases whose activity resides in the HAD domain and that the At5g32470 TenA domain has the expected thiamin salvage activity. In vitro and in vivo experiments showed that alternative translation start sites direct the At5g32470 protein to the cytosol and potentially also to mitochondria. Our findings establish that plants have a dedicated ThMP phosphatase and indicate that modest (50%) ThDP depletion can produce severe deficiency symptoms. PMID:27677881

Involvement of plasma membrane peroxidases and oxylipin pathway in the recovery from phytoplasma disease in apple (Malus domestica).

PubMed

Patui, Sonia; Bertolini, Alberto; Clincon, Luisa; Ermacora, Paolo; Braidot, Enrico; Vianello, Angelo; Zancani, Marco

2013-06-01

Apple trees (Malus domestica Borkh.) may be affected by apple proliferation (AP), caused by 'Candidatus Phytoplasma mali'. Some plants can spontaneously recover from the disease, which implies the disappearance of symptoms through a phenomenon known as recovery. In this article it is shown that NAD(P)H peroxidases of leaf plasma membrane-enriched fractions exhibited a higher activity in samples from both AP-diseased and recovered plants. In addition, an increase in endogenous SA was characteristic of the symptomatic plants, since its content increased in samples obtained from diseased apple trees. In agreement, phenylalanine ammonia lyase (PAL) activity, a key enzyme of the phenylpropanoid pathway, was increased too. Jasmonic acid (JA) increased only during recovery, in a phase subsequent to the pathological state, and in concomitance to a decline of salicylic acid (SA). Oxylipin pathway, responsible for JA synthesis, was not induced during the development of AP-disease, but it appeared to be stimulated when the recovery occurred. Accordingly, lipoxygenase (LOX) activity, detected in plasma membrane-enriched fractions, showed an increase in apple leaves obtained from recovered plants. This enhancement was paralleled by an increase of hydroperoxide lyase (HPL) activity, detected in leaf microsomes, albeit the latter enzyme was activated in either the disease or recovery conditions. Hence, a reciprocal antagonism between SA- and JA-pathways could be suggested as an effective mechanism by which apple plants react to phytoplasma invasions, thereby providing a suitable defense response leading to the establishment of the recovery phenomenon. Copyright © Physiologia Plantarum 2012.

The antiproliferative activity of di-2-pyridylketone dithiocarbamate is partly attributed to catalase inhibition: detailing the interaction by spectroscopic methods.

PubMed

Li, Cuiping; Liu, Youxun; Fu, Yun; Huang, Tengfei; Kang, Lixia; Li, Changzheng

2017-08-22

The bioactivity of drugs is attributed to their interaction with biological molecules, embodied in either their direct or indirect influence on enzyme activity and conformation. Di-2-pyridylketone hydrazine dithiocarbamate (DpdtC) exhibits significant antitumor activity in our preliminary study. We speculated that its activity may partly stem from enzyme inhibition due to strong metal chelating ability. To this end, we assessed its effect on catalase from erythrocytes and found evidence of inhibition, which was further confirmed by ROS determination in vivo. Thus, detailing the interaction between the agent and catalase via spectroscopic methods and molecular docking was required to obtain information on both the dynamics and thermodynamic parameters. The Lineweaver-Burk plot implied an uncompetitive pattern between DpdtC and catalase from beef liver, and IC 50 = ∼7 μM. The thermodynamic parameters from fluorescence quenching measurements indicated that DpdtC could bind to catalase with moderate affinity (K a = approximately 10 4 M -1 ). CD spectra revealed that DpdtC could significantly disrupt the secondary structure of catalase. Docking studies indicated that DpdtC bound to a flexible region of catalase, involving hydrogen bonds and salt bond; this was consistent with thermodynamic results from spectral investigations. Our data clearly showed that catalase inhibition of DpdtC was not due to direct chelation of iron from heme (killing), but through an allosteric effect. Thus, it can be concluded that the antiproliferative activity of DpdtC is partially attributed to its catalase inhibition.

Association of angiotensin-converting enzyme (ACE) gene polymorphism with elevated serum ACE activity and major depression in an Iranian population.

PubMed

Firouzabadi, Negar; Shafiei, Massoumeh; Bahramali, Ehsan; Ebrahimi, Soltan Ahmed; Bakhshandeh, Hooman; Tajik, Nader

2012-12-30

Genetic factors contribute substantially to the likelihood of developing major depressive disorder (MDD). The importance of renin-angiotensin system (RAS) elements in cognition and behaviour and their involvement in aetiology and treatment of depression imply that RAS gene polymorphism(s) associated with RAS overactivity might also be associated with depression. In the present study, genotype and allele frequencies of six common polymorphisms of genes encoding for RAS components were determined in DNAs extracted from venous blood of 191 depressed and 104 healthy individuals using polymerase chain reaction (PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and serum angiotensin-converting enzyme (ACE) activity was assayed using a high-performance liquid chromatography (HPLC) method. The results showed, for the first time, that GG genotype of ACE A2350G was significantly associated with MDD among Iranian participants (P=0.001; odds ratio (OR)=6.2; 95% confidence interval (CI)=2.1-18.3). Significant higher serum ACE activity (P=0.0001) as well as higher diastolic blood pressure (P=0.036) were observed in depressed patients compared to the healthy control group. Depressed patients carrying GG genotype of the A2350G polymorphism had a significantly higher serum ACE activity (P=0.02) than individuals with either AA or AG genotype. In conclusion, this study supports the hypothesis of RAS overactivity in depression in that the genotype associated with higher serum ACE activity in an Iranian population was also associated with MDD. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Effects of memantine on soluble Alphabeta(25-35)-induced changes in peptidergic and glial cells in Alzheimer's disease model rat brain regions.

PubMed

Arif, M; Chikuma, T; Ahmed, Md M; Nakazato, M; Smith, M A; Kato, T

2009-12-15

Soluble forms of amyloid-beta (Abeta) have been considered responsible for cognitive dysfunction prior to senile plaque formation in Alzheimer's disease (AD). As its mechanism is not well understood, we examined the effects of repeated i.c.v. infusion of soluble Alphabeta(25-35) on peptidergic system and glial cells in the pathogenesis of AD. The present study aims to investigate the protective effects of memantine on Abeta(25-35)-induced changes in peptidergic and glial systems. Infusion of Alphabeta(25-35) decreased the level of immunoreactive somatostatin (SS) and substance P (SP) in the hippocampus prior to neuronal loss or caspase activation, which is correlated with the loss of spine density and activation of inducible nitric-oxide synthase (iNOS). Biochemical experiment with peptide-degrading enzymes, prolyl oligopeptidase (POP) and endopeptidase 24.15 (EP 24.15) activities demonstrated a concomitant increase with the activation of glial marker proteins, glial fibrillary acidic protein (GFAP) and CD11b in the Abeta-treated hippocampus. Double immunostaining experiments of EP 24.15 and GFAP/CD11b antibodies clearly demonstrated the co-localization of neuro peptidases with astrocytes and microglia. Treatment with memantine, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist significantly attenuated Abeta(25-35)-induced changes of neuropeptides, their metabolizing enzymes, glial marker proteins, and activation of iNOS. Taken together, the data implies that memantine exerts its protective effects by modulating the neuropeptide system as a consequence of suppressing the glial cells and oxidative stress in AD model rat brain regions.

Relationships between the Efficiencies of Photosystems I and II and Stromal Redox State in CO2-Free Air 1

PubMed Central

Harbinson, Jeremy; Foyer, Christine H.

1991-01-01

The responses of the efficiencies of photosystems I and II, stromal redox state (as indicated by NADP-malate dehydrogenase activation state), and activation of the Benson-Calvin cycle enzymes ribulose 1,5-bisphosphate carboxylase and fructose 1,6-bisphosphatase to varying irradiance were measured in pea (Pisum sativum L.) leaves operating close to the CO2 compensation point. A comparison of the relationships among these parameters obtained from leaves in air was made with those obtained when the leaves were maintained in air from which the CO2 had been removed. P700 was more oxidized at any measured irradiance in CO2-free air than in air. The relationship between the quantum efficiencies of the photosystems in CO2-free air was distinctly curvilinear in contrast to the predominantly linear relationship obtained with leaves in air. This nonlinearity may be consistent with the operation of cyclic electron flow around photosystem I because the quantum efficiency of photosystem II was much more restricted than the quantum efficiency of photosystem I. In CO2-free air, measured NADP-malate dehydrogenase activities varied considerably at low irradiances. However, at high irradiance the activity of the enzyme was low, implying that the stroma was oxidized. In contrast, fructose-1,6-bisphosphatase activities tended to increase with increasing electron flux through the photosystems. Ribulose-1,5-bisphosphate carboxylase activity remained relatively constant with respect to irradiance in CO2-free air, with an activation state 50% of maximum. We conclude that, at the CO2 compensation point and high irradiance, low redox states are favored and that cyclic electron flow may be substantial. These two features may be the requirements necessary to trigger and maintain the dissipative processes in the thylakoid membrane. PMID:16668401

Evaluation of IgE reactivity of active and thermally inactivated actinidin, a biomarker of kiwifruit allergy.

PubMed

Grozdanovic, Milica; Popovic, Milica; Polovic, Natalija; Burazer, Lidija; Vuckovic, Olga; Atanaskovic-Markovic, Marina; Lindner, Buko; Petersen, Arnd; Gavrovic-Jankulovic, Marija

2012-03-01

Actinidin, an abundant cysteine protease from kiwifruit, is a specific biomarker of isolated allergy to kiwifruit. This study evaluates the IgE-binding properties of biologically active and thermally inactivated actinidin. Employing two different activity assays (caseinolytic assay and zymogram with gelatin) we showed that actinidin obtained from kiwifruit extract under native conditions represents a mixture of inactive and active enzyme. The structural integrity of actinidin was confirmed by SDS-PAGE, Edman degradation, mass fingerprint and Western blot with polyclonal antibodies. Although it was capable of inducing positive skin prick test reactions, we failed to detect IgE reactivity of active actinidin in Western blot with patient sera. Thermally inactivated actinidin exhibited IgE reactivity both in vivo and in vitro, indicating that heat processed kiwifruit products may induce clinical reactivity. These findings imply that apart from the allergenic epitopes on its surface, actinidin also contains hidden epitopes inside the protein which become accessible to IgE upon thermal treatment. Copyright © 2011 Elsevier Ltd. All rights reserved.

Evaluation of the impact on food safety of a Lactobacillus coryniformis strain from pickled vegetables with degradation activity against nitrite and other undesirable compounds.

PubMed

Fang, Fang; Feng, Tingting; Du, Guocheng; Chen, Jian

2016-01-01

Four strains of lactic acid bacteria showing antimicrobial activity against some food-spoilage microorganisms or pathogens, including both Gram-negative and -positive strains, were isolated from naturally fermented pickled vegetables and a traditional cheese product. Among these isolates, Lactobacillus coryniformis strain BBE-H3, characterised previously to be a non-biogenic amine producer, showed a high level of activity in degrading sodium nitrite and exhibited the ability to eliminate ethyl carbamate and one of its precursors, urea. The antimicrobial substance produced by L. coryniformis BBE-H3 was found to be active at an acidic pH range of 4.0-4.5. The antimicrobial activity of this strain decreased differentially after treatment with proteolytic enzymes (pepsin, papain, trypsin and proteinase K), implying this growth inhibitory compound is either a protein or a polypeptide. The results of this study show the suitability of L. coryniformis BBE-H3 as a starter in food manufacturing processes, and demonstrate its potential role in eliminating food origin carcinogens such as sodium nitrite and ethyl carbamate.

Chlorophyll catabolism in olive fruits (var. Arbequina and Hojiblanca) during maturation.

PubMed

Vergara-Domínguez, Honorio; Ríos, José Julían; Gandul-Rojas, Beatriz; Roca, María

2016-12-01

The central reaction of chlorophyll (chl) breakdown pathway occurring during olive fruits maturation is the cleavage of the macrocycle pheophorbide a to a primary fluorescent chl catabolite (pFCC) and it is catalyzed by two enzymes: pheophorbide a oxygenase (PaO) and red chl catabolite reductase (RCCR). In subsequent steps, pFCC is converted to different fluorescent chlorophyll catabolites (FCCs) and nonfluorescent chlorophyll catabolites (NCCs). This work demonstrated that RCCR activity of olive fruits is type II. During the study of evolution of PaO and RCCR activities through the olive fruits maturation in two varieties: Hojiblanca and Arbequina, a significant increase in PaO and RCCR activity was found in ripening stage. In addition, the profile and structure of NCCs present in epicarp of this fruit was studied using HPLC/ESI-TOF-MS. Five different NCCs were defined and for the first time the enzymatic reactions implied in chlorophyll degradations in olive fruits elucidated. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections.

PubMed

Jiang, Dong; Weidner, Jessica M; Qing, Min; Pan, Xiao-Ben; Guo, Haitao; Xu, Chunxiao; Zhang, Xianchao; Birk, Alex; Chang, Jinhong; Shi, Pei-Yong; Block, Timothy M; Guo, Ju-Tao

2010-08-01

Interferons (IFNs) are key mediators of the host innate antiviral immune response. To identify IFN-stimulated genes (ISGs) that instigate an antiviral state against two medically important flaviviruses, West Nile virus (WNV) and dengue virus (DENV), we tested 36 ISGs that are commonly induced by IFN-alpha for antiviral activity against the two viruses. We discovered that five ISGs efficiently suppressed WNV and/or DENV infection when they were individually expressed in HEK293 cells. Mechanistic analyses revealed that two structurally related cell plasma membrane proteins, IFITM2 and IFITM3, disrupted early steps (entry and/or uncoating) of the viral infection. In contrast, three IFN-induced cellular enzymes, viperin, ISG20, and double-stranded-RNA-activated protein kinase, inhibited steps in viral proteins and/or RNA biosynthesis. Our results thus imply that the antiviral activity of IFN-alpha is collectively mediated by a panel of ISGs that disrupt multiple steps of the DENV and WNV life cycles.

Cloning, expression and characterization of glycerol dehydrogenase involved in 2,3-butanediol formation in Serratia marcescens H30.

PubMed

Zhang, Liaoyuan; Xu, Quanming; Peng, Xiaoqian; Xu, Boheng; Wu, Yuehao; Yang, Yulong; Sun, Shujing; Hu, Kaihui; Shen, Yaling

2014-09-01

The meso-2,3-butanediol dehydrogenase (meso-BDH) from S. marcescens H30 is responsible for converting acetoin into 2,3-butanediol during sugar fermentation. Inactivation of the meso-BDH encoded by budC gene does not completely abolish 2,3-butanediol production, which suggests that another similar enzyme involved in 2,3-butanediol formation exists in S. marcescens H30. In the present study, a glycerol dehydrogenase (GDH) encoded by gldA gene from S. marcescens H30 was expressed in Escherichia coli BL21(DE3), purified and characterized for its properties. In vitro conversion indicated that the purified GDH could catalyze the interconversion of (3S)-acetoin/meso-2,3-butanediol and (3R)-acetoin/(2R,3R)-2,3-butanediol. (2S,3S)-2,3-Butanediol was not a substrate for the GDH at all. Kinetic parameters of the GDH enzyme showed lower K m value and higher catalytic efficiency for (3S/3R)-acetoin in comparison to those for (2R,3R)-2,3-butanediol and meso-2,3-butanediol, implying its physiological role in favor of 2,3-butanediol formation. Maximum activity for reduction of (3S/3R)-acetoin and oxidations of meso-2,3-butanediol and glycerol was observed at pH 8.0, while it was pH 7.0 for diacetyl reduction. The enzyme exhibited relative high thermotolerance with optimum temperature of 60 °C in the oxidation-reduction reactions. Over 60 % of maximum activity was retained at 70 °C. Additionally, the GDH activity was significantly enhanced for meso-2,3-BD oxidation in the presence of Fe(2+) and for (3S/3R)-acetoin reduction in the presence of Mn(2+), while several cations inhibited its activity, particularly Fe(2+) and Fe(3+) for (3S/3R)-acetoin reduction. The properties provided potential application for single configuration production of acetoin and 2,3-butanediol .

Separating neural activity associated with emotion and implied motion: An fMRI study.

PubMed

Kolesar, Tiffany A; Kornelsen, Jennifer; Smith, Stephen D

2017-02-01

Previous research provides evidence for an emo-motoric neural network allowing emotion to modulate activity in regions of the nervous system related to movement. However, recent research suggests that these results may be due to the movement depicted in the stimuli. The purpose of the current study was to differentiate the unique neural activity of emotion and implied motion using functional MRI. Thirteen healthy participants viewed 4 sets of images: (a) negative stimuli implying movement, (b) negative stimuli not implying movement, (c) neutral stimuli implying movement, and (d) neutral stimuli not implying movement. A main effect for implied motion was found, primarily in regions associated with multimodal integration (bilateral insula and cingulate), and visual areas that process motion (bilateral middle temporal gyrus). A main effect for emotion was found primarily in occipital and parietal regions, indicating that emotion enhances visual perception. Surprisingly, emotion also activated the left precentral gyrus, a motor region. These results demonstrate that emotion elicits activity above and beyond that evoked by the perception of implied movement, but that the neural representations of these characteristics overlap. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

New Insights into the Function and Global Distribution of Polyethylene Terephthalate (PET)-Degrading Bacteria and Enzymes in Marine and Terrestrial Metagenomes.

PubMed

Danso, Dominik; Schmeisser, Christel; Chow, Jennifer; Zimmermann, Wolfgang; Wei, Ren; Leggewie, Christian; Li, Xiangzhen; Hazen, Terry; Streit, Wolfgang R

2018-04-15

Polyethylene terephthalate (PET) is one of the most important synthetic polymers used today. Unfortunately, the polymers accumulate in nature and to date no highly active enzymes are known that can degrade it at high velocity. Enzymes involved in PET degradation are mainly α- and β-hydrolases, like cutinases and related enzymes (EC 3.1.1). Currently, only a small number of such enzymes are well characterized. In this work, a search algorithm was developed that identified 504 possible PET hydrolase candidate genes from various databases. A further global search that comprised more than 16 Gb of sequence information within 108 marine and 25 terrestrial metagenomes obtained from the Integrated Microbial Genome (IMG) database detected 349 putative PET hydrolases. Heterologous expression of four such candidate enzymes verified the function of these enzymes and confirmed the usefulness of the developed search algorithm. In this way, two novel and thermostable enzymes with high potential for downstream application were partially characterized. Clustering of 504 novel enzyme candidates based on amino acid similarities indicated that PET hydrolases mainly occur in the phyla of Actinobacteria , Proteobacteria , and Bacteroidetes Within the Proteobacteria , the Betaproteobacteria , Deltaproteobacteria , and Gammaproteobacteria were the main hosts. Remarkably enough, in the marine environment, bacteria affiliated with the phylum Bacteroidetes appear to be the main hosts of PET hydrolase genes, rather than Actinobacteria or Proteobacteria , as observed for the terrestrial metagenomes. Our data further imply that PET hydrolases are truly rare enzymes. The highest occurrence of 1.5 hits/Mb was observed in sequences from a sample site containing crude oil. IMPORTANCE Polyethylene terephthalate (PET) accumulates in our environment without significant microbial conversion. Although a few PET hydrolases are already known, it is still unknown how frequently they appear and with which main bacterial phyla they are affiliated. In this study, deep sequence mining of protein databases and metagenomes demonstrated that PET hydrolases indeed occur at very low frequencies in the environment. Furthermore, it was possible to link them to phyla that were previously not known to harbor such enzymes. This work contributes novel knowledge on the phylogenetic relationships, the recent evolution, and the global distribution of PET hydrolases. Finally, we describe the biochemical traits of four novel PET hydrolases. Copyright © 2018 Danso et al.

New Insights into the Function and Global Distribution of Polyethylene Terephthalate (PET)-Degrading Bacteria and Enzymes in Marine and Terrestrial Metagenomes

PubMed Central

Danso, Dominik; Schmeisser, Christel; Chow, Jennifer; Wei, Ren; Leggewie, Christian; Li, Xiangzhen

2018-01-01

ABSTRACT Polyethylene terephthalate (PET) is one of the most important synthetic polymers used today. Unfortunately, the polymers accumulate in nature and to date no highly active enzymes are known that can degrade it at high velocity. Enzymes involved in PET degradation are mainly α- and β-hydrolases, like cutinases and related enzymes (EC 3.1.1). Currently, only a small number of such enzymes are well characterized. In this work, a search algorithm was developed that identified 504 possible PET hydrolase candidate genes from various databases. A further global search that comprised more than 16 Gb of sequence information within 108 marine and 25 terrestrial metagenomes obtained from the Integrated Microbial Genome (IMG) database detected 349 putative PET hydrolases. Heterologous expression of four such candidate enzymes verified the function of these enzymes and confirmed the usefulness of the developed search algorithm. In this way, two novel and thermostable enzymes with high potential for downstream application were partially characterized. Clustering of 504 novel enzyme candidates based on amino acid similarities indicated that PET hydrolases mainly occur in the phyla of Actinobacteria, Proteobacteria, and Bacteroidetes. Within the Proteobacteria, the Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria were the main hosts. Remarkably enough, in the marine environment, bacteria affiliated with the phylum Bacteroidetes appear to be the main hosts of PET hydrolase genes, rather than Actinobacteria or Proteobacteria, as observed for the terrestrial metagenomes. Our data further imply that PET hydrolases are truly rare enzymes. The highest occurrence of 1.5 hits/Mb was observed in sequences from a sample site containing crude oil. IMPORTANCE Polyethylene terephthalate (PET) accumulates in our environment without significant microbial conversion. Although a few PET hydrolases are already known, it is still unknown how frequently they appear and with which main bacterial phyla they are affiliated. In this study, deep sequence mining of protein databases and metagenomes demonstrated that PET hydrolases indeed occur at very low frequencies in the environment. Furthermore, it was possible to link them to phyla that were previously not known to harbor such enzymes. This work contributes novel knowledge on the phylogenetic relationships, the recent evolution, and the global distribution of PET hydrolases. Finally, we describe the biochemical traits of four novel PET hydrolases. PMID:29427431

Human dipeptidyl peptidase III acts as a post-proline-cleaving enzyme on endomorphins.

PubMed

Barsun, Marina; Jajcanin, Nina; Vukelić, Bojana; Spoljarić, Jasminka; Abramić, Marija

2007-03-01

Dipeptidyl peptidase III (DPP III) is a zinc exopeptidase with an implied role in the mammalian pain-modulatory system owing to its high affinity for enkephalins and localisation in the superficial laminae of the spinal cord dorsal horn. Our study revealed that this human enzyme hydrolyses opioid peptides belonging to three new groups, endomorphins, hemorphins and exorphins. The enzymatic hydrolysis products of endomorphin-1 were separated and quantified by capillary electrophoresis and the kinetic parameters were determined for human DPP III and rat DPP IV. Both peptidases cleave endomorphin-1 at comparable rates, with liberation of the N-terminal Tyr-Pro. This is the first evidence of DPP III acting as an endomorphin-cleaving enzyme.

Substrate-induced inactivation of the OXA2 beta-lactamase.

PubMed Central

Ledent, P; Frère, J M

1993-01-01

The hydrolysis time courses of 22 beta-lactam antibiotics by the class D OXA2 beta-lactamase were studied. Among these, only three appeared to correspond to the integrated Henri-Michaelis equation. 'Burst' kinetics, implying branched pathways, were observed with most penicillins, cephalosporins and with flomoxef and imipenem. Kinetic parameters characteristic of the different phases of the hydrolysis were determined for some substrates. Mechanisms generally accepted to explain such reversible partial inactivations involving branches at either the free enzyme or the acyl-enzyme were inadequate to explain the enzyme behaviour. The hydrolysis of imipenem was characterized by the occurrence of two 'bursts', and that of nitrocefin by a partial substrate-induced inactivation complicated by a competitive inhibition by the hydrolysis product. PMID:8240304

Intracellular postsynaptic cannabinoid receptors link thyrotropin-releasing hormone receptors to TRPC-like channels in thalamic paraventricular nucleus neurons.

PubMed

Zhang, L; Kolaj, M; Renaud, L P

2015-12-17

In rat thalamic paraventricular nucleus of thalamus (PVT) neurons, activation of thyrotropin-releasing hormone (TRH) receptors enhances excitability via concurrent decrease in G protein-coupled inwardly-rectifying potassium (GIRK)-like and activation of transient receptor potential cation (TRPC)4/5-like cationic conductances. An exploration of intracellular signaling pathways revealed the TRH-induced current to be insensitive to phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitors, but reduced by D609, an inhibitor of phosphatidylcholine-specific PLC (PC-PLC). A corresponding change in the I-V relationship implied suppression of the cationic component of the TRH-induced current. Diacylglycerol (DAG) is a product of the hydrolysis of PC. Studies focused on the isolated cationic component of the TRH-induced response revealed a reduction by RHC80267, an inhibitor of DAG lipase, the enzyme involved in the hydrolysis of DAG to the endocannabinoid 2-arachidonoylglycerol (2-AG). Further investigation revealed enhancement of the cationic component in the presence of either JZL184 or WWL70, inhibitors of enzymes involved in the hydrolysis of 2-AG. A decrease in the TRH-induced response was noted in the presence of rimonabant or SR144528, membrane permeable CB1 and CB2 receptor antagonists, respectively. A decrease in the TRH-induced current by intracellular, but not by bath application of the membrane impermeable peptide hemopressin, selective for CB1 receptors, suggests a postsynaptic intracellular localization of these receptors. The TRH-induced current was increased in the presence of arachidonyl-2'-chloroethylamide (ACEA) or JWH133, CB1 and CB2 receptor agonists, respectively. The PI3-kinase inhibitor LY294002, known to inhibit TRPC translocation, decreased the response to TRH. In addition, a TRH-induced enhancement of the low-threshold spike was prevented by both rimonabant, and SR144528. TRH had no influence on excitatory or inhibitory miniature postsynaptic currents, suggesting presynaptic CB receptors are not involved in this situation. Collectively, the data imply that activation of TRH receptors in these midline thalamic neurons engages novel signaling pathways that include postsynaptic intracellular CB1 and CB2 receptors in the activation of TRPC4/5-like channels. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Oxidative stress response of the aquatic macrophyte Hydrilla verticillata exposed to TiO2 nanoparticles.

PubMed

Okupnik, Annette; Pflugmacher, Stephan

2016-11-01

The present study investigated the effects of titanium dioxide nanoparticles (TiO 2 -NPs) on the oxidative stress response in Hydrilla verticillata. Macrophytes were exposed to different concentrations of TiO 2 -NPs (0 mg/L, 0.01 mg/L, 0.1 mg/L, 1 mg/L, and 10 mg/L) for 24 h, based on currently predicted levels of nano-TiO 2 in surface waters. In addition, TiO 2 -NPs with varying crystalline status were used to assess the potential influence of crystalline phases on oxidative stress responses. The level of hydrogen peroxide (H 2 O 2 ), reduced and oxidized glutathione (GSH and GSSG), and activities of the antioxidative enzymes peroxidase (POD), catalase (CAT), and glutathione reductase (GR) were measured and compared with a bulk counterpart. Although POD was not considered to be active, the results imply an activation of the enzymatic defense system, because increased CAT and GR activities were observed. Exposure to bulk TiO 2 revealed lower enzyme activities at all exposure concentrations, suggesting a nano-specific influence on the antioxidative defense mechanisms in H. verticillata. Moreover, all TiO 2 -NP concentrations resulted in a decreased GSH/GSSG ratio, indicating high GSH-dependent metabolic activity to protect against the destructive effects of reactive oxygen species (ROS) generated during nano-TiO 2 exposure. As the level of H 2 O 2 was solely elevated after exposure to 10 mg/L of P25, it appears plausible that the adaptive metabolic mechanisms of H. verticillata are able to cope with environmentally relevant concentrations of TiO 2 -NPs. Environ Toxicol Chem 2016;35:2859-2866. © 2016 SETAC. © 2016 SETAC.

Slow domain reconfiguration causes power-law kinetics in a two-state enzyme.

PubMed

Grossman-Haham, Iris; Rosenblum, Gabriel; Namani, Trishool; Hofmann, Hagen

2018-01-16

Protein dynamics are typically captured well by rate equations that predict exponential decays for two-state reactions. Here, we describe a remarkable exception. The electron-transfer enzyme quiescin sulfhydryl oxidase (QSOX), a natural fusion of two functionally distinct domains, switches between open- and closed-domain arrangements with apparent power-law kinetics. Using single-molecule FRET experiments on time scales from nanoseconds to milliseconds, we show that the unusual open-close kinetics results from slow sampling of an ensemble of disordered domain orientations. While substrate accelerates the kinetics, thus suggesting a substrate-induced switch to an alternative free energy landscape of the enzyme, the power-law behavior is also preserved upon electron load. Our results show that the slow sampling of open conformers is caused by a variety of interdomain interactions that imply a rugged free energy landscape, thus providing a generic mechanism for dynamic disorder in multidomain enzymes.

Effects of dietary quercetin on female fertility in mice: implication of transglutaminase 2

PubMed Central

Beazley, Kelly E.; Nurminskaya, Maria

2016-01-01

Use of the dietary supplement quercetin is on the rise. Because previous studies imply an inhibitory effect of quercetin on male fertility, we explored the effects of this flavonoid on fertility in female mice. Birth outcomes, and ovarian morphology in 4-week-old offspring, were assessed in mice receiving dietary quercetin (5 mg kg−1 day−1) for 9 months during two breeding periods: from 2 to 6 months (prime reproductive age) and 8 to11 months of age. Quercetin increased birth spacing, leading to a 60% reduction in the number of litters, but enhanced folliculogenesis in ovaries of female offspring. While in young females quercetin caused an almost 70% increase in litter size, in older animals this effect was reversed. Consistent with the inhibitory activity of quercetin on the enzyme transglutaminase 2 (TG2), genetic ablation of TG2 in mice mirrors the effects of quercetin on birth outcomes and follicular development. Further, TG2-null mice lack responsiveness to quercetin ingestion. Our study shows for the first time that dietary quercetin can cause reduced reproductive potential in female mice and implies that TG2 may regulate ovarian ageing. PMID:25557047

Oxidative stress mediated toxicity of TiO2 nanoparticles after a concentration and time dependent exposure of the aquatic macrophyte Hydrilla verticillata.

PubMed

Spengler, Annette; Wanninger, Lena; Pflugmacher, Stephan

2017-09-01

The present study focused on oxidative stress effects in the aquatic macrophyte Hydrilla verticillata after exposure to titanium dioxide nanoparticles (TiO 2 -NPs). Experiments were conducted with different TiO 2 -NPs and concentrations (0.1 mg/L and 10 mg/L) in a time-dependent manner (0 h, 24 h, 48 h, 96 h, 168 h). To assess various levels of the oxidative stress response in H. verticillata, the level of hydrogen peroxide (H 2 O 2 ), the ratio of reduced to oxidized glutathione (GSH/GSSG), and activities of the antioxidative enzymes catalase (CAT) and glutathione reductase (GR) were evaluated. Study results imply oxidative stress effects after TiO 2 -NP exposure as adaptations in plant metabolism became apparent to counteract increased ROS formation. All TiO 2 -NPs caused elevated activities of the enzymes CAT and GR. Moreover, decreased ratios of GSH/GSSG indicated an activation of GSH-dependent pathways counteracting ROS formation. Plants exposed to a bulk-sized control revealed a size-dependent influence on the antioxidative stress response. As H 2 O 2 level increases were solely detected after exposure to 10 mg/L TiO 2 -NPs and nano-exposed plants showed normalization in its antioxidative stress response after 168h of exposure, it can be suggested that macrophytes are able to cope with currently predicted low-level exposures to TiO 2 -NPs. Copyright © 2017 Elsevier B.V. All rights reserved.

Metabolic and cellular stress responses of catfish, Horabagrus brachysoma (Günther) acclimated to increasing temperatures.

PubMed

Dalvi, Rishikesh S; Das, Tilak; Debnath, Dipesh; Yengkokpam, Sona; Baruah, Kartik; Tiwari, Lalchand R; Pal, Asim K

2017-04-01

We investigated the metabolic and cellular stress responses in an endemic catfish Horabagrus brachysoma acclimated to ambient (26°C), 31, 33 and 36°C for 30 days. After acclimation, fish were sampled to investigate changes in the levels of blood glucose, tissue glycogen and ascorbic acid, activities of enzymes involved in glycolysis (LDH), citric acid cycle (MDH), gluconeogenesis (FBPase and G6Pase), pentose phosphate pathway (G6PDH), protein metabolism (AST and ALT), phosphate metabolism (ACP and ALP) and energy metabolism (ATPase), and HSP70 levels in various tissues. Acclimation to higher temperatures (33 and 36°C) significantly increased activities of LDH, MDH, ALP, ACP, AST, ALT and ATPase and blood glucose levels, whereas decreased the G6PDH enzyme activity and, tissue glycogen and ascorbic acid. Results indicated an overall increase in the carbohydrate, protein and lipid metabolism implying increased metabolic demands for maintaining homeostasis in fish acclimated to higher temperatures (33 and 36°C). We observed tissue specific response of HSP70 in H. brachysoma, with significant increase in gill and liver at 33 and 36°C, and in brain and muscle at 36°C, enabling cellular protection at higher acclimation temperatures. In conclusion, H. brachysoma adjusted metabolic and cellular responses to withstand increased temperatures, however, these responses suggest that the fish was under stress at 33°C or higher temperature. Copyright © 2017 Elsevier Ltd. All rights reserved.

Large-scale variation in subsurface stream biofilms: a cross-regional comparison of metabolic function and community similarity.

PubMed

Findlay, S; Sinsabaugh, R L

2006-10-01

We examined bacterial metabolic activity and community similarity in shallow subsurface stream sediments distributed across three regions of the eastern United States to assess whether there were parallel changes in functional and structural attributes at this large scale. Bacterial growth, oxygen consumption, and a suite of extracellular enzyme activities were assayed to describe functional variability. Community similarity was assessed using randomly amplified polymorphic DNA (RAPD) patterns. There were significant differences in streamwater chemistry, metabolic activity, and bacterial growth among regions with, for instance, twofold higher bacterial production in streams near Baltimore, MD, compared to Hubbard Brook, NH. Five of eight extracellular enzymes showed significant differences among regions. Cluster analyses of individual streams by metabolic variables showed clear groups with significant differences in representation of sites from different regions among groups. Clustering of sites based on randomly amplified polymorphic DNA banding resulted in groups with generally less internal similarity although there were still differences in distribution of regional sites. There was a marginally significant (p = 0.09) association between patterns based on functional and structural variables. There were statistically significant but weak (r2 approximately 30%) associations between landcover and measures of both structure and function. These patterns imply a large-scale organization of biofilm communities and this structure may be imposed by factor(s) such as landcover and covariates such as nutrient concentrations, which are known to also cause differences in macrobiota of stream ecosystems.

Molecular Basis for the Catalytic Specificity of the CTX-M Extended-Spectrum β-Lactamases

DOE PAGES

Adamski, Carolyn J.; Cardenas, Ana Maria; Brown, Nicholas G.; ...

2014-12-09

We report that extended-spectrum β-lactamases (ESBLs) pose a threat to public health because of their ability to confer resistance to extended-spectrum cephalosporins such as cefotaxime. The CTX-M β-lactamases are the most widespread ESBL enzymes among antibiotic resistant bacteria. Many of the active site residues are conserved between the CTX-M family and non-ESBL β-lactamases such as TEM-1, but the residues Ser237 and Arg276 are specific to the CTX-M family, suggesting that they may help to define the increased specificity for cefotaxime hydrolysis. To test this hypothesis, site-directed mutagenesis of these positions was performed in the CTX-M-14 β-lactamase. Substitutions of Ser237 andmore » Arg276 with their TEM-1 counterparts, Ala237 and Asn276, had a modest effect on cefotaxime hydrolysis, as did removal of the Arg276 side chain in an R276A mutant. The S237A:R276N and S237A:R276A double mutants, however, exhibited 29- and 14-fold losses in catalytic efficiency for cefotaxime hydrolysis, respectively, while the catalytic efficiency for benzylpenicillin hydrolysis was unchanged. Therefore, together, the Ser237 and Arg276 residues are important contributors to the cefotaximase substrate profile of the enzyme. High-resolution crystal structures of the CTX-M-14 S70G, S70G:S237A, and S70G:S237A:R276A variants alone and in complex with cefotaxime show that residues Ser237 and Arg276 in the wild-type enzyme promote the expansion of the active site to accommodate cefotaxime and favor a conformation of cefotaxime that allows optimal contacts between the enzyme and substrate. In conclusion, the conservation of these residues, linked to their effects on structure and catalysis, imply that their coevolution is an important specificity determinant in the CTX-M family.« less

A proof for substitution of endogenous iron (II) in lipoxygenase by exogenous Cu2+.

PubMed

Cai, Yan; Xu, Hong; Xia, Yongmei; Su, Yafen; Fang, Yun

2010-10-01

Soybean lipoxygenase (LOX) contains endogenous iron (II) at the active site, which is important for the enzyme activity. The activity of LOX can be accelerated by some exogenous metal ions including Cu2+. However, the mechanism of the activity improvement caused by exogenous metal ions remains unclear, not only for LOX but for most other metalloenzymes. Meanwhile, the possibility that exogenous metal ions can displace endogenous iron (II) is still in discussion for a lack of a direct and quantitative proof. In this paper, a quantitative proof of replacing iron (II) inside LOX by exogenous Cu2+ was provided, simply using UV-Vis spectrometry with two indicators p-carboxylantipyrylazo and 9-(4-carboxyphenyl)-2,3,7-trihydroxyl-6-fluorine. A 0.56 microM free iron (II) was observed in the bulk solution after incubating 9.45 microM Cu2+ with 16.10 microM LOX at 20 degrees C for 5 min, which is in coincidence with the decrement of Cu2+ in the bulk solution (0.53 microM), implying that iron (II) was replaced by Cu2+.

The role of quantum effects in proton transfer reactions in enzymes: quantum tunneling in a noisy environment?

NASA Astrophysics Data System (ADS)

Bothma, Jacques P.; Gilmore, Joel B.; McKenzie, Ross H.

2010-05-01

We consider the role of quantum effects in the transfer of hydrogen-like species in enzyme-catalyzed reactions. This review is stimulated by claims that the observed magnitude and temperature dependence of kinetic isotope effects (KIEs) implies that quantum tunneling below the energy barrier associated with the transition state significantly enhances the reaction rate in many enzymes. We review the path integral approach and the Caldeira-Leggett model, which provides a general framework to describe and understand tunneling in a quantum system that interacts with a noisy environment at nonzero temperature. Here the quantum system is the active site of the enzyme, and the environment is the surrounding protein and water. Tunneling well below the barrier only occurs for temperatures less than a temperature T0, which is determined by the curvature of the potential energy surface near the top of the barrier. We argue that for most enzymes this temperature is less than room temperature. We review typical values for the parameters in the Caldeira-Leggett Hamiltonian, including the frequency-dependent friction and noise due to the environment. For physically reasonable parameters, we show that quantum transition state theory gives a quantitative description of the temperature dependence and magnitude of KIEs for two classes of enzymes that have been claimed to exhibit signatures of quantum tunneling. The only quantum effects are those associated with the transition state, both reflection at the barrier top and tunneling just below the barrier. We establish that the friction and noise due to the environment are weak and only slightly modify the reaction rate. Furthermore, at room temperature and for typical energy barriers environmental fluctuations with frequencies much less than 1000 cm-1 do not have a significant effect on quantum corrections to the reaction rate. This is essentially because the time scales associated with the dynamics of proton transfer are faster than much of the low-frequency noise associated with the protein and solvent.

Lack of association of human immunodeficiency virus seroconversion with visits to foreign ports in US Navy personnel.

PubMed

Garland, F C; Garland, C F; Gorham, E D; Miller, M R; Cunnion, S O; Berg, S W; Balazs, L L

1993-12-13

The US Navy visits ports on all continents and many islands of the world, many of which are reported to have a high endemicity of human immunodeficiency virus (HIV) infection. The objective of this study was to determine whether visits to foreign ports by active-duty navy personnel were associated with increased risk of HIV infection. The Naval Health Research Center in San Diego, Calif, maintains records of all HIV enzyme-linked immunosorbent assay and Western blot tests given in the navy. This information, along with career histories and ship movement data, was used in a nested case-control design to examine the relationship between visits to the 100 foreign ports most frequently visited by the navy and risk of HIV seroconversion. All visits to a port and total time in each port during the study period were examined. A total of 813 seroconverters were matched to 6993 seronegative active-duty controls by age, race, sex, occupational group, home port, and year of test. Estimated relative risks of seroconversion associated with visits to foreign ports showed no statistically significant excess risk of HIV infection for navy personnel after visits to any foreign port. These results do not imply that an individual's risk of acquisition of HIV would be less in a foreign port if the individual engaged in high-risk activity there. Rather, they imply that despite the mobility of the US Navy and the large variation in HIV seroprevalence rates throughout the world, navy personnel generally do not appear to be acquiring HIV infections abroad.

Actinobacillus pleuropneumoniae Iron Transport and Urease Activity: Effects on Bacterial Virulence and Host Immune Response

PubMed Central

Baltes, Nina; Tonpitak, Walaiporn; Gerlach, Gerald-F.; Hennig-Pauka, Isabel; Hoffmann-Moujahid, Astrid; Ganter, Martin; Rothkötter, Hermann-J.

2001-01-01

Actinobacillus pleuropneumoniae, a porcine respiratory tract pathogen, has been shown to express transferrin-binding proteins and urease during infection. Both activities have been associated with virulence; however, their functional role for infection has not yet been elucidated. We used two isogenic A. pleuropneumoniae single mutants (ΔexbB and ΔureC) and a newly constructed A. pleuropneumoniae double (ΔureC ΔexbB) mutant in aerosol infection experiments. Neither the A. pleuropneumoniae ΔexbB mutant nor the double ΔureC ΔexbB mutant was able to colonize sufficiently long to initiate a detectable humoral immune response. These results imply that the ability to utilize transferrin-bound iron is required for multiplication and persistence of A. pleuropneumoniae in the porcine respiratory tract. The A. pleuropneumoniae ΔureC mutant and the parent strain both caused infections that were indistinguishable from one another in the acute phase of disease; however, 3 weeks postinfection the A. pleuropneumoniae ΔureC mutant, in contrast to the parent strain, could not be isolated from healthy lung tissue. In addition, the local immune response—as assessed by fluorescence-activated cell sorter and enzyme-linked immunosorbent spot analyses—revealed a significantly higher number of A. pleuropneumoniae-specific B cells in the bronchoalveolar lavage fluid (BALF) of pigs infected with the A. pleuropneumoniae ΔureC mutant than in the BALF of those infected with the parent strain. These results imply that A. pleuropneumoniae urease activity may cause sufficient impairment of the local immune response to slightly improve the persistence of the urease-positive A. pleuropneumoniae parent strain. PMID:11119539

CYP1A1 induction and CYP3A4 inhibition by the fungicide imazalil in the human intestinal Caco-2 cells-comparison with other conazole pesticides.

PubMed

Sergent, Thérèse; Dupont, Isabelle; Jassogne, Coralie; Ribonnet, Laurence; van der Heiden, Edwige; Scippo, Marie-Louise; Muller, Marc; McAlister, Dan; Pussemier, Luc; Larondelle, Yvan; Schneider, Yves-Jacques

2009-02-10

Imazalil (IMA) is a widely used imidazole-antifungal pesticide and, therefore, a food contaminant. This compound is also used as a drug (enilconazole). As intestine is the first site of exposure to ingested drugs and pollutants, we have investigated the effects of IMA, at realistic intestinal concentrations, on xenobiotic-metabolizing enzymes and efflux pumps by using Caco-2 cells, as a validated in vitro model of the human intestinal absorptive epithelium. For comparison, other conazole fungicides, i.e. ketoconazole, propiconazole and tebuconazole, were also studied. IMA induced cytochrome P450 (CYP) 1A1 activity to the same extent as benzo(a)pyrene (B(a)P) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in a dose- and time-dependent manner. Cell-free aryl hydrocarbon receptor (AhR) binding assay and reporter gene assay suggested that IMA is not an AhR-ligand, implying that IMA-mediated induction should involve an AhR-independent pathway. Moreover, IMA strongly inhibited the CYP3A4 activity in 1,25-vitamin D(3)-induced Caco-2 cells. The other fungicides had weak or nil effects on CYP activities. Study of the apical efflux pump activities revealed that ketoconazole inhibited both P-glycoprotein (Pgp) and multidrug resistance-associated protein 2 (MRP-2) or breast cancer resistance protein (BCRP), whereas IMA and other fungicides did not. Our results imply that coingestion of IMA-contaminated food and CYP3A4- or CYP1A1-metabolizable drugs or chemicals could lead to drug bioavailability modulation or toxicological interactions, with possible adverse effects for human health.

Phosphorylation Reaction in cAPK Protein Kinase - Free Energy Quantum Mechanic/Molecular Mechanics Simulations.

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

Valiev, Marat; Yang, Jie; Adams, Joseph

2007-11-29

Protein kinases catalyze the transfer of the γ-phosphoryl group from ATP, a key regulatory process governing signalling pathways in eukaryotic cells. The structure of the active site in these enzymes is highly conserved implying common catalytic mechanism. In this work we investigate the reaction process in cAPK protein kinase (PKA) using a combined quantum mechanics and molecular mechanics approach. The novel computational features of our work include reaction pathway determination with nudged elastic band methodology and calculation of free energy profiles of the reaction process taking into account finite temperature fluctuations of the protein environment. We find that the transfermore » of the γ-phosphoryl group in the protein environment is an exothermic reaction with the reaction barrier of 15 kcal/mol.« less

Anti-thrombosis Repertoire of Blood-feeding Horsefly Salivary Glands*

PubMed Central

Ma, Dongying; Wang, Yipeng; Yang, Hailong; Wu, Jing; An, Shu; Gao, Li; Xu, Xueqing; Lai, Ren

2009-01-01

Blood-feeding arthropods rely heavily on the pharmacological properties of their saliva to get a blood meal and suppress immune reactions of hosts. Little information is available on antihemostatic substances in horsefly salivary glands although their saliva has been thought to contain wide range of physiologically active molecules. In traditional Eastern medicine, horseflies are used as anti-thrombosis material for hundreds of years. By proteomics coupling transcriptome analysis with pharmacological testing, several families of proteins or peptides, which exert mainly on anti-thrombosis functions, were identified and characterized from 60,000 pairs of salivary glands of the horsefly Tabanus yao Macquart (Diptera, Tabanidae). They are: (I) ten fibrin(ogen)olytic enzymes, which hydrolyze specially alpha chain of fibrin(ogen) and are the first family of fibrin(ogen)olytic enzymes purified and characterized from arthropods; (II) another fibrin(ogen)olytic enzyme, which hydrolyzes both alpha and beta chain of fibrin(ogen); (III) ten Arg-Gly-Asp-motif containing proteins acting as platelet aggregation inhibitors; (IV) five thrombin inhibitor peptides; (V) three vasodilator peptides; (VI) one apyrase acting as platelet aggregation inhibitor; (VII) one peroxidase with both platelet aggregation inhibitory and vasodilator activities. The first three families are belonging to antigen five proteins, which show obvious similarity with insect allergens. They are the first members of the antigen 5 family found in salivary glands of blood sucking arthropods to have anti-thromobosis function. The current results imply a possible evolution from allergens of blood-sucking insects to anti-thrombosis agents. The extreme diversity of horsefly anti-thrombosis components also reveals the anti-thrombosis molecular mechanisms of the traditional Eastern medicine insect material. PMID:19531497

Oxidative bioactivation of abacavir in subcellular fractions of human antigen presenting cells.

PubMed

Bell, Catherine C; Santoyo Castelazo, Anahi; Yang, Emma L; Maggs, James L; Jenkins, Rosalind E; Tugwood, Jonathan; O'Neill, Paul M; Naisbitt, Dean J; Park, B Kevin

2013-07-15

Human exposure to abacavir, a primary alcohol antiretroviral, is associated with the development of immunological drug reactions in individuals carrying the HLA risk allele B*57:01. Interaction of abacavir with antigen presenting cells results in cell activation through an Hsp70-mediated Toll-like receptor pathway and the provision of T-cell antigenic determinants. Abacavir's electrophilic aldehyde metabolites are potential precursors of neoantigens. Herein, we have used mass spectrometry to study the oxidative metabolism of abacavir in EBV-transformed human B-cells. RNA and protein were isolated from the cells and subjected to transcriptomic and mass spectrometric analyses to identify the redox enzymes expressed. Low levels of isomeric abacavir carboxylic acids were detected in subcellular fractions of EBV-transformed human B-cells incubated with abacavir. Metabolite formation was time-dependent but was not reduced by an inhibitor of Class I alcohol dehydrogenases. Relatively high levels of mRNA were detected for several redox enzymes, including alcohol dehydrogenase 5 (Class III), aldehyde dehydrogenases (ALDH3A2, ALDH6A1, and ALDH9A1), CYP1B1, CYP2R1, CYP7B1, and hydroxysteroid dehydrogenase 10. Over 2600 proteins were identified by mass spectrometry. More than 1000 of these proteins exhibited catalytic activity, and 80 were oxido-reductases. This is the first proteomic inventory of enzymes in antigen presenting cells. However, neither of the hepatic alcohol dehydrogenases of Class I which metabolize abacavir in vitro was expressed at the protein level. Nevertheless the metabolic production of abacavir carboxylic acids by B-cell fractions implies abacavir-treated immune cells might be exposed to the drug's protein-reactive aldehyde metabolites in vivo.

Quantitative metabolome analysis profiles activation of glutaminolysis in glioma with IDH1 mutation.

PubMed

Ohka, Fumiharu; Ito, Maki; Ranjit, Melissa; Senga, Takeshi; Motomura, Ayako; Motomura, Kazuya; Saito, Kaori; Kato, Keiko; Kato, Yukinari; Wakabayashi, Toshihiko; Soga, Tomoyoshi; Natsume, Atsushi

2014-06-01

Isocitrate dehydrogenase 1 (IDH1), which localizes to the cytosol and peroxisomes, catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) and in parallel converts NADP(+) to NADPH. IDH1 mutations are frequently detected in grades 2-4 gliomas and in acute myeloid leukemias (AML). Mutations of IDH1 have been identified at codon 132, with arginine being replaced with histidine in most cases. Mutant IDH1 gains novel enzyme activity converting α-KG to D-2-hydroxyglutarate (2-HG) which acts as a competitive inhibitor of α-KG. As a result, the activity of α-KG-dependent enzyme is reduced. Based on these findings, 2-HG has been proposed to be an oncometabolite. In this study, we established HEK293 and U87 cells that stably expressed IDH1-WT and IDH1-R132H and investigated the effect of glutaminase inhibition on cell proliferation with 6-diazo-5-oxo-L-norleucine (DON). We found that cell proliferation was suppressed in IDH1-R132H cells. The addition of α-KG restored cell proliferation. The metabolic features of 33 gliomas with wild type IDH1 (IDH1-WT) and with IDH1-R132H mutation were examined by global metabolome analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We showed that the 2-HG levels were highly elevated in gliomas with IDH1-R132H mutation. Intriguingly, in gliomas with IDH1-R132H, glutamine and glutamate levels were significantly reduced which implies replenishment of α-KG by glutaminolysis. Based on these results, we concluded that glutaminolysis is activated in gliomas with IDH1-R132H mutation and that development of novel therapeutic approaches targeting activated glutaminolysis is warranted.

Antioxidant defense system and oxidative status in Antarctic fishes: The sluggish rockcod Notothenia coriiceps versus the active marbled notothen Notothenia rossii.

PubMed

Klein, Roberta Daniele; Rosa, Carlos Eduardo; Colares, Elton Pinto; Robaldo, Ricardo Berteaux; Martinez, Pablo Elias; Bianchini, Adalto

2017-08-01

Adaptive responses of antioxidant defense systems (ADS) to changes in increased levels of activity are critical, especially in Antarctic fishes. The benthopelagic marbled notothen (Notothenia rossii) shows higher spontaneous activity than the benthonic and sluggish rockcod (N. coriiceps). Therefore, we hypothesize that species-related responses of ADS would occur to counteract different rates of reactive oxygen species formation in these two Antarctic fish. Here we evaluated ADS and oxidative damage in tissues (brain, gills, liver and white muscle) of the two Antarctic fish. Despite no significant differences in lipid and protein oxidative damage were observed, we actually found species- and tissue-specific differences in ADS. Gill metallothionein-like proteins (MTLP) and liver reduced glutathione (GSH) concentrations were higher in N. coriiceps than in N. rossii. Brain and gill antioxidant capacity against peroxyl radicals (ACAP); gill enzyme [glutamate-cysteine ligase (GSL), superoxide dismutase (SOD) and catalase (CAT)] activity; liver GCL and SOD activity; and white muscle CAT activity were higher in N. rossii than in N. coriiceps. Therefore, the more active fish (N. rossii) maintains higher activities of enzymes involved in superoxide ions (O 2 .- ) detoxification and GSH production in peripheral tissues (gills, liver and white muscle). This allows the more active fish (N. rossii) to keep levels of lipid and protein oxidative damage similar to those observed in the sluggish fish (N. coriiceps). It is worth noting that the more active fish also shows a higher brain antioxidant capacity, which could involve other non-enzymatic antioxidants like vitamins C and E. In contrast, N. coriiceps shows lower consumption of non-enzymatic antioxidants in peripheral tissues than N. coriiceps. As hypothesized, our results indicate that differences in ADS profiles between fish species are likely related to their habits and metabolic rates. This would imply in different fish abilities to deal with oxidative stress associated with increasing seawater temperature. Copyright © 2017 Elsevier Ltd. All rights reserved.

Redox properties of transitional milk from mothers of preterm infants.

PubMed

Minić, Simeon; Ješić, Miloš; Đurović, Dijana; Miletić, Srdjan; Lugonja, Nikoleta; Marinković, Vesna; Nikolić-Kokić, Aleksandra; Spasić, Snežana; Vrvić, Miroslav M

2018-02-01

There is a discrepancy between the amount of transitional milk produced by mothers of preterm infants and the low capacity of premature infants to consume it. This milk can be used in milk banks, but previous studies found that there are large variations in the level of host-defence proteins in individual samples of milk from mothers of premature infants, which implies that large individual variations in antioxidative defence composition are also possible. Milk samples were collected from 20 healthy mothers of preterm infants. We determined the values for non-enzymatic antioxidative capacity parameters (oxygen radical absorbance capacity (ORAC)), static oxidation-reduction potential (ORP), activities of antioxidant defence enzymes and the amount of vitamin C in whole milk, skim and whey fractions of transitional milk. The main low-molecular-weight antioxidant in transitional milk is vitamin C and most of it is contained in whey. ORAC is higher in whole transitional milk than in skim milk and whey, and ORP is lower in whole transitional milk than that in skim milk and whey. Antioxidative enzyme activities are similar in all individual samples of transitional milk from mothers of preterm infants. Our results indicate that transitional milk of mothers of preterm infants shows slow individual variations in antioxidative defence composition; therefore, it can be used in human milk banks. © 2017 Paediatrics and Child Health Division (The Royal Australasian College of Physicians).

Encodings of implied motion for animate and inanimate object categories in the two visual pathways.

PubMed

Lu, Zhengang; Li, Xueting; Meng, Ming

2016-01-15

Previous research has proposed two separate pathways for visual processing: the dorsal pathway for "where" information vs. the ventral pathway for "what" information. Interestingly, the middle temporal cortex (MT) in the dorsal pathway is involved in representing implied motion from still pictures, suggesting an interaction between motion and object related processing. However, the relationship between how the brain encodes implied motion and how the brain encodes object/scene categories is unclear. To address this question, fMRI was used to measure activity along the two pathways corresponding to different animate and inanimate categories of still pictures with different levels of implied motion speed. In the visual areas of both pathways, activity induced by pictures of humans and animals was hardly modulated by the implied motion speed. By contrast, activity in these areas correlated with the implied motion speed for pictures of inanimate objects and scenes. The interaction between implied motion speed and stimuli category was significant, suggesting different encoding mechanisms of implied motion for animate-inanimate distinction. Further multivariate pattern analysis of activity in the dorsal pathway revealed significant effects of stimulus category that are comparable to the ventral pathway. Moreover, still pictures of inanimate objects/scenes with higher implied motion speed evoked activation patterns that were difficult to differentiate from those evoked by pictures of humans and animals, indicating a functional role of implied motion in the representation of object categories. These results provide novel evidence to support integrated encoding of motion and object categories, suggesting a rethink of the relationship between the two visual pathways. Copyright © 2015 Elsevier Inc. All rights reserved.

Interaction of renin-angiotensin system and adenosine monophosphate-activated protein kinase signaling pathway in renal carcinogenesis of uninephrectomized rats.

PubMed

Yang, Ke-Ke; Sui, Yi; Zhou, Hui-Rong; Zhao, Hai-Lu

2017-05-01

Renin-angiotensin system and adenosine monophosphate-activated protein kinase signaling pathway both play important roles in carcinogenesis, but the interplay of renin-angiotensin system and adenosine monophosphate-activated protein kinase in carcinogenesis is not clear. In this study, we researched the interaction of renin-angiotensin system and adenosine monophosphate-activated protein kinase in renal carcinogenesis of uninephrectomized rats. A total of 96 rats were stratified into four groups: sham, uninephrectomized, and uninephrectomized treated with angiotensin-converting enzyme inhibitor or angiotensin receptor blocker. Renal adenosine monophosphate-activated protein kinase and its downstream molecule acetyl coenzyme A carboxylase were detected by immunohistochemistry and western blot at 10 months after uninephrectomy. Meanwhile, we examined renal carcinogenesis by histological transformation and expressions of Ki67 and mutant p53. During the study, fasting lipid profiles were detected dynamically at 3, 6, 8, and 10 months. The results indicated that adenosine monophosphate-activated protein kinase expression in uninephrectomized rats showed 36.8% reduction by immunohistochemistry and 89.73% reduction by western blot. Inversely, acetyl coenzyme A carboxylase expression increased 83.3% and 19.07% in parallel to hyperlipidemia at 6, 8, and 10 months. The histopathology of carcinogenesis in remnant kidneys was manifested by atypical proliferation and carcinoma in situ, as well as increased expressions of Ki67 and mutant p53. Intervention with angiotensin-converting enzyme inhibitor or angiotensin receptor blocker significantly prevented the inhibition of adenosine monophosphate-activated protein kinase signaling pathway and renal carcinogenesis in uninephrectomized rats. In conclusion, the novel findings suggest that uninephrectomy-induced disturbance in adenosine monophosphate-activated protein kinase signaling pathway resulted in hyperlipidemia and carcinogenesis in tubular epithelial cells, which may be largely attenuated by renin-angiotensin system blockade, implying the interaction of renin-angiotensin system and adenosine monophosphate-activated protein kinase signaling pathway in renal carcinogenesis of uninephrectomized rats.

Implied motion because of instability in Hokusai Manga activates the human motion-sensitive extrastriate visual cortex: an fMRI study of the impact of visual art.

PubMed

Osaka, Naoyuki; Matsuyoshi, Daisuke; Ikeda, Takashi; Osaka, Mariko

2010-03-10

The recent development of cognitive neuroscience has invited inference about the neurosensory events underlying the experience of visual arts involving implied motion. We report functional magnetic resonance imaging study demonstrating activation of the human extrastriate motion-sensitive cortex by static images showing implied motion because of instability. We used static line-drawing cartoons of humans by Hokusai Katsushika (called 'Hokusai Manga'), an outstanding Japanese cartoonist as well as famous Ukiyoe artist. We found 'Hokusai Manga' with implied motion by depicting human bodies that are engaged in challenging tonic posture significantly activated the motion-sensitive visual cortex including MT+ in the human extrastriate cortex, while an illustration that does not imply motion, for either humans or objects, did not activate these areas under the same tasks. We conclude that motion-sensitive extrastriate cortex would be a critical region for perception of implied motion in instability.

A biomimetic colorimetric logic gate system based on multi-functional peptide-mediated gold nanoparticle assembly

NASA Astrophysics Data System (ADS)

Li, Yong; Li, Wang; He, Kai-Yu; Li, Pei; Huang, Yan; Nie, Zhou; Yao, Shou-Zhuo

2016-04-01

In natural biological systems, proteins exploit various functional peptide motifs to exert target response and activity switch, providing a functional and logic basis for complex cellular activities. Building biomimetic peptide-based bio-logic systems is highly intriguing but remains relatively unexplored due to limited logic recognition elements and complex signal outputs. In this proof-of-principle work, we attempted to address these problems by utilizing multi-functional peptide probes and the peptide-mediated nanoparticle assembly system. Here, the rationally designed peptide probes function as the dual-target responsive element specifically responsive to metal ions and enzymes as well as the mediator regulating the assembly of gold nanoparticles (AuNPs). Taking advantage of Zn2+ ions and chymotrypsin as the model inputs of metal ions and enzymes, respectively, we constructed the peptide logic system computed by the multi-functional peptide probes and outputted by the readable colour change of AuNPs. In this way, the representative binary basic logic gates (AND, OR, INHIBIT, NAND, IMPLICATION) have been achieved by delicately coding the peptide sequence, demonstrating the versatility of our logic system. Additionally, we demonstrated that the three-input combinational logic gate (INHIBIT-OR) could also be successfully integrated and applied as a multi-tasking biosensor for colorimetric detection of dual targets. This nanoparticle-based peptide logic system presents a valid strategy to illustrate peptide information processing and provides a practical platform for executing peptide computing or peptide-related multiplexing sensing, implying that the controllable nanomaterial assembly is a promising and potent methodology for the advancement of biomimetic bio-logic computation.In natural biological systems, proteins exploit various functional peptide motifs to exert target response and activity switch, providing a functional and logic basis for complex cellular activities. Building biomimetic peptide-based bio-logic systems is highly intriguing but remains relatively unexplored due to limited logic recognition elements and complex signal outputs. In this proof-of-principle work, we attempted to address these problems by utilizing multi-functional peptide probes and the peptide-mediated nanoparticle assembly system. Here, the rationally designed peptide probes function as the dual-target responsive element specifically responsive to metal ions and enzymes as well as the mediator regulating the assembly of gold nanoparticles (AuNPs). Taking advantage of Zn2+ ions and chymotrypsin as the model inputs of metal ions and enzymes, respectively, we constructed the peptide logic system computed by the multi-functional peptide probes and outputted by the readable colour change of AuNPs. In this way, the representative binary basic logic gates (AND, OR, INHIBIT, NAND, IMPLICATION) have been achieved by delicately coding the peptide sequence, demonstrating the versatility of our logic system. Additionally, we demonstrated that the three-input combinational logic gate (INHIBIT-OR) could also be successfully integrated and applied as a multi-tasking biosensor for colorimetric detection of dual targets. This nanoparticle-based peptide logic system presents a valid strategy to illustrate peptide information processing and provides a practical platform for executing peptide computing or peptide-related multiplexing sensing, implying that the controllable nanomaterial assembly is a promising and potent methodology for the advancement of biomimetic bio-logic computation. Electronic supplementary information (ESI) available: Additional figures (Tables S1-S3 and Fig. S1-S6). See DOI: 10.1039/c6nr01072e

Structure and Mechanism of ArnA: Conformational Change Implies Ordered Dehydrogenase Mechanism in Key Enzyme for Polymyxin Resistance

PubMed Central

Gatzeva-Topalova, Petia Z.; May, Andrew P.; Sousa, Marcelo C.

2010-01-01

Summary The modification of lipid A with 4-amino-4-deoxy-L-arabinose (Ara4N) allows gram-negative bacteria to resist the antimicrobial activity of cationic antimicrobial peptides and antibiotics such as polymyxin. ArnA is the first enzyme specific to the lipid A-Ara4N pathway. It contains two functionally and physically separable domains: a dehydrogenase domain (ArnA_DH) catalyzing the NAD+-dependent oxidative decarboxylation of UDP-Glucuronic acid (UDP-GlcA), and a transformylase domain that formylates UDP-Ara4N. Here, we describe the crystal structure of the full-length bifunctional ArnA with UDP-GlcA and ATP bound to the dehydrogenase domain. Binding of UDP-GlcA triggers a 17 Å conformational change in ArnA_DH that opens the NAD+ binding site while trapping UDP-GlcA. We propose an ordered mechanism of substrate binding and product release. Mutation of residues R619 and S433 demonstrates their importance in catalysis and suggests that R619 functions as a general acid in catalysis. The proposed mechanism for ArnA_DH has important implications for the design of selective inhibitors. PMID:15939024

A mitochondrial-like aconitase in the bacterium Bacteroides fragilis: implications for the evolution of the mitochondrial Krebs cycle.

PubMed

Baughn, Anthony D; Malamy, Michael H

2002-04-02

Aconitase and isocitrate dehydrogenase (IDH) enzyme activities were detected in anaerobically prepared cell extracts of the obligate anaerobe Bacteroides fragilis. The aconitase gene was located upstream of the genes encoding the other two components of the oxidative branch of the Krebs cycle, IDH and citrate synthase. Mutational analysis indicates that these genes are cotranscribed. A nonpolar in-frame deletion of the acnA gene that encodes the aconitase prevented growth in glucose minimal medium unless heme or succinate was added to the medium. These results imply that B. fragilis has two pathways for alpha-ketoglutarate biosynthesis-one from isocitrate and the other from succinate. Homology searches indicated that the B. fragilis aconitase is most closely related to aconitases of two other Cytophaga-Flavobacterium-Bacteroides (CFB) group bacteria, Cytophaga hutchinsonii and Fibrobacter succinogenes. Phylogenetic analysis indicates that the CFB group aconitases are most closely related to mitochondrial aconitases. In addition, the IDH of C. hutchinsonii was found to be most closely related to the mitochondrial/cytosolic IDH-2 group of eukaryotic organisms. These data suggest a common origin for these Krebs cycle enzymes in mitochondria and CFB group bacteria.

Nucleosome breathing and remodeling constrain CRISPR-Cas9 function

PubMed Central

Isaac, R Stefan; Jiang, Fuguo; Doudna, Jennifer A; Lim, Wendell A; Narlikar, Geeta J; Almeida, Ricardo

2016-01-01

The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo. DOI: http://dx.doi.org/10.7554/eLife.13450.001 PMID:27130520

Iron limitation of microbial phosphorus acquisition in the tropical North Atlantic

PubMed Central

Browning, T. J.; Achterberg, E. P.; Yong, J. C.; Rapp, I.; Utermann, C.; Engel, A.; Moore, C. M.

2017-01-01

In certain regions of the predominantly nitrogen limited ocean, microbes can become co-limited by phosphorus. Within such regions, a proportion of the dissolved organic phosphorus pool can be accessed by microbes employing a variety of alkaline phosphatase (APase) enzymes. In contrast to the PhoA family of APases that utilize zinc as a cofactor, the recent discovery of iron as a cofactor in the more widespread PhoX and PhoD implies the potential for a biochemically dependant interplay between oceanic zinc, iron and phosphorus cycles. Here we demonstrate enhanced natural community APase activity following iron amendment within the low zinc and moderately low iron Western North Atlantic. In contrast we find no evidence for trace metal limitation of APase activity beneath the Saharan dust plume in the Eastern Atlantic. Such intermittent iron limitation of microbial phosphorus acquisition provides an additional facet in the argument for iron controlling the coupling between oceanic nitrogen and phosphorus cycles. PMID:28524880

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.

Attenuation of Oxidative Damage by Boerhaavia diffusa L. Against Different Neurotoxic Agents in Rat Brain Homogenate.

PubMed

Ayyappan, Prathapan; Palayyan, Salin Raj; Kozhiparambil Gopalan, Raghu

2016-01-01

Due to a high rate of oxidative metabolic activity in the brain, intense production of reactive oxygen metabolite occurs, and the subsequent generation of free radicals is implicated in the pathogenesis of traumatic brain injury, epilepsy, and ischemia as well as chronic neurodegenerative diseases. In the present study, protective effects of polyphenol rich ethanolic extract of Boerhaavia diffusa (BDE), a neuroprotective edible medicinal plant against oxidative stress induced by different neurotoxic agents, were evaluated. BDE was tested against quinolinic acid (QA), 3-nitropropionic acid (NPA), sodium nitroprusside (SNP), and Fe (II)/EDTA complex induced oxidative stress in rat brain homogenates. QA, NPA, SNP, and Fe (II)/EDTA treatment caused an increased level of thiobarbituric acid reactive substances (TBARS) in brain homogenates along with a decline in the activities of antioxidant enzymes. BDE treatment significantly decreased the production of TBARS (p < .05) and increased the activities of antioxidant enzymes like catalase and superoxide dismutase along with increased concentration of non-enzymatic antioxidant, reduced glutathione (GSH). Similarly, BDE caused a significant decrease in the lipid peroxidation (LPO) in the cerebral cortex. Inhibitory potential of BDE against deoxyribose degradation (IC50 value 38.91 ± 0.12 μg/ml) shows that BDE can protect hydroxyl radical induced DNA damage in the tissues. Therefore, B. diffusa had high antioxidant potential that could inhibit the oxidative stress induced by different neurotoxic agents in brain. Since many of the neurological disorders are associated with free radical injury, these data may imply that B. diffusa, functioning as an antioxidant agent, may be beneficial for reducing various neurodegenerative complications.

Structural and Biochemical Characterization of the Type II Fructose-1,6-bisphosphatase GlpX from Escherichia coli

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

Brown, G.; Singer, A.; Lunin, V. V.

2009-02-06

Gluconeogenesis is an important metabolic pathway, which produces glucose from noncarbohydrate precursors such as organic acids, fatty acids, amino acids, or glycerol. Fructose-1,6-bisphosphatase, a key enzyme of gluconeogenesis, is found in all organisms, and five different classes of these enzymes have been identified. Here we demonstrate that Escherichia coli has two class II fructose-1,6-bisphosphatases, GlpX and YggF, which show different catalytic properties. We present the first crystal structure of a class II fructose-1,6-bisphosphatase (GlpX) determined in a free state and in the complex with a substrate (fructose 1,6-bisphosphate) or inhibitor (phosphate). The crystal structure of the ligand-free GlpX revealed amore » compact, globular shape with two {alpha}/{beta}-sandwich domains. The core fold of GlpX is structurally similar to that of Li{sup +}-sensitive phosphatases implying that they have a common evolutionary origin and catalytic mechanism. The structure of the GlpX complex with fructose 1,6-bisphosphate revealed that the active site is located between two domains and accommodates several conserved residues coordinating two metal ions and the substrate. The third metal ion is bound to phosphate 6 of the substrate. Inorganic phosphate strongly inhibited activity of both GlpX and YggF, and the crystal structure of the GlpX complex with phosphate demonstrated that the inhibitor molecule binds to the active site. Alanine replacement mutagenesis of GlpX identified 12 conserved residues important for activity and suggested that Thr{sup 90} is the primary catalytic residue. Our data provide insight into the molecular mechanisms of the substrate specificity and catalysis of GlpX and other class II fructose-1,6-bisphosphatases.« less

Biochemical and genotoxic effect of triclosan on earthworms (Eisenia fetida) using contact and soil tests.

PubMed

Lin, Dasong; Xie, Xiujie; Zhou, Qixing; Liu, Yao

2012-07-01

Triclosan (TCS) is a broad-spectrum bactericide that is used for a variety of antimicrobial functions. TCS is frequently detected in the terrestrial environment due to application of sewage sludge to agricultural land. In the present study, 48-h paper contact and 28-day spiked soil tests were conducted to examine the toxic effects of TCS on the antioxidative and genetic indices of earthworms (Eisenia fetida). The activity of antioxidative enzymes (superoxide dismutase, SOD; catalase, CAT) and the content of the lipid peroxidation product (malondialdehyde, MDA) were determined as biomarkers of oxidative stress in E. fetida. Moreover, single cell gel electrophoresis (SCGE) was used as a biomarker of genotoxicity. The results showed that triclosan induced a significant increase (P < 0.05) in antioxidative enzyme activities and MDA content. Of all of the biomarkers examined, CAT activity was most sensitive to TCS, and the CAT activity increased significantly (P < 0.05) at bactericidal concentrations of 7.86 ng cm⁻² in the contact test and 10 mg kg⁻¹ in the spiked soil test. The comet assay showed that TCS treatments significantly induced (P < 0.05) DNA damage in E. fetida, and that 78.6 ng cm⁻² caused significant genotoxic effects in the acute test (48 h). Clear dose-dependent DNA damage to E. fetida was observed both in contact and spiked soil tests. These results imply that TCS may have potential biochemical and genetic toxicity toward earthworms (E. fetida). A battery of biomarkers covering multiple molecular targets of acute toxicity can be combined to better understand the impacts of TCS on E. fetida. Copyright © 2010 Wiley Periodicals, Inc.

Alterations of erythrocyte rheology and cellular susceptibility in end stage renal disease: Effects of peritoneal dialysis.

PubMed

Ertan, Nesrin Zeynep; Bozfakioglu, Semra; Ugurel, Elif; Sinan, Mukaddes; Yalcin, Ozlem

2017-01-01

In this study, we investigated the effects of peritoneal dialysis on hemorheological and hematological parameters and their relations with oxidant and antioxidant status of uremic patients. Hemorheological parameters (erythrocyte deformability, erythrocyte aggregation, osmotic deformability, blood and plasma viscosity) were measured in patients with renal insufficiency undergoing peritoneal dialysis (PD) and volunteers. Erythrocyte deformability, osmotic deformability and aggregation in both autologous plasma and 3% dextran 70 were measured by laser diffraction ektacytometry. Enzyme activities of glutathione peroxidase, superoxide dismutase and catalase were studied in erythrocytes; lipid peroxidation was studied by measuring the amount of malondialdehyde in both erythrocytes and plasma samples. Blood viscosity at native hematocrit was significantly lower in PD patients at all measured shear rates compared to controls, but it was high in PD patients at corrected (45%) hematocrit. Erythrocyte deformability did not show any difference between the two groups. Osmotic deformability was significantly lower in PD patients compared to controls. Aggregation index values were significantly high in PD patients in plasma Catalase and glutathione peroxidase activities in erythrocytes were decreased in PD patients whereas superoxide dismutase activity was increased compared to controls. Malondialdehyde was significantly increased in erythrocytes and plasma samples of PD patients which also shows correlations with aggregation parameters. It has been concluded that erythrocytes in PD patients are more prone to aggregation and this tendency could be influenced by lipid peroxidation activity in patient's plasma. These results imply that uremic conditions, loss of plasma proteins and an increased risk of oxidative stress because of decreasing levels of antioxidant enzymes affect erythrocyte rheology during peritoneal dialysis. This level of distortion may have crucial effects, impairing the blood flow dynamics and causing inadequate microcirculatory perfusion.

Induction of Phosphoenolpyruvate Carboxykinase (PEPCK) during Acute Acidosis and Its Role in Acid Secretion by V-ATPase-Expressing Ionocytes.

PubMed

Furukawa, Fumiya; Tseng, Yung-Che; Liu, Sian-Tai; Chou, Yi-Ling; Lin, Ching-Chun; Sung, Po-Hsuan; Uchida, Katsuhisa; Lin, Li-Yih; Hwang, Pung-Pung

2015-01-01

Vacuolar-Type H(+)-ATPase (V-ATPase) takes the central role in pumping H(+) through cell membranes of diverse organisms, which is essential for surviving acid-base fluctuating lifestyles or environments. In mammals, although glucose is believed to be an important energy source to drive V-ATPase, and phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme for gluconeogenesis, is known to be activated in response to acidosis, the link between acid secretion and PEPCK activation remains unclear. In the present study, we used zebrafish larva as an in vivo model to show the role of acid-inducible PEPCK activity in glucose production to support higher rate of H(+) secretion via V-ATPase, by utilizing gene knockdown, glucose supplementation, and non-invasive scanning ion-selective electrode technique (SIET). Zebrafish larvae increased V-ATPase-mediated acid secretion and transiently expression of Pck1, a zebrafish homolog of PEPCK, in response to acid stress. When pck1 gene was knocked down by specific morpholino, the H(+) secretion via V-ATPase decreased, but this effect was rescued by supplementation of glucose into the yolk. By assessing changes in amino acid content and gene expression of respective enzymes, glutamine and glutamate appeared to be the major source for replenishment of Krebs cycle intermediates, which are subtracted by Pck1 activity. Unexpectedly, pck1 knockdown did not affect glutamine/glutamate catalysis, which implies that Pck1 does not necessarily drive this process. The present study provides the first in vivo evidence that acid-induced PEPCK provides glucose for acid-base homeostasis at an individual level, which is supported by rapid pumping of H(+) via V-ATPase at the cellular level.

Comparison of a homology model and the crystallographic structure of human 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) in a structure-based identification of inhibitors

NASA Astrophysics Data System (ADS)

Miguet, Laurence; Zhang, Ziding; Barbier, Maryse; Grigorov, Martin G.

2006-02-01

Human 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) catalyzes the interconversion of cortisone into active cortisol. 11βHSD1 inhibition is a tempting target for the treatment of a host of human disorders that might benefit from blockade of glucocorticoid action, such as obesity, metabolic syndrome, and diabetes type 2. Here, we report an in silico screening study aimed at identifying new selective inhibitors of human 11βHSD1 enzyme. In the first step, homology modeling was employed to build the 3D structure of 11βHSD1. Further, molecular docking was used to validate the predicted model by showing that it was able to discriminate between known 11βHSD1 inhibitors or substrates and non-inhibitors. The homology model was found to reproduce closely the crystal structure that became publicly available in the final stages of this work. Finally, we carried out structure-based virtual screening experiments on both the homology model and the crystallographic structure with a database of 114'000 natural molecules. Among these, 15 molecules were consistently selected as inhibitors based on both the model and crystal structures of the enzyme, implying a good quality for the homology model. Among these putative 11βHSD1 inhibitors, two were flavonone derivatives that have already been shown to be potent inhibitors of the enzyme.

Selective activators of protein phosphatase 5 target the auto-inhibitory mechanism.

PubMed

Haslbeck, Veronika; Drazic, Adrian; Eckl, Julia M; Alte, Ferdinand; Helmuth, Martin; Popowicz, Grzegorz; Schmidt, Werner; Braun, Frank; Weiwad, Matthias; Fischer, Gunter; Gemmecker, Gerd; Sattler, Michael; Striggow, Frank; Groll, Michael; Richter, Klaus

2015-04-20

Protein phosphatase 5 (PP5) is an evolutionary conserved serine/threonine phosphatase. Its dephosphorylation activity modulates a diverse set of cellular factors including protein kinases and the microtubule-associated tau protein involved in neurodegenerative disorders. It is auto-regulated by its heat-shock protein (Hsp90)-interacting tetratricopeptide repeat (TPR) domain and its C-terminal α-helix. In the present study, we report the identification of five specific PP5 activators [PP5 small-molecule activators (P5SAs)] that enhance the phosphatase activity up to 8-fold. The compounds are allosteric modulators accelerating efficiently the turnover rate of PP5, but do barely affect substrate binding or the interaction between PP5 and the chaperone Hsp90. Enzymatic studies imply that the compounds bind to the phosphatase domain of PP5. For the most promising compound crystallographic comparisons of the apo PP5 and the PP5-P5SA-2 complex indicate a relaxation of the auto-inhibited state of PP5. Residual electron density and mutation analyses in PP5 suggest activator binding to a pocket in the phosphatase/TPR domain interface, which may exert regulatory functions. These compounds thus may expose regulatory mechanisms in the PP5 enzyme and serve to develop optimized activators based on these scaffolds. © 2015 Authors.

Relationships between the Efficiencies of Photosystems I and II and Stromal Redox State in CO(2)-Free Air : Evidence for Cyclic Electron Flow in Vivo.

PubMed

Harbinson, J; Foyer, C H

1991-09-01

The responses of the efficiencies of photosystems I and II, stromal redox state (as indicated by NADP-malate dehydrogenase activation state), and activation of the Benson-Calvin cycle enzymes ribulose 1,5-bisphosphate carboxylase and fructose 1,6-bisphosphatase to varying irradiance were measured in pea (Pisum sativum L.) leaves operating close to the CO(2) compensation point. A comparison of the relationships among these parameters obtained from leaves in air was made with those obtained when the leaves were maintained in air from which the CO(2) had been removed. P700 was more oxidized at any measured irradiance in CO(2)-free air than in air. The relationship between the quantum efficiencies of the photosystems in CO(2)-free air was distinctly curvilinear in contrast to the predominantly linear relationship obtained with leaves in air. This nonlinearity may be consistent with the operation of cyclic electron flow around photosystem I because the quantum efficiency of photosystem II was much more restricted than the quantum efficiency of photosystem I. In CO(2)-free air, measured NADP-malate dehydrogenase activities varied considerably at low irradiances. However, at high irradiance the activity of the enzyme was low, implying that the stroma was oxidized. In contrast, fructose-1,6-bisphosphatase activities tended to increase with increasing electron flux through the photosystems. Ribulose-1,5-bisphosphate carboxylase activity remained relatively constant with respect to irradiance in CO(2)-free air, with an activation state 50% of maximum. We conclude that, at the CO(2) compensation point and high irradiance, low redox states are favored and that cyclic electron flow may be substantial. These two features may be the requirements necessary to trigger and maintain the dissipative processes in the thylakoid membrane.

N5-(L-1-carboxyethyl)-L-ornithine synthase: physical and spectral characterization of the enzyme and its unusual low pKa fluorescent tyrosine residues.

PubMed Central

Sackett, D. L.; Ruvinov, S. B.; Thompson, J.

1999-01-01

N5-(L-1-carboxyethyl)-L-ornithine synthase [E.C. 1.5.1.24] (CEOS) from Lactococcus lactis has been cloned, expressed, and purified from Escherichia coli in quantities sufficient for characterization by biophysical methods. The NADPH-dependent enzyme is a homotetramer (Mr approximately equal to 140,000) and in the native state is stabilized by noncovalent interactions between the monomers. The far-ultraviolet circular dichroism spectrum shows that the folding pattern of the enzyme is typical of the alpha,beta family of proteins. CEOS contains one tryptophan (Trp) and 19 tyrosines (Tyr) per monomer, and the fluorescence spectrum of the protein shows emission from both Trp and Tyr residues. Relative to N-acetyltyrosinamide, the Tyr quantum yield of the native enzyme is about 0.5. All 19 Tyr residues are titratable and, of these, two exhibit the uncommonly low pKa of approximately 8.5, 11 have pKa approximately 10.75, and the remaining six titrate with pKa approximately 11.3. The two residues with pKa approximately 8.5 contribute approximately 40% of the total tyrosine emission, implying a relative quantum yield >1, probably indicating Tyr-Tyr energy transfer. In the presence of NADPH, Tyr fluorescence is reduced by 40%, and Trp fluorescence is quenched completely. The latter result suggests that the single Trp residue is either at the active site, or in proximity to the sequence GSGNVA, that constitutes the beta alphabeta fold of the nucleotide-binding domain. Chymotrypsin specifically cleaves native CEOS after Phe255. Although inactivated by this single-site cleavage of the subunit, the enzyme retains the capacity to bind NADPH and tetramer stability is maintained. Possible roles in catalysis for the chymotrypsin sensitive loop and for the low pKa Tyr residues are discussed. PMID:10548058

Structure of YciA from Haemophilus influenzae (HI0827), a Hexameric Broad Specificity Acyl-Coenzyme A Thioesterase

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

Willis, Mark A.; Zhuang, Zhihao; Song, Feng

2008-04-02

The crystal structure of HI0827 from Haemophilus influenzae Rd KW20, initially annotated 'hypothetical protein' in sequence databases, exhibits an acyl-coenzyme A (acyl-CoA) thioesterase 'hot dog' fold with a trimer of dimers oligomeric association, a novel assembly for this enzyme family. In studies described in the preceding paper [Zhuang, Z., Song, F., Zhao, H., Li, L., Cao, J., Eisenstein, E., Herzberg, O., and Dunaway-Mariano, D. (2008) Biochemistry 47, 2789-2796], HI0827 is shown to be an acyl-CoA thioesterase that acts on a wide range of acyl-CoA compounds. Two substrate binding sites are located across the dimer interface. The binding sites are occupiedmore » by two CoA molecules, one with full occupancy and the second only partially occupied. The CoA molecules, acquired from HI0827-expressing Escherichia coli cells, remained tightly bound to the enzyme through the protein purification steps. The difference in CoA occupancies indicates a different substrate affinity for each of the binding sites, which in turn implies that the enzyme might be subject to allosteric regulation. Mutagenesis studies have shown that the replacement of the putative catalytic carboxylate Asp44 with an alanine residue abolishes activity. The impact of this mutation is seen in the crystal structure of D44A HI0827. Whereas the overall fold and assembly of the mutant protein are the same as those of the wild-type enzyme, the CoA ligands are absent. The dimer interface is perturbed, and the channel that accommodates the thioester acyl chain is more open and wider than that observed in the wild-type enzyme. A model of intact substrate bound to wild-type HI0827 provides a structural rationale for the broad substrate range.« less

The Large Hydrophilic Loop of Presenilin 1 Is Important for Regulating γ-Secretase Complex Assembly and Dictating the Amyloid β Peptide (Aβ) Profile without Affecting Notch Processing*

PubMed Central

Wanngren, Johanna; Frånberg, Jenny; Svensson, Annelie I.; Laudon, Hanna; Olsson, Fredrik; Winblad, Bengt; Liu, Frank; Näslund, Jan; Lundkvist, Johan; Karlström, Helena

2010-01-01

γ-Secretase is an enzyme complex that mediates both Notch signaling and β-amyloid precursor protein (APP) processing, resulting in the generation of Notch intracellular domain, APP intracellular domain, and the amyloid β peptide (Aβ), the latter playing a central role in Alzheimer disease (AD). By a hitherto undefined mechanism, the activity of γ-secretase gives rise to Aβ peptides of different lengths, where Aβ42 is considered to play a particular role in AD. In this study we have examined the role of the large hydrophilic loop (amino acids 320–374, encoded by exon 10) of presenilin 1 (PS1), the catalytic subunit of γ-secretase, for γ-secretase complex formation and activity on Notch and APP processing. Deletion of exon 10 resulted in impaired PS1 endoproteolysis, γ-secretase complex formation, and had a differential effect on Aβ-peptide production. Although the production of Aβ38, Aβ39, and Aβ40 was severely impaired, the effect on Aβ42 was affected to a lesser extent, implying that the production of the AD-related Aβ42 peptide is separate from the production of the Aβ38, Aβ39, and Aβ40 peptides. Interestingly, formation of the intracellular domains of both APP and Notch was intact, implying a differential cleavage activity between the ϵ/S3 and γ sites. The most C-terminal amino acids of the hydrophilic loop were important for regulating APP processing. In summary, the large hydrophilic loop of PS1 appears to differentially regulate the relative production of different Aβ peptides without affecting Notch processing, two parameters of significance when considering γ-secretase as a target for pharmaceutical intervention in AD. PMID:20106965

Comparison of telomere length and telomerase activation between breast fibroadenoma and infiltrating ductal carcinoma in Malaysian women.

PubMed

Looi, Lai-Meng; Cheah, Phaik-Leng; Ng, Min-Hwei; Yip, Cheng-Har; Mun, Kein-Seong; Rahman, Nazarina Abdul

2010-01-01

A study was initiated to explore possible differences in handling telomere attrition in the most common lignant and benign tumours of the breast in Malaysian women. Infiltrating ductal carcinoma (IDC) and fibroadenoma (FA) represented the malignant and benign prototypes respectively. 29 IDC, 28 FA and 22 benign non-lesional control (BNL) breast tissue samples were analysed for telomerase activation using a Telomerase PCR ELISA kit (Boehringer Mannheim). In addition, 23 IDC, 12 FA and 14 BNL were subjected to telomere length determination with a TeloTAGGG Telomere Length Assay Kit (Roche Diagnostic GmbH, Germany), following digestion of genomic DNA by frequently cutting restriction enzymes RsaI and HinfI. Mean telomerase activity in IDC (A450nm=0.3338), but not FA (A450nm=0.0003) was significantly raised (p<0.05) compared with BNL (A450nm=0.0031). Similarly IDC (1.2 kb), but not FA (2.2 kb), showed significant telomere shortening (p<0.05) relative to BNL (2.9 kb). The findings imply that telomere attrition and telomerase activation differ between malignant and benign tumours of the breast and may be important for targeted therapy.

High-resolution physical and functional mapping of the template adjacent DNA binding site in catalytically active telomerase.

PubMed

Romi, Erez; Baran, Nava; Gantman, Marina; Shmoish, Michael; Min, Bosun; Collins, Kathleen; Manor, Haim

2007-05-22

Telomerase is a cellular reverse transcriptase, which utilizes an integral RNA template to extend single-stranded telomeric DNA. We used site-specific photocrosslinking to map interactions between DNA primers and the catalytic protein subunit (tTERT) of Tetrahymena thermophila telomerase in functional enzyme complexes. Our assays reveal contact of the single-stranded DNA adjacent to the primer-template hybrid and tTERT residue W187 at the periphery of the N-terminal domain. This contact was detected in complexes with three different registers of template in the active site, suggesting that it is maintained throughout synthesis of a complete telomeric repeat. Substitution of nearby residue Q168, but not W187, alters the K(m) for primer elongation, implying that it plays a role in the DNA recognition. These findings are the first to directly demonstrate the physical location of TERT-DNA contacts in catalytically active telomerase and to identify amino acid determinants of DNA binding affinity. Our data also suggest a movement of the TERT active site relative to the template-adjacent single-stranded DNA binding site within a cycle of repeat synthesis.

Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B1.

PubMed Central

Doehmer, J; Dogra, S; Friedberg, T; Monier, S; Adesnik, M; Glatt, H; Oesch, F

1988-01-01

V79 Chinese hamster fibroblasts are widely used for mutagenicity testing but have the serious limitation that they do not express cytochromes P-450, which are needed for the activation of many promutagens to mutagenic metabolites. A full-length cDNA clone encoding the monooxygenase cytochrome P-450IIB1 under control of the simian virus 40 early promoter was constructed and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to G418) into V79 Chinese hamster cells. G418-resistant cells were selected, established as cell lines, and tested for cytochrome P-450IIB1 expression and enzymatic activity. Two cell lines (SD1 and SD3) were found that stably produce cytochrome P-450IIB1. Although purified cytochromes P-450 possess monooxygenase activity only after reconstitution with cytochrome P-450 reductase and phospholipid, the gene product of the construct exhibited this activity. This implies that the gene product is intracellularly localized in a way that allows access to the required components. If compared with V79 cells, the mutation rate for the hypoxanthine phosphoribosyltransferase (HPRT) locus in SD1 cells is markedly increased when exposed to aflatoxin B1, which is activated by this enzyme. Images PMID:3137560

Potential for bioremediation of agro-industrial effluents with high loads of pesticides by selected fungi.

PubMed

Karas, Panagiotis A; Perruchon, Chiara; Exarhou, Katerina; Ehaliotis, Constantinos; Karpouzas, Dimitrios G

2011-02-01

Wastewaters from the fruit packaging industry contain a high pesticide load and require treatment before their environmental discharge. We provide first evidence for the potential bioremediation of these wastewaters. Three white rot fungi (WRF) (Phanerochaete chrysosporium, Trametes versicolor, Pleurotus ostreatus) and an Aspergillus niger strain were tested in straw extract medium (StEM) and soil extract medium (SEM) for degrading the pesticides thiabendazole (TBZ), imazalil (IMZ), thiophanate methyl (TM), ortho-phenylphenol (OPP), diphenylamine (DPA) and chlorpyrifos (CHL). Peroxidase (LiP, MnP) and laccase (Lac) activity was also determined to investigate their involvement in pesticide degradation. T. versicolor and P. ostreatus were the most efficient degraders and degraded all pesticides (10 mg l⁻¹) except TBZ, with maximum efficiency in StEM. The phenolic pesticides OPP and DPA were rapidly degraded by these two fungi with a concurrent increase in MnP and Lac activity. In contrast, these enzymes were not associated with the degradation of CHL, IMZ and TM implying the involvement of other enzymes. T. versicolor degraded spillage-level pesticide concentrations (50 mg l⁻¹) either fully (DPA, OPP) or partially (TBZ, IMZ). The fungus was also able to rapidly degrade a mixture of TM/DPA (50 mg l⁻¹), whereas it failed to degrade IMZ and TBZ when supplied in a mixture with OPP. Overall, T. versicolor and P. ostreatus showed great potential for the bioremediation of wastewaters from the fruit packaging industry. However, degradation of TBZ should be also achieved before further scaling up.

Development of the radical-stable Coprinus cinereus peroxidase (CiP) by blocking the radical attack.

PubMed

Kim, Su Jin; Joo, Jeong Chan; Kim, Han Sang; Kwon, Inchan; Song, Bong Keun; Yoo, Young Je; Kim, Yong Hwan

2014-11-10

Despite the potential use of peroxidases as industrial biocatalysts, their practical application is often impeded due to suicide inactivation by radicals generated in oxidative reactions. Using a peroxidase from Coprinus cinereus (CiP) as a model enzyme, we revealed a dominant factor for peroxidase inactivation during phenol oxidation, and we engineered radical-stable mutants by site-directed mutagenesis of an amino acid residue susceptible to modification by phenoxyl radical. Mass spectrometry analysis of inactivated CiP identified an adduct between F230 and a phenoxyl radical, and subsequently, the F230 residue was mutated to amino acids that resisted radical coupling. Of the F230 mutants, the F230A mutant showed the highest stability against radical inactivation, retaining 80% of its initial activity, while the wild-type protein was almost completely inactivated. The F230A mutant also exhibited a 16-fold higher turnover of the phenol substrate compared with the wild-type enzyme. Furthermore, the F230A mutant was stable during the oxidation of other phenolic compounds, including m-cresol and 3-methoxyphenol. No structural changes were observed by UV-vis and CD spectra of CiP after radical coupling, implying that the F230-phenol radical adduct inactivated CiP by blocking substrate access to the active site. Our novel strategy can be used to improve the stability of other peroxidases inactivated by radicals. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

Deglycosylation of serum vitamin D3-binding protein leads to immunosuppression in cancer patients.

PubMed

Yamamoto, N; Naraparaju, V R; Asbell, S O

1996-06-15

Serum vitamin D3-binding protein (Gc protein) can be converted by beta-galactosidase of B cells and sialidase of T cells to a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor of the macrophage activating factor (MAF). Treatment of Gc protein with immobilized beta-galactosidase and sialidase generates an extremely high titered MAF, Gc-MAF. When peripheral blood monocytes/macrophages of 52 patients bearing various types of cancer were incubated with 100 pg/ml of GcMAF, the monocytes/macrophages of all patients were efficiently activated. However, the MAF precursor activity of patient plasma Gc protein was found to be severely reduced in about 25% of this patient population. About 45% of the patients had moderately reduced MAF precursor activities. Loss of the precursor activity was found to be due to deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase detected in the patient's bloodstream. The source of the enzyme appeared to be cancerous cells. Radiation therapy decreased plasma alpha-N-acetylgalactosaminidase activity with concomitant increase of precursor activity. This implies that radiation therapy decreases the number of cancerous cells capable of secreting alpha-N-acetylgalactosaminidase. Both alpha-N-acetylgalactosaminidase activity and MAF precursor activity of Gc protein in patient bloodstream can serve as diagnostic and prognostic indices.

Prediction of enzymatic pathways by integrative pathway mapping

PubMed Central

Wichelecki, Daniel J; San Francisco, Brian; Zhao, Suwen; Rodionov, Dmitry A; Vetting, Matthew W; Al-Obaidi, Nawar F; Lin, Henry; O'Meara, Matthew J; Scott, David A; Morris, John H; Russel, Daniel; Almo, Steven C; Osterman, Andrei L

2018-01-01

The functions of most proteins are yet to be determined. The function of an enzyme is often defined by its interacting partners, including its substrate and product, and its role in larger metabolic networks. Here, we describe a computational method that predicts the functions of orphan enzymes by organizing them into a linear metabolic pathway. Given candidate enzyme and metabolite pathway members, this aim is achieved by finding those pathways that satisfy structural and network restraints implied by varied input information, including that from virtual screening, chemoinformatics, genomic context analysis, and ligand -binding experiments. We demonstrate this integrative pathway mapping method by predicting the L-gulonate catabolic pathway in Haemophilus influenzae Rd KW20. The prediction was subsequently validated experimentally by enzymology, crystallography, and metabolomics. Integrative pathway mapping by satisfaction of structural and network restraints is extensible to molecular networks in general and thus formally bridges the gap between structural biology and systems biology. PMID:29377793

The genes and enzymes of sucrose metabolism in moderately thermophilic methanotroph Methylocaldum szegediense O12.

PubMed

But, Sergey Y; Solntseva, Natalia P; Egorova, Svetlana V; Mustakhimov, Ildar I; Khmelenina, Valentina N; Reshetnikov, Alexander; Trotsenko, Yuri A

2018-05-01

Four enzymes involved in sucrose metabolism: sucrose phosphate synthase (Sps), sucrose phosphate phosphatase (Spp), sucrose synthase (Sus) and fructokinase (FruK), were obtained as his-tagged proteins from the moderately thermophilic methanotroph Methylocaldum szegediense O12. Sps, Spp, FruK and Sus demonstrated biochemical properties similar to those of other bacterial counterparts, but the translated amino acid sequences of Sps and Spp displayed high divergence from the respective microbial enzymes. The Sus of M. szegediense O12 catalyzed the reversible reaction of sucrose cleavage in the presence of ADP or UDP and preferred ADP as a substrate, thus implying a connection between sucrose and glycogen metabolism. Sus-like genes were found only in a few methanotrophs, whereas amylosucrase was generally used in sucrose cleavage in this group of bacteria. Like other microbial fructokinases, FruK of M. szegediense O12 showed a high specificity to fructose.

Time-scale dynamics of proteome and transcriptome of the white-rot fungus Phlebia radiata: growth on spruce wood and decay effect on lignocellulose.

PubMed

Kuuskeri, Jaana; Häkkinen, Mari; Laine, Pia; Smolander, Olli-Pekka; Tamene, Fitsum; Miettinen, Sini; Nousiainen, Paula; Kemell, Marianna; Auvinen, Petri; Lundell, Taina

2016-01-01

The white-rot Agaricomycetes species Phlebia radiata is an efficient wood-decaying fungus degrading all wood components, including cellulose, hemicellulose, and lignin. We cultivated P. radiata in solid state cultures on spruce wood, and extended the experiment to 6 weeks to gain more knowledge on the time-scale dynamics of protein expression upon growth and wood decay. Total proteome and transcriptome of P. radiata were analyzed by peptide LC-MS/MS and RNA sequencing at specific time points to study the enzymatic machinery on the fungus' natural growth substrate. According to proteomics analyses, several CAZy oxidoreductase class-II peroxidases with glyoxal and alcohol oxidases were the most abundant proteins produced on wood together with enzymes important for cellulose utilization, such as GH7 and GH6 cellobiohydrolases. Transcriptome additionally displayed expression of multiple AA9 lytic polysaccharide monooxygenases indicative of oxidative cleavage of wood carbohydrate polymers. Large differences were observed for individual protein quantities at specific time points, with a tendency of enhanced production of specific peroxidases on the first 2 weeks of growth on wood. Among the 10 class-II peroxidases, new MnP1-long, characterized MnP2-long and LiP3 were produced in high protein abundances, while LiP2 and LiP1 were upregulated at highest level as transcripts on wood together with the oxidases and one acetyl xylan esterase, implying their necessity as primary enzymes to function against coniferous wood lignin to gain carbohydrate accessibility and fungal growth. Majority of the CAZy encoding transcripts upregulated on spruce wood represented activities against plant cell wall and were identified in the proteome, comprising main activities of white-rot decay. Our data indicate significant changes in carbohydrate-active enzyme expression during the six-week surveillance of P. radiata growing on wood. Response to wood substrate is seen already during the first weeks. The immediate oxidative enzyme action on lignin and wood cell walls is supported by detected lignin substructure sidechain cleavages, release of phenolic units, and visual changes in xylem cell wall ultrastructure. This study contributes to increasing knowledge on fungal genetics and lignocellulose bioconversion pathways, allowing us to head for systems biology, development of biofuel production, and industrial applications on plant biomass utilizing wood-decay fungi.

Antioxidant and anti-inflammatory effects of pigeon pea (Cajanus cajan L.) extracts on hydrogen peroxide- and lipopolysaccharide-treated RAW264.7 macrophages.

PubMed

Lai, Yi-Syuan; Hsu, Wei-Hsuan; Huang, Jan-Jeng; Wu, She-Ching

2012-12-01

Chronic inflammation has been linked to a wide range of progressive diseases, including cancer, neurological disease, metabolic disorder, and cardiovascular disease. Epidemiological studies have provided convincing evidence that natural dietary compounds, which humans consume as food, possess many biological activities, including chemopreventative activities against various chronic inflammatory diseases. Here, we investigated the effect of 50% ethanol extracts of pigeon pea, as well as its major component, cyanidin-3-monoglucoside, an anthocyanin, on DNA damage, the activity of antioxidant enzymes, and free radical scavenging capacity in hydrogen peroxide (H(2)O(2))-treated RAW264.7 macrophages. High-pressure liquid chromatography results indicated that 2 mg of the 50% ethanol extracts of pigeon pea contained 45 μg of cyanidin-3-monoglucoside. A comet assay indicated that 50% ethanol extracts of pigeon pea (2 mg mL(-1)) and of cyanidin-3-monoglucoside (10 μM) protected RAW264.7 cells from DNA damage induced by a 24 h H(2)O(2) treatment. These results can be attributed to the prevention of reduction in antioxidant enzyme activity and lipid peroxidation in H(2)O(2)-treated murine RAW264.7 macrophages by the 50% ethanol extracts of pigeon pea. Moreover, as there is an active interplay between oxidative stress and inflammation, we also evaluated the anti-inflammatory activity of the 50% ethanol extracts of pigeon pea and cyanidin-3-monoglucoside in lipopolysaccharide-treated RAW264.7 macrophages. We found that the 50% ethanol extracts of pigeon pea and of cyanidin-3-monoglucoside suppressed the production of inflammatory cytokines, including TNF-α, IL-1β, and IL-6, in these macrophages. These results imply that pigeon pea could be developed as a functional food by the food industry, or could be utilized for the commercial production of anthocyanins as antioxidants.

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

Gamma rays induce DNA damage and oxidative stress associated with impaired growth and reproduction in the copepod Tigriopus japonicus.

PubMed

Han, Jeonghoon; Won, Eun-Ji; Lee, Bo-Young; Hwang, Un-Ki; Kim, Il-Chan; Yim, Joung Han; Leung, Kenneth Mei Yee; Lee, Yong Sung; Lee, Jae-Seong

2014-07-01

Nuclear radioisotope accidents are potentially ecologically devastating due to their impact on marine organisms. To examine the effects of exposure of a marine organism to radioisotopes, we irradiated the intertidal copepod Tigriopus japonicus with several doses of gamma radiation and analyzed the effects on mortality, fecundity, and molting by assessing antioxidant enzyme activities and gene expression patterns. No mortality was observed at 96h, even in response to exposure to a high dose (800Gy) of radiation, but mortality rate was significantly increased 120h (5 days) after exposure to 600 or 800Gy gamma ray radiation. We observed a dose-dependent reduction in fecundity of ovigerous females; even the group irradiated with 50Gy showed a significant reduction in fecundity, suggesting that gamma rays are likely to have a population level effect. In addition, we observed growth retardation, particularly at the nauplius stage, in individuals after gamma irradiation. In fact, nauplii irradiated with more than 200Gy, though able to molt to copepodite stage 1, did not develop into adults. Upon gamma radiation, T. japonicus showed a dose-dependent increase in reactive oxygen species (ROS) levels, the activities of several antioxidant enzymes, and expression of double-stranded DNA break damage genes (e.g. DNA-PK, Ku70, Ku80). At a low level (sub-lethal dose) of gamma irradiation, we found dose-dependent upregulation of p53, implying cellular damage in T. japonicus in response to sub-lethal doses of gamma irradiation, suggesting that T. japonicus is not susceptible to sub-lethal doses of gamma irradiation. Additionally, antioxidant genes, phase II enzyme (e.g. GSTs), and cellular chaperone genes (e.g. Hsps) that are involved in cellular defense mechanisms also showed the same expression patterns for sublethal doses of gamma irradiation (50-200Gy). These findings indicate that sublethal doses of gamma radiation can induce oxidative stress-mediated DNA damage and increase the expression of antioxidant enzymes and proteins with chaperone-related functions, thereby significantly affecting life history parameters such as fecundity and molting in the copepod T. japonicus. Copyright © 2014 Elsevier B.V. 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

Circulating Matrix Metalloproteinase-2 and -9 Enzyme Activities in the Children with Ventricular Septal Defect

PubMed Central

Cheng, Kun-Shan; Liao, Yan-Chiou; Chen, Mu-Yuan; Kuan, Tang-Ching; Hong, Yi-Han; Ko, Li; Hsieh, Wen-Yeh; Wu, Chien-Liang; Chen, Ming-Ren; Lin, Chih-Sheng

2013-01-01

Ventricular septal defect (VSD) is the most common form of congenital heart diseases. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases involved in causal cardiac tissue remodeling. We studied the changes of circulating MMP-2 and MMP-9 activities in the patients with VSD severity and closure. There were 96 children with perimembranous VSD enrolled in this study. We assigned the patients into three groups according to the ratio of VSD diameter/diameter of aortic root (Ao). They were classified as below: Trivial (VSD/Ao ratio ≤ 0.2), Small (0.2 < VSD/Ao ≤ 0.3) and Median (0.3 < VSD/Ao) group. Plasma MMP-2 and MMP-9 activities were assayed by gelatin zymography. There was a significant higher MMP-2 activity in the VSD (Trivial, Small and Median) groups compared with that in Control group. The plasma MMP-9 activity showed a similar trend as the findings in MMP-2 activity. After one year follow-up, a significant difference in the MMP-9 activity was found between VSD spontaneous closure and non-closure groups. In conclusion, a positive trend between the severity of VSD and activities of MMP-2 and MMP-9 was found. Our data imply that MMP-2 and MMP-9 activities may play a role in the pathogenesis of VSD. PMID:23847438

Circulating matrix metalloproteinase-2 and -9 enzyme activities in the children with ventricular septal defect.

PubMed

Cheng, Kun-Shan; Liao, Yan-Chiou; Chen, Mu-Yuan; Kuan, Tang-Ching; Hong, Yi-Han; Ko, Li; Hsieh, Wen-Yeh; Wu, Chien-Liang; Chen, Ming-Ren; Lin, Chih-Sheng

2013-01-01

Ventricular septal defect (VSD) is the most common form of congenital heart diseases. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases involved in causal cardiac tissue remodeling. We studied the changes of circulating MMP-2 and MMP-9 activities in the patients with VSD severity and closure. There were 96 children with perimembranous VSD enrolled in this study. We assigned the patients into three groups according to the ratio of VSD diameter/diameter of aortic root (Ao). They were classified as below: Trivial (VSD/Ao ratio ≤ 0.2), Small (0.2 < VSD/Ao ≤ 0.3) and Median (0.3 < VSD/Ao) group. Plasma MMP-2 and MMP-9 activities were assayed by gelatin zymography. There was a significant higher MMP-2 activity in the VSD (Trivial, Small and Median) groups compared with that in Control group. The plasma MMP-9 activity showed a similar trend as the findings in MMP-2 activity. After one year follow-up, a significant difference in the MMP-9 activity was found between VSD spontaneous closure and non-closure groups. In conclusion, a positive trend between the severity of VSD and activities of MMP-2 and MMP-9 was found. Our data imply that MMP-2 and MMP-9 activities may play a role in the pathogenesis of VSD.

[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.

Unusual isotopic composition of C-CO2 from sterilized soil microcosms: a new way to separate intracellular from extracellular respiratory metabolisms.

NASA Astrophysics Data System (ADS)

Kéraval, Benoit; Alvarez, Gaël; Lehours, Anne Catherine; Amblard, Christian; Fontaine, Sebastien

2015-04-01

The mineralization of organic C requires two main steps. First, microorganisms secrete exoenzymes in soil in order to depolymerize plant and microbial cell walls and release soluble substrates for microbial assimilation. The second step of mineralization, during which C is released as CO2, implies the absorption and utilization of solubilized substrates by microbial cells with the aim to produce energy (ATP). In cells, soluble substrates are carried out by a cascade of respiratory enzymes, along which protons and electrons are transferred from a substrate to oxygen. Given the complexity of this oxidative metabolism and the typical fragility of respiratory enzymes, it is traditionally considered that respiration (second step of C mineralization process) is strictly an intracellular metabolism process. The recurrent observations of substantial CO2 emissions in soil microcosms where microbial cells have been reduced to extremely low levels challenges this paradigm. In a recent study where some respiratory enzymes have shown to function in an extracellular context in soils, Maire et al. (2013) suggested that an extracellular oxidative metabolism (EXOMET) substantially contributes to CO2 emission from soils. This idea is supported by the recent publication of Blankinship et al., 2014 who showed the presence of active enzymes involved in the Krebs cycle on soil particles. Many controversies subsist in the scientific community due to the presence of non-proliferating but morphologically intact cells after irradiation that could substantially contribute to those soil CO2 emissions. To test whether a purely extracellular oxidative metabolism contribute to soil CO2 emissions, we combined high doses of gamma irradiations to different time of soil autoclaving. The presence of active and non-active cells in soil was checked by DNA and RNA extraction and by electronic microscopy. None active cells (RNA-containing cells) were detectable after irradiation, but some morphological intact cells were observed by microscopy. These "ghost" cells were completely destroyed by the irradiation-autoclaving combination releasing large amount of soluble C. The soil respiration (O2 consumption and CO2 production) was reduced by irradiation and autoclaving but not stopped, suggesting the presence of an EXOMET. The delta 13C of CO2 released in the irradiated-autoclaved soil was strongly depleted (-70‰) indicating that this extracellular metabolism induced a substantial isotopic fractionation. Our findings suggest that two main oxidative metabolisms co-occur in soils: cell respiration and EXOMET. The isotopic fractionation induced by the EXOMET open perspectives for its quantification in non-sterilized living soils.

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

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.

Purification and characterization of the enzyme cholesterol oxidase from a new isolate of Streptomyces sp.

PubMed

Praveen, Vandana; Srivastava, Akanksha; Tripathi, C K M

2011-11-01

An extracellular cholesterol oxidase (cho) enzyme was isolated from the Streptomyces parvus, a new source and purified 18-fold by ion exchange and gel filtration chromatography. Specific activity of the purified enzyme was found to be 20 U/mg with a 55 kDa molecular mass. The enzyme was stable at pH 7.2 and 50 °C. The enzyme activity was inhibited in the presence of Pb(2+), Ag(2+), Hg(2+), and Zn(2+) and enhanced in the presence of Mn(2+). The enzyme activity was inhibited by the thiol-reducing reagents (DTT, β-mercaptoethanol), suggesting that disulfide linkage is essential for the enzyme activity. The enzyme activity was found to be maximum in the presence of Triton X-100 and X-114 detergents whereas sodium dodecyl sulfate fully inactivated the enzyme. The enzyme showed moderate stability towards all organic solvents except acetone, benzene, chloroform and the activity increased in the presence of isopropanol and ethanol. The K(m) value for the oxidation of cholesterol by this enzyme was 0.02 mM.

Esterase 22 and beta-glucuronidase hydrolyze retinoids in mouse liver

PubMed Central

Schreiber, Renate; Taschler, Ulrike; Wolinski, Heimo; Seper, Andrea; Tamegger, Stefanie N.; Graf, Maria; Kohlwein, Sepp D.; Haemmerle, Guenter; Zimmermann, Robert; Zechner, Rudolf; Lass, Achim

2009-01-01

Excess dietary vitamin A is esterified with fatty acids and stored in the form of retinyl ester (RE) predominantly in the liver. According to the requirements of the body, liver RE stores are hydrolyzed and retinol is delivered to peripheral tissues. The controlled mobilization of retinol ensures a constant supply of the body with the vitamin. Currently, the enzymes catalyzing liver RE hydrolysis are unknown. In this study, we identified mouse esterase 22 (Es22) as potent RE hydrolase highly expressed in the liver, particularly in hepatocytes. The enzyme is located exclusively at the endoplasmic reticulum (ER), implying that it is not involved in the mobilization of RE present in cytosolic lipid droplets. Nevertheless, cell culture experiments revealed that overexpression of Es22 attenuated the formation of cellular RE stores, presumably by counteracting retinol esterification at the ER. Es22 was previously shown to form a complex with β-glucuronidase (Gus). Our studies revealed that Gus colocalizes with Es22 at the ER but does not affect its RE hydrolase activity. Interestingly, however, Gus was capable of hydrolyzing the naturally occurring vitamin A metabolite retinoyl β-glucuronide. In conclusion, our observations implicate that both Es22 and Gus play a role in liver retinoid metabolism. PMID:19723663

Rhizobium meliloti anthranilate synthase gene: cloning, sequence, and expression in Escherichia coli.

PubMed Central

Bae, Y M; Holmgren, E; Crawford, I P

1989-01-01

We determined the DNA sequence of the Rhizobium meliloti gene encoding anthranilate synthase, the first enzyme of the tryptophan pathway. Sequences similar to those seen for the two subunits of the enzyme as found in all other procaryotic species studied are present in a single open reading frame of 729 codons. This apparent gene fusion joins the C terminus of the large subunit (TrpE) to the N terminus of the small subunit (TrpG) through a short connecting segment. We designate the fused gene trpE(G). The gene is flanked by a typical rho-independent terminator at the 3' end and a complex regulatory region at the 5' end resembling those of operons under transcriptional attenuation control. The location of the promoter was determined by S1 nuclease protection, using Rhizobium mRNA. Although this promoter was inactive in Escherichia coli, mutations eliciting activity were easily obtained. One of these was a C----T change at position -9 in the -10 region. The +1 position of the mRNA is the first base of the initiation codon of the leader peptide, implying that unlike trpE(G), which has a normal Shine-Dalgarno sequence, the leader peptide gene lacks a ribosome-binding site. Images PMID:2656657

Functional Characterization of a Flavonoid Glycosyltransferase in Sweet Orange (Citrus sinensis).

PubMed

Liu, Xiaogang; Lin, Cailing; Ma, Xiaodi; Tan, Yan; Wang, Jiuzhao; Zeng, Ming

2018-01-01

Fruits of sweet orange ( Citrus sinensis ), a popular commercial Citrus species, contain high concentrations of flavonoids beneficial to human health. These fruits predominantly accumulate O -glycosylated flavonoids, in which the disaccharides [neohesperidose (rhamnosyl-α-1,2-glucose) or rutinose (rhamnosyl-α-1,6-glucose)] are linked to the flavonoid aglycones through the 3- or 7-hydroxyl sites. The biotransformation of the flavonoid aglycones into O -rutinosides or O -neohesperidosides in the Citrus plants usually consists of two glycosylation reactions involving a series of uridine diphosphate-sugar dependent glycosyltransferases (UGTs). Although several genes encoding flavonoid UGTs have been functionally characterized in the Citrus plants, full elucidation of the flavonoid glycosylation process remains elusive. Based on the available genomic and transcriptome data, we isolated a UGT with a high expression level in the sweet orange fruits that possibly encodes a flavonoid glucosyltransferase and/or rhamnosyltransferase. Biochemical analyses revealed that a broad range of flavonoid substrates could be glucosylated at their 3- and/or 7-hydrogen sites by the recombinant enzyme, including hesperetin, naringenin, diosmetin, quercetin, and kaempferol. Furthermore, overexpression of the gene could significantly increase the accumulations of quercetin 7- O -rhamnoside, quercetin 7- O -glucoside, and kaempferol 7- O -glucoside, implying that the enzyme has flavonoid 7- O -glucosyltransferase and 7- O -rhamnosyltransferase activities in vivo .

WRINKLED Transcription Factors Orchestrate Tissue-Specific Regulation of Fatty Acid Biosynthesis in Arabidopsis[W

PubMed Central

To, Alexandra; Joubès, Jérôme; Barthole, Guillaume; Lécureuil, Alain; Scagnelli, Aurélie; Jasinski, Sophie; Lepiniec, Loïc; Baud, Sébastien

2012-01-01

Acyl lipids are essential constituents of all cells, but acyl chain requirements vary greatly and depend on the cell type considered. This implies a tight regulation of fatty acid production so that supply fits demand. Isolation of the Arabidopsis thaliana WRINKLED1 (WRI1) transcription factor established the importance of transcriptional regulation for modulating the rate of acyl chain production. Here, we report the isolation of two additional regulators of the fatty acid biosynthetic pathway, WRI3 and WRI4, which are closely related to WRI1 and belong to the APETALA2–ethylene-responsive element binding protein family of transcription factors. These three WRIs define a family of regulators capable of triggering sustained rates of acyl chain synthesis. However, expression patterns of the three WRIs differ markedly. Whereas only WRI1 activates fatty acid biosynthesis in seeds for triacylglycerol production, the three WRIs are required in floral tissues to provide acyl chains for cutin biosynthesis and prevent adherence of these developing organs and subsequent semisterility. The targets of these WRIs encode enzymes providing precursors (acyl chain and glycerol backbones) for various lipid biosynthetic pathways, but not the subsequent lipid-assembling enzymes. These results provide insights into the developmental regulation of fatty acid production in plants. PMID:23243127

Functional Characterization of a Flavonoid Glycosyltransferase in Sweet Orange (Citrus sinensis)

PubMed Central

Liu, Xiaogang; Lin, Cailing; Ma, Xiaodi; Tan, Yan; Wang, Jiuzhao; Zeng, Ming

2018-01-01

Fruits of sweet orange (Citrus sinensis), a popular commercial Citrus species, contain high concentrations of flavonoids beneficial to human health. These fruits predominantly accumulate O-glycosylated flavonoids, in which the disaccharides [neohesperidose (rhamnosyl-α-1,2-glucose) or rutinose (rhamnosyl-α-1,6-glucose)] are linked to the flavonoid aglycones through the 3- or 7-hydroxyl sites. The biotransformation of the flavonoid aglycones into O-rutinosides or O-neohesperidosides in the Citrus plants usually consists of two glycosylation reactions involving a series of uridine diphosphate-sugar dependent glycosyltransferases (UGTs). Although several genes encoding flavonoid UGTs have been functionally characterized in the Citrus plants, full elucidation of the flavonoid glycosylation process remains elusive. Based on the available genomic and transcriptome data, we isolated a UGT with a high expression level in the sweet orange fruits that possibly encodes a flavonoid glucosyltransferase and/or rhamnosyltransferase. Biochemical analyses revealed that a broad range of flavonoid substrates could be glucosylated at their 3- and/or 7-hydrogen sites by the recombinant enzyme, including hesperetin, naringenin, diosmetin, quercetin, and kaempferol. Furthermore, overexpression of the gene could significantly increase the accumulations of quercetin 7-O-rhamnoside, quercetin 7-O-glucoside, and kaempferol 7-O-glucoside, implying that the enzyme has flavonoid 7-O-glucosyltransferase and 7-O-rhamnosyltransferase activities in vivo. PMID:29497429

Real-time dynamics of RNA Polymerase II clustering in live human cells

NASA Astrophysics Data System (ADS)

Cisse, Ibrahim

2014-03-01

Transcription is the first step in the central dogma of molecular biology, when genetic information encoded on DNA is made into messenger RNA. How this fundamental process occurs within living cells (in vivo) is poorly understood,[1] despite extensive biochemical characterizations with isolated biomolecules (in vitro). For high-order organisms, like humans, transcription is reported to be spatially compartmentalized in nuclear foci consisting of clusters of RNA Polymerase II, the enzyme responsible for synthesizing all messenger RNAs. However, little is known of when these foci assemble or their relative stability. We developed an approach based on photo-activation localization microscopy (PALM) combined with a temporal correlation analysis, which we refer to as tcPALM. The tcPALM method enables the real-time characterization of biomolecular spatiotemporal organization, with single-molecule sensitivity, directly in living cells.[2] Using tcPALM, we observed that RNA Polymerase II clusters form transiently, with an average lifetime of 5.1 (+/- 0.4) seconds. Stimuli affecting transcription regulation yielded orders of magnitude changes in the dynamics of the polymerase clusters, implying that clustering is regulated and plays a role in the cells ability to effect rapid response to external signals. Our results suggest that the transient crowding of enzymes may aid in rate-limiting steps of genome regulation.

Novel protein-protein interaction between spermidine synthase and S-adenosylmethionine decarboxylase from Leishmania donovani.

PubMed

Mishra, Arjun K; Agnihotri, Pragati; Srivastava, Vijay Kumar; Pratap, J Venkatesh

2015-01-09

Polyamine biosynthesis pathway has long been considered an essential drug target for trypanosomatids including Leishmania. S-adenosylmethionine decarboxylase (AdoMetDc) and spermidine synthase (SpdSyn) are enzymes of this pathway that catalyze successive steps, with the product of the former, decarboxylated S-adenosylmethionine (dcSAM), acting as an aminopropyl donor for the latter enzyme. Here we have explored the possibility of and identified the protein-protein interaction between SpdSyn and AdoMetDc. The protein-protein interaction has been identified using GST pull down assay. Isothermal titration calorimetry reveals that the interaction is thermodynamically favorable. Fluorescence spectroscopy studies also confirms the interaction, with SpdSyn exhibiting a change in tertiary structure with increasing concentrations of AdoMetDc. Size exclusion chromatography suggests the presence of the complex as a hetero-oligomer. Taken together, these results suggest that the enzymes indeed form a heteromer. Computational analyses suggest that this complex differs significantly from the corresponding human complex, implying that this complex could be a better therapeutic target than the individual enzymes. Copyright © 2014 Elsevier Inc. All rights reserved.

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.

Nitric oxide is an obligate bacterial nitrification intermediate produced by hydroxylamine oxidoreductase.

PubMed

Caranto, Jonathan D; Lancaster, Kyle M

2017-08-01

Ammonia (NH 3 )-oxidizing bacteria (AOB) emit substantial amounts of nitric oxide (NO) and nitrous oxide (N 2 O), both of which contribute to the harmful environmental side effects of large-scale agriculture. The currently accepted model for AOB metabolism involves NH 3 oxidation to nitrite (NO 2 - ) via a single obligate intermediate, hydroxylamine (NH 2 OH). Within this model, the multiheme enzyme hydroxylamine oxidoreductase (HAO) catalyzes the four-electron oxidation of NH 2 OH to NO 2 - We provide evidence that HAO oxidizes NH 2 OH by only three electrons to NO under both anaerobic and aerobic conditions. NO 2 - observed in HAO activity assays is a nonenzymatic product resulting from the oxidation of NO by O 2 under aerobic conditions. Our present study implies that aerobic NH 3 oxidation by AOB occurs via two obligate intermediates, NH 2 OH and NO, necessitating a mediator of the third enzymatic step.

Multi-platform ’Omics Analysis of Human Ebola Virus Disease Pathogenesis

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

Eisfeld, Amie J.; Halfmann, Peter J.; Wendler, Jason P.

The pathogenesis of human Ebola virus disease (EVD) is complex. EVD is characterized by high levels of virus replication and dissemination, dysregulated immune responses, extensive virus- and host-mediated tissue damage, and disordered coagulation. To clarify how host responses contribute to EVD pathophysiology, we performed multi-platform ’omics analysis of peripheral blood mononuclear cells and plasma from EVD patients. Our results indicate that EVD molecular signatures overlap with those of sepsis, imply that pancreatic enzymes contribute to tissue damage in fatal EVD, and suggest that Ebola virus infection may induce aberrant neutrophils whose activity could explain hallmarks of fatal EVD. Moreover, integratedmore » biomarker prediction identified putative biomarkers from different data platforms that differentiated survivors and fatalities early after infection. This work reveals insight into EVD pathogenesis, suggests an effective approach for biomarker identification, and provides an important community resource for further analysis of human EVD severity.« less

Red-Emitting Fluorescent Probe for Detection of γ-Glutamyltranspeptidase and Its Application of Real-Time Imaging under Oxidative Stress in Cells and in Vivo.

PubMed

Liu, Feiyan; Wang, Zhen; Wang, Wenli; Luo, Jian-Guang; Kong, Lingyi

2018-06-19

γ-Glutamyltranspeptidase (GGT) plays critical roles in regulating various physiological/pathophysiological processes including the intracellular redox homeostasis. However, an effective fluorescent probe for dissecting the relationships between GGT and oxidative stress in vivo remains largely unexplored. Herein, we present a light-up fluorescent probe (DCDHF-Glu) with long wavelength emission (613 nm) for the highly sensitive and selective detection of GGT using dicyanomethylenedihydrofuran derivative as the fluorescent reporter and γ-glutamyl group as the enzyme-active trigger. DCDHF-Glu is competent to real-time image endogenous GGT in live cells and mice. In particular, DCDHF-Glu enables the direct real-time visualization of the upregulation of GGT under drug-induced oxidative stress in the HepG2 cells and the LO2 cells, as well as in vivo, vividly implying its excellent capacity in elucidation of GGT function in GGT-related biological events.

Multi-platform 'Omics Analysis of Human Ebola Virus Disease Pathogenesis.

PubMed

Eisfeld, Amie J; Halfmann, Peter J; Wendler, Jason P; Kyle, Jennifer E; Burnum-Johnson, Kristin E; Peralta, Zuleyma; Maemura, Tadashi; Walters, Kevin B; Watanabe, Tokiko; Fukuyama, Satoshi; Yamashita, Makoto; Jacobs, Jon M; Kim, Young-Mo; Casey, Cameron P; Stratton, Kelly G; Webb-Robertson, Bobbie-Jo M; Gritsenko, Marina A; Monroe, Matthew E; Weitz, Karl K; Shukla, Anil K; Tian, Mingyuan; Neumann, Gabriele; Reed, Jennifer L; van Bakel, Harm; Metz, Thomas O; Smith, Richard D; Waters, Katrina M; N'jai, Alhaji; Sahr, Foday; Kawaoka, Yoshihiro

2017-12-13

The pathogenesis of human Ebola virus disease (EVD) is complex. EVD is characterized by high levels of virus replication and dissemination, dysregulated immune responses, extensive virus- and host-mediated tissue damage, and disordered coagulation. To clarify how host responses contribute to EVD pathophysiology, we performed multi-platform 'omics analysis of peripheral blood mononuclear cells and plasma from EVD patients. Our results indicate that EVD molecular signatures overlap with those of sepsis, imply that pancreatic enzymes contribute to tissue damage in fatal EVD, and suggest that Ebola virus infection may induce aberrant neutrophils whose activity could explain hallmarks of fatal EVD. Moreover, integrated biomarker prediction identified putative biomarkers from different data platforms that differentiated survivors and fatalities early after infection. This work reveals insight into EVD pathogenesis, suggests an effective approach for biomarker identification, and provides an important community resource for further analysis of human EVD severity. Copyright © 2017 Elsevier Inc. All rights reserved.

The Molecular Basis of TnrA Control by Glutamine Synthetase in Bacillus subtilis*

PubMed Central

Hauf, Ksenia; Kayumov, Airat; Gloge, Felix; Forchhammer, Karl

2016-01-01

TnrA is a master regulator of nitrogen assimilation in Bacillus subtilis. This study focuses on the mechanism of how glutamine synthetase (GS) inhibits TnrA function in response to key metabolites ATP, AMP, glutamine, and glutamate. We suggest a model of two mutually exclusive GS conformations governing the interaction with TnrA. In the ATP-bound state (A-state), GS is catalytically active but unable to interact with TnrA. This conformation was stabilized by phosphorylated l-methionine sulfoximine (MSX), fixing the enzyme in the transition state. When occupied by glutamine (or its analogue MSX), GS resides in a conformation that has high affinity for TnrA (Q-state). The A- and Q-state are mutually exclusive, and in agreement, ATP and glutamine bind to GS in a competitive manner. At elevated concentrations of glutamine, ATP is no longer able to bind GS and to bring it into the A-state. AMP efficiently competes with ATP and prevents formation of the A-state, thereby favoring GS-TnrA interaction. Surface plasmon resonance analysis shows that TnrA bound to a positively regulated promoter fragment binds GS in the Q-state, whereas it rapidly dissociates from a negatively regulated promoter fragment. These data imply that GS controls TnrA activity at positively controlled promoters by shielding the transcription factor in the DNA-bound state. According to size exclusion and multiangle light scattering analysis, the dodecameric GS can bind three TnrA dimers. The highly interdependent ligand binding properties of GS reveal this enzyme as a sophisticated sensor of the nitrogen and energy state of the cell to control the activity of DNA-bound TnrA. PMID:26635369

Osteoimmunology: Influence of the Immune System on Bone Regeneration and Consumption.

PubMed

Limmer, Andreas; Wirtz, Dieter C

2017-06-01

Background Stimulating bone regeneration is a central aim in orthopaedic and trauma surgery. Although the replacement of bone with artificial materials like cement or apatite helps to keep up bone stability, new bone often cannot be regenerated. Increasing research efforts have led to the clinical application of growth factors stimulating bone growth (e.g. bone morphogenic protein, BMP) and inhibitors preventing bone consumption (e.g. RANKL blocking antibodies). These factors mostly concentrate on stimulating osteoblast or preventing osteoclast activity. Current Situation It is widely accepted that osteoblasts and osteoclasts are central players in bone regeneration. This concept assumes that osteoblasts are responsible for bone growth while osteoclasts cause bone consumption by secreting matrix-degrading enzymes such as cathepsin K and matrix metalloproteinases (MMP). However, according to new research results, bone growth or consumption are not regulated by single cell types. It is rather the interaction of various cell types that regulates bone metabolism. While factors secreted by osteoblasts are essential for osteoclast differentiation and activation, factors secreted by activated osteoclasts are essential for osteoblast activity. In addition, recent research results imply that the influence of the immune system on bone metabolism has long been neglected. Factors secreted by macrophages or T cells strongly influence bone growth or degradation, depending on the bone microenvironment. Infections, sterile inflammation or tumour metastases not only affect bone cells directly, but also influence immune cells such as T cells indirectly. Furthermore, immune cells and bone are mechanistically regulated by similar factors such as cytokines, chemokines and transcription factors, suggesting that the definition of bone and immune cells has to be thought over. Outlook Bone and the immune system are regulated by similar mechanisms. These newly identified similarities between bone and the immune system imply that medication developed for tumour and autoimmune patients could also be applied in bone diseases. Georg Thieme Verlag KG Stuttgart · New York.

Biodegradation of cyanide by acetonitrile-induced cells of Rhodococcus sp. UKMP-5M.

PubMed

Nallapan Maniyam, Maegala; Sjahrir, Fridelina; Ibrahim, Abdul Latif; Cass, Anthony E G

2013-01-01

A Rhodococcus sp. UKMP-5M isolate was shown to detoxify cyanide successfully, suggesting the presence of an intrinsic property in the bacterium which required no prior cyanide exposure for induction of this property. However, in order to promote growth, Rhodococcus sp. UKMP-5M was fully acclimatized to cyanide after 7 successive subcultures in 0.1 mM KCN for 30 days. To further shorten the lag phase and simultaneously increase the tolerance towards higher cyanide concentrations, the bacterium was induced with various nitrile compounds sharing a similar degradatory pathway to cyanide. Acetonitrile emerged as the most favored inducer and the induced cells were able to degrade 0.1 mM KCN almost completely within 18 h. With the addition of subsequent aliquots of 0.1 mM KCN a shorter period for complete removal of cyanide was required, which proved to be advantageous economically. Both resting cells and crude enzyme of Rhodococcus sp. UKMP-5M were able to biodegrade cyanide to ammonia and formate without the formation of formamide, implying the identification of a simple hydrolytic cyanide degradation pathway involving the enzyme cyanidase. Further verification with SDS-PAGE revealed that the molecular weight of the active enzyme was estimated to be 38 kDa, which is consistent with previously reported cyanidases. Since the recent advancement in the application of biological methods in treating cyanide-bearing wastewater has been promising, the discovery of this new bacterium will add value by diversifying the existing microbial populations capable of cyanide detoxification.

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.

Isolated and mixed effects of diuron and its metabolites on biotransformation enzymes and oxidative stress response of Nile tilapia (Oreochromis niloticus).

PubMed

Felício, Andréia Arantes; Freitas, Juliane Silberschmidt; Scarin, Jéssica Bolpeti; de Souza Ondei, Luciana; Teresa, Fabrício Barreto; Schlenk, Daniel; de Almeida, Eduardo Alves

2018-03-01

Diuron is one of the most used herbicide in the world, and its field application has been particularly increased in Brazil due to the expansion of sugarcane crops. Diuron has often been detected in freshwater ecosystems and it can be biodegraded into three main metabolites in the environment, the 3,4-dichloroaniline (DCA), 3,4-dichlorophenylurea (DCPU) and 3,4-dichlorophenyl-N-methylurea (DCPMU). Negative effects under aquatic biota are still not well established for diuron, especially when considering its presence in mixture with its different metabolites. In this study, we evaluated the effects of diuron alone or in combination with its metabolites, DCPMU, DCPU and 3,4-DCA on biochemical stress responses and biotransformation activity of the fish Oreochromis niloticus. Results showed that diuron and its metabolites caused significant but dispersed alterations in oxidative stress markers and biotransformation enzymes, except for ethoxyresorufin-O-deethylase (EROD) activity, that presented a dose-dependent increase after exposure to either diuron or its metabolites. Glutathione S-transferase (GST) activity was significant lower in gills after exposure to diuron metabolites, but not diuron. Diuron, DCPMU and DCA also decreased the multixenobiotic resistance (MXR) activity. Lipid peroxidation levels were increased in gill after exposure to all compounds, indicating that the original compound and diuron metabolites can induce oxidative stress in fish. The integration of all biochemical responses by the Integrated Biomarker Response (IBR) model indicated that all compounds caused significant alterations in O. niloticus, but DCPMU caused the higher alterations in both liver and gill. Our findings imply that diuron and its metabolites may impair the physiological response related to biotransformation and antioxidant activity in fish at field concentrations. Such alterations could interfere with the ability of aquatic animals to adapt to environments contaminated by agriculture. Copyright © 2017 Elsevier Inc. All rights reserved.

A Patatin-Like Protein Associated with the Polyhydroxyalkanoate (PHA) Granules of Haloferax mediterranei Acts as an Efficient Depolymerase in the Degradation of Native PHA

PubMed Central

Liu, Guiming; Hou, Jing; Cai, Shuangfeng; Zhao, Dahe; Cai, Lei; Han, Jing; Zhou, Jian

2015-01-01

The key enzymes and pathways involved in polyhydroxyalkanoate (PHA) biosynthesis in haloarchaea have been identified in recent years, but the haloarchaeal enzymes for PHA degradation remain unknown. In this study, a patatin-like PHA depolymerase, PhaZh1, was determined to be located on the PHA granules in the haloarchaeon Haloferax mediterranei. PhaZh1 hydrolyzed the native PHA (nPHA) [including native polyhydroxybutyrate (nPHB) and native poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (nPHBV) in this study] granules in vitro with 3-hydroxybutyrate (3HB) monomer as the primary product. The site-directed mutagenesis of PhaZh1 indicated that Gly16, Ser47 (in a classical lipase box, G-X-S47-X-G), and Asp195 of this depolymerase were essential for its activity in nPHA granule hydrolysis. Notably, phaZh1 and bdhA (encoding putative 3HB dehydrogenase) form a gene cluster (HFX_6463 to _6464) in H. mediterranei. The 3HB monomer generated from nPHA degradation by PhaZh1 could be further converted into acetoacetate by BdhA, indicating that PhaZh1-BdhA may constitute the first part of a PHA degradation pathway in vivo. Interestingly, although PhaZh1 showed efficient activity and was most likely the key enzyme in nPHA granule hydrolysis in vitro, the knockout of phaZh1 had no significant effect on the intracellular PHA mobilization, implying the existence of an alternative PHA mobilization pathway(s) that functions effectively within the cells of H. mediterranei. Therefore, identification of this patatin-like depolymerase of haloarchaea may provide a new strategy for producing the high-value-added chiral compound (R)-3HB and may also shed light on the PHA mobilization in haloarchaea. PMID:25710370

Noncompetitive inhibition of indolethylamine-N-methyltransferase by N,N-dimethyltryptamine and N,N-dimethylaminopropyltryptamine.

PubMed

Chu, Uyen B; Vorperian, Sevahn K; Satyshur, Kenneth; Eickstaedt, Kelsey; Cozzi, Nicholas V; Mavlyutov, Timur; Hajipour, Abdol R; Ruoho, Arnold E

2014-05-13

Indolethylamine-N-methyltransferase (INMT) is a Class 1 transmethylation enzyme known for its production of N,N-dimethyltryptamine (DMT), a hallucinogen with affinity for various serotonergic, adrenergic, histaminergic, dopaminergic, and sigma-1 receptors. DMT is produced via the action of INMT on the endogenous substrates tryptamine and S-adenosyl-l-methionine (SAM). The biological, biochemical, and selective small molecule regulation of INMT enzyme activity remain largely unknown. Kinetic mechanisms for inhibition of rabbit lung INMT (rabINMT) by the product, DMT, and by a new novel tryptamine derivative were determined. After Michaelis-Menten and Lineweaver-Burk analyses had been applied to study inhibition, DMT was found to be a mixed competitive and noncompetitive inhibitor when measured against tryptamine. The novel tryptamine derivative, N-[2-(1H-indol-3-yl)ethyl]-N',N'-dimethylpropane-1,3-diamine (propyl dimethyl amino tryptamine or PDAT), was shown to inhibit rabINMT by a pure noncompetitive mechanism when measured against tryptamine with a Ki of 84 μM. No inhibition by PDAT was observed at 2 mM when it was tested against structurally similar Class 1 methyltransferases, such as human phenylethanolamine-N-methyltransferase (hPNMT) and human nicotinamide-N-methyltransferase (hNNMT), indicating selectivity for INMT. The demonstration of noncompetitive mechanisms for INMT inhibition implies the presence of an inhibitory allosteric site. In silico analyses using the computer modeling software Autodock and the rabINMT sequence threaded onto the human INMT (hINMT) structure (Protein Data Bank entry 2A14 ) identified an N-terminal helix-loop-helix non-active site binding region of the enzyme. The energies for binding of DMT and PDAT to this region of rabINMT, as determined by Autodock, were -6.34 and -7.58 kcal/mol, respectively. Assessment of the allosteric control of INMT may illuminate new biochemical pathway(s) underlying the biology of INMT.

Noncompetitive Inhibition of Indolethylamine-N-methyltransferase by N,N-Dimethyltryptamine and N,N-Dimethylaminopropyltryptamine

PubMed Central

2015-01-01

Indolethylamine-N-methyltransferase (INMT) is a Class 1 transmethylation enzyme known for its production of N,N-dimethyltryptamine (DMT), a hallucinogen with affinity for various serotonergic, adrenergic, histaminergic, dopaminergic, and sigma-1 receptors. DMT is produced via the action of INMT on the endogenous substrates tryptamine and S-adenosyl-l-methionine (SAM). The biological, biochemical, and selective small molecule regulation of INMT enzyme activity remain largely unknown. Kinetic mechanisms for inhibition of rabbit lung INMT (rabINMT) by the product, DMT, and by a new novel tryptamine derivative were determined. After Michaelis–Menten and Lineweaver–Burk analyses had been applied to study inhibition, DMT was found to be a mixed competitive and noncompetitive inhibitor when measured against tryptamine. The novel tryptamine derivative, N-[2-(1H-indol-3-yl)ethyl]-N′,N′-dimethylpropane-1,3-diamine (propyl dimethyl amino tryptamine or PDAT), was shown to inhibit rabINMT by a pure noncompetitive mechanism when measured against tryptamine with a Ki of 84 μM. No inhibition by PDAT was observed at 2 mM when it was tested against structurally similar Class 1 methyltransferases, such as human phenylethanolamine-N-methyltransferase (hPNMT) and human nicotinamide-N-methyltransferase (hNNMT), indicating selectivity for INMT. The demonstration of noncompetitive mechanisms for INMT inhibition implies the presence of an inhibitory allosteric site. In silico analyses using the computer modeling software Autodock and the rabINMT sequence threaded onto the human INMT (hINMT) structure (Protein Data Bank entry 2A14) identified an N-terminal helix–loop–helix non-active site binding region of the enzyme. The energies for binding of DMT and PDAT to this region of rabINMT, as determined by Autodock, were −6.34 and −7.58 kcal/mol, respectively. Assessment of the allosteric control of INMT may illuminate new biochemical pathway(s) underlying the biology of INMT. PMID:24730580

Active subsite properties, subsite residues and targeting to lysosomes or midgut lumen of cathepsins L from the beetle Tenebrio molitor.

PubMed

Damasceno, Ticiane F; Dias, Renata O; de Oliveira, Juliana R; Salinas, Roberto K; Juliano, Maria A; Ferreira, Clelia; Terra, Walter R

2017-10-01

Cathepsins L are the major digestive peptidases in the beetle Tenebrio molitor. Two digestive cathepsins L (TmCAL2 and TmCAL3) from it had their 3D structures solved. The aim of this paper was to study in details TmCAL3 specificity and properties and relate them to its 3D structure. Recombinant TmCAL3 was assayed with 64 oligopeptides with different amino acid replacements in positions P2, P1, P1' and P2'. Results showed that TmCAL3 S2 specificity differs from the human enzyme and that its specificities also explain why on autoactivation two propeptide residues remain in the enzyme. Data on free energy of binding and of activation showed that S1 and S2' are mainly involved in substrate binding, S1' acts in substrate binding and catalysis, whereas S2 is implied mainly in catalysis. Enzyme subsite residues were identified by docking with the same oligopeptide used for kinetics. The subsite hydrophobicities were calculated from the efficiency of hydrolysis of different amino acid replacements in the peptide and from docking data. The results were closer for S1 and S2' than for S1' and S2, indicating that the residue subsites that were more involved in transition state binding are different from those binding the substrate seen in docking. Besides TmCAL1-3, there are nine other cathepsins L, most of them more expressed at midgut. They are supposed to be directed to lysosomes by a Drosophila-like Lerp receptor and/or motifs in their prodomains. The mannose 6-phosphate lysosomal sorting machinery is absent from T. molitor transcriptome. Cathepsin L direction to midgut contents seems to depend on overexpression. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

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.

Novel cinnamic acid/4-aminoquinoline conjugates bearing non-proteinogenic amino acids: towards the development of potential dual action antimalarials.

PubMed

Pérez, Bianca C; Teixeira, Cátia; Figueiras, Marta; Gut, Jiri; Rosenthal, Philip J; Gomes, José R B; Gomes, Paula

2012-08-01

A series of cinnamic acid/4-aminoquinoline conjugates conceived to link, through a proper retro-enantio dipeptide, a heterocyclic core known to prevent hemozoin formation, to a trans-cinnamic acid motif capable of inhibiting enzyme catalytic Cys residues, were synthesized as potential dual-action antimalarials. The effect of amino acid configuration and the absence of the dipeptide spacer were also assessed. The replacement of the D-amino acids by their natural L counterparts led to a decrease in both anti-plasmodial and falcipain-inhibitory activity, suggesting that the former are preferable. Molecules with such spacer were active against blood-stage Plasmodium falciparum, in vitro, and hemozoin formation, implying that the dipeptide has a key role in mediating these two activities. In turn, compounds without spacer were better falcipain-2 inhibitors, likely because these compounds are smaller and have their vinyl bonds in closer vicinity to the catalytic Cys, as suggested by molecular modeling calculations. These novel conjugates constitute promising leads for the development of new antiplasmodials targeted at blood-stage malaria parasites. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Cardiac-specific overexpression of aldehyde dehydrogenase 2 exacerbates cardiac remodeling in response to pressure overload.

PubMed

Dassanayaka, Sujith; Zheng, Yuting; Gibb, Andrew A; Cummins, Timothy D; McNally, Lindsey A; Brittian, Kenneth R; Jagatheesan, Ganapathy; Audam, Timothy N; Long, Bethany W; Brainard, Robert E; Jones, Steven P; Hill, Bradford G

2018-06-01

Pathological cardiac remodeling during heart failure is associated with higher levels of lipid peroxidation products and lower abundance of several aldehyde detoxification enzymes, including aldehyde dehydrogenase 2 (ALDH2). An emerging idea that could explain these findings concerns the role of electrophilic species in redox signaling, which may be important for adaptive responses to stress or injury. The purpose of this study was to determine whether genetically increasing ALDH2 activity affects pressure overload-induced cardiac dysfunction. Mice subjected to transverse aortic constriction (TAC) for 12 weeks developed myocardial hypertrophy and cardiac dysfunction, which were associated with diminished ALDH2 expression and activity. Cardiac-specific expression of the human ALDH2 gene in mice augmented myocardial ALDH2 activity but did not improve cardiac function in response to pressure overload. After 12 weeks of TAC, ALDH2 transgenic mice had larger hearts than their wild-type littermates and lower capillary density. These findings show that overexpression of ALDH2 augments the hypertrophic response to pressure overload and imply that downregulation of ALDH2 may be an adaptive response to certain forms of cardiac pathology. Copyright © 2018. Published by Elsevier B.V.

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

Protective effect of Ocimum sanctum on 3-methylcholanthrene, 7,12-dimethylbenz(a)anthracene and aflatoxin B1 induced skin tumorigenesis in mice

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

Rastogi, Shipra; Shukla, Yogeshwer; Paul, Bhola N.

A study on the protective effect of alcoholic extract of the leaves of Ocimum sanctum on 3-mthylcholanthrene (MCA), 7,12-dimethylbenzanthracene (DMBA) and aflatoxin B{sub 1} (AFB{sub 1}) induced skin tumorigenesis in a mouse model has been investigated. The study involved pretreatment of mice with the leaf extract prior to either MCA application or tetradecanoyl phorbol acetate (TPA) treatment in a two-stage tumor protocol viz a viz, DMBA/TPA and AFB1/TPA. The results of the present study indicate that the pretreatment with alcoholic extract of the leaves of O. sanctum decreased the number of tumors in MCA, DMBA/TPA and AFB1/TPA treated mice. Themore » skin tumor induced animals pretreated with alcoholic extract led to a decrease in the expression of cutaneous {gamma}-glutamyl transpeptidase (GGT) and glutathione-S-transferase-P (GST-P) protein. The histopathological examination of skin tumors treated with leaf extract showed increased infiltration of polymorphonuclear, mononuclear and lymphocytic cells, decreased ornithine decarboxylase activity with concomitant enhancement of interleukin-1{beta} (IL-1{beta}) and tumor necrosis factor-{alpha} (TNF-{alpha}) in the serum, implying the in vivo antiproliferative and immunomodulatory activity of leaf extract. The decrease in cutaneous phase I enzymes and elevation of phase II enzymes in response to topical application of leaf extract prior to MCA, AFB1, DMBA/TPA and AFB1/TPA treatment indicate the possibility of impairment in reactive metabolite(s) formation and thereby reducing skin carcinogenicity. Furthermore, pretreatment of leaf extract in the carcinogen induced animals resulted in elevation of glutathione levels and decrease in lipid peroxidation along with heat shock protein expression, indicating a scavenging or antioxidant potential of the extract during chemical carcinogenesis. Thus it can be concluded that leaf extract of O. sanctum provides protection against chemical carcinogenesis in one or more of the following mechanisms: (i) by acting as an antioxidant; (ii) by modulating phase I and II enzymes; (iii) by exhibiting antiproliferative activity.« less

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.

SAR11 lipid renovation in response to phosphate starvation

PubMed Central

Carini, Paul; Van Mooy, Benjamin A. S.; Thrash, J. Cameron; White, Angelicque; Zhao, Yanlin; Campbell, Emily O.; Fredricks, Helen F.; Giovannoni, Stephen J.

2015-01-01

Phytoplankton inhabiting oligotrophic ocean gyres actively reduce their phosphorus demand by replacing polar membrane phospholipids with those lacking phosphorus. Although the synthesis of nonphosphorus lipids is well documented in some heterotrophic bacterial lineages, phosphorus-free lipid synthesis in oligotrophic marine chemoheterotrophs has not been directly demonstrated, implying they are disadvantaged in phosphate-deplete ecosystems, relative to phytoplankton. Here, we show the SAR11 clade chemoheterotroph Pelagibacter sp. str. HTCC7211 renovates membrane lipids when phosphate starved by replacing a portion of its phospholipids with monoglucosyl- and glucuronosyl-diacylglycerols and by synthesizing new ornithine lipids. Lipid profiles of cells grown with excess phosphate consisted entirely of phospholipids. Conversely, up to 40% of the total lipids were converted to nonphosphorus lipids when cells were starved for phosphate, or when growing on methylphosphonate. Cells sequentially limited by phosphate and methylphosphonate transformed >75% of their lipids to phosphorus-free analogs. During phosphate starvation, a four-gene cluster was significantly up-regulated that likely encodes the enzymes responsible for lipid renovation. These genes were found in Pelagibacterales strains isolated from a phosphate-deficient ocean gyre, but not in other strains from coastal environments, suggesting alternate lipid synthesis is a specific adaptation to phosphate scarcity. Similar gene clusters are found in the genomes of other marine α-proteobacteria, implying lipid renovation is a common strategy used by heterotrophic cells to reduce their requirement for phosphorus in oligotrophic habitats. PMID:26056292

[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.

Removal of pigments from molasses wastewater by combining micro-electrolysis with biological treatment method.

PubMed

Chen, Ben; Tian, Xiaofei; Yu, Lian; Wu, Zhenqiang

2016-12-01

Pigments in molasses wastewater (MWW) effluent, such as melanoidins, were considered as kinds of the most recalcitrant and hazardous colorant contaminants to the environment. In this study, de-coloring the MWW by a synergistic combination of micro-electrolysis with bio-treatment was performed. Aiming to a high de-colorization yield, levels of nutrition source supplies, MWW dilution ratio, and micro-electrolysis reaction time were optimized accordingly. For a diluted (50 %, v/v) MWW, an maximum overall de-colorization yield (97.1 ± 0.5 %, for absorbance at 475 nm) was achieved through the bio-electrolysis treatment. In electrolysis bio-treatment, the positive effect of micro-electrolysis was also revealed by a promoted growth of fungal biomass as well as activities of ligninolytic enzymes. Activities of lignin peroxidase, manganese peroxidase, and laccase were promoted by 111.2, 103.9, and 7.7 %, respectively. This study also implied that the bio-treatment and the micro-electrolysis had different efficiencies on removal of pigments with distinct polarities.

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.

Biochemistry of Catabolic Reductive Dehalogenation.

PubMed

Fincker, Maeva; Spormann, Alfred M

2017-06-20

A wide range of phylogenetically diverse microorganisms couple the reductive dehalogenation of organohalides to energy conservation. Key enzymes of such anaerobic catabolic pathways are corrinoid and Fe-S cluster-containing, membrane-associated reductive dehalogenases. These enzymes catalyze the reductive elimination of a halide and constitute the terminal reductases of a short electron transfer chain. Enzymatic and physiological studies revealed the existence of quinone-dependent and quinone-independent reductive dehalogenases that are distinguishable at the amino acid sequence level, implying different modes of energy conservation in the respective microorganisms. In this review, we summarize current knowledge about catabolic reductive dehalogenases and the electron transfer chain they are part of. We review reaction mechanisms and the role of the corrinoid and Fe-S cluster cofactors and discuss physiological implications.

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.

Analytic derivation of bacterial growth laws from a simple model of intracellular chemical dynamics.

PubMed

Pandey, Parth Pratim; Jain, Sanjay

2016-09-01

Experiments have found that the growth rate and certain other macroscopic properties of bacterial cells in steady-state cultures depend upon the medium in a surprisingly simple manner; these dependencies are referred to as 'growth laws'. Here we construct a dynamical model of interacting intracellular populations to understand some of the growth laws. The model has only three population variables: an amino acid pool, a pool of enzymes that transport an external nutrient and produce the amino acids, and ribosomes that catalyze their own and the enzymes' production from the amino acids. We assume that the cell allocates its resources between the enzyme sector and the ribosomal sector to maximize its growth rate. We show that the empirical growth laws follow from this assumption and derive analytic expressions for the phenomenological parameters in terms of the more basic model parameters. Interestingly, the maximization of the growth rate of the cell as a whole implies that the cell allocates resources to the enzyme and ribosomal sectors in inverse proportion to their respective 'efficiencies'. The work introduces a mathematical scheme in which the cellular growth rate can be explicitly determined and shows that two large parameters, the number of amino acid residues per enzyme and per ribosome, are useful for making approximations.

[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.

Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines

PubMed Central

López-Cruz, Roberto I.; Crocker, Daniel E.; Gaxiola-Robles, Ramón; Bernal, Jaime A.; Real-Valle, Roberto A.; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

2016-01-01

Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements. PMID:27375492

Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines.

PubMed

López-Cruz, Roberto I; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal, Jaime A; Real-Valle, Roberto A; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

2016-01-01

Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements.

A defence-related Olea europaea β-glucosidase hydrolyses and activates oleuropein into a potent protein cross-linking agent

PubMed Central

Koudounas, Konstantinos; Banilas, Georgios; Michaelidis, Christos; Demoliou, Catherine; Rigas, Stamatis; Hatzopoulos, Polydefkis

2015-01-01

Oleuropein, the major secoiridoid compound in olive, is involved in a sophisticated two-component defence system comprising a β-glucosidase enzyme that activates oleuropein into a toxic glutaraldehyde-like structure. Although oleuropein deglycosylation studies have been monitored extensively, an oleuropein β-glucosidase gene has not been characterized as yet. Here, we report the isolation of OeGLU cDNA from olive encoding a β-glucosidase belonging to the defence-related group of terpenoid-specific glucosidases. In planta recombinant protein expression assays showed that OeGLU deglycosylated and activated oleuropein into a strong protein cross-linker. Homology and docking modelling predicted that OeGLU has a characteristic (β/α)8 TIM barrel conformation and a typical construction of a pocket-shaped substrate recognition domain composed of conserved amino acids supporting the β-glucosidase activity and non-conserved residues associated with aglycon specificity. Transcriptional analysis in various olive organs revealed that the gene was developmentally regulated, with its transcript levels coinciding well with the spatiotemporal patterns of oleuropein degradation and aglycon accumulation in drupes. OeGLU upregulation in young organs reflects its prominent role in oleuropein-mediated defence system. High gene expression during drupe maturation implies an additional role in olive secondary metabolism, through the degradation of oleuropein and reutilization of hydrolysis products. PMID:25697790

Functional metagenomics reveals novel β-galactosidases not predictable from gene sequences.

PubMed

Cheng, Jiujun; Romantsov, Tatyana; Engel, Katja; Doxey, Andrew C; Rose, David R; Neufeld, Josh D; Charles, Trevor C

2017-01-01

The techniques of metagenomics have allowed researchers to access the genomic potential of uncultivated microbes, but there remain significant barriers to determination of gene function based on DNA sequence alone. Functional metagenomics, in which DNA is cloned and expressed in surrogate hosts, can overcome these barriers, and make important contributions to the discovery of novel enzymes. In this study, a soil metagenomic library carried in an IncP cosmid was used for functional complementation for β-galactosidase activity in both Sinorhizobium meliloti (α-Proteobacteria) and Escherichia coli (γ-Proteobacteria) backgrounds. One β-galactosidase, encoded by six overlapping clones that were selected in both hosts, was identified as a member of glycoside hydrolase family 2. We could not identify ORFs obviously encoding possible β-galactosidases in 19 other sequenced clones that were only able to complement S. meliloti. Based on low sequence identity to other known glycoside hydrolases, yet not β-galactosidases, three of these ORFs were examined further. Biochemical analysis confirmed that all three encoded β-galactosidase activity. Lac36W_ORF11 and Lac161_ORF7 had conserved domains, but lacked similarities to known glycoside hydrolases. Lac161_ORF10 had neither conserved domains nor similarity to known glycoside hydrolases. Bioinformatic and structural modeling implied that Lac161_ORF10 protein represented a novel enzyme family with a five-bladed propeller glycoside hydrolase domain. By discovering founding members of three novel β-galactosidase families, we have reinforced the value of functional metagenomics for isolating novel genes that could not have been predicted from DNA sequence analysis alone.

Expression of lysozyme in the life history of the house fly (Musca domestica l.).

PubMed

Nayduch, Dana; Joyner, Chester

2013-07-01

From egg to adult, all life history stages of house flies associate with septic environments teeming with bacteria. House fly lysozyme was first identified in the larval midgut, where it is used for digestion of microbe-rich meals because of its broad-spectrum activity against gram-positive and gram-negative bacteria as well as fungi. This study aimed to determine the temporal expression of lysozyme in the life history of house flies (from egg through adults) on both the mRNA and protein level, and to determine the tissue-specific expression of lysozyme in adult flies induced by feeding Staphylococcus aureus. From 30-min postoviposition through adulthood, all life history stages of the house fly express lysozyme on the mRNA level. In adult flies, lysozyme is expressed both locally in the alimentary canal and systemically in the fat body. Interestingly, we found that during the normal life history of flies, lysozyme protein was only detected in larval stages and older adults, likely because of ingestion of immune-stimulating levels of bacteria, not experienced during egg, pupa, and teneral adult stages. Constitutive expression on the mRNA level implies that this effector is a primary defense molecule in all stages of the house fly life history, and that a mechanism for posttranscriptional control of mature lysozyme enzyme expression may be present. Lysozyme active enzyme primarily serves both a digestive and defensive function in larval and adult flies, and may be a key player in the ability of Musca domestica L. to thrive in microbe-rich environments.

Structural and Biochemical Characterization of a Novel Aminopeptidase from Human Intestine

DOE PAGES

Tykvart, Jan; Bařinka, Cyril; Svoboda, Michal; ...

2015-03-09

N-acetylated α-linked acidic dipeptidase-like protein (NAALADase L), encoded by the NAALADL1 gene, is a close homolog of glutamate carboxypeptidase II, a metallopeptidase that has been intensively studied as a target for imaging and therapy of solid malignancies and neuropathologies. However, neither the physiological functions nor structural features of NAALADase L are known at present. In this paper, we report a thorough characterization of the protein product of the human NAALADL1 gene, including heterologous overexpression and purification, structural and biochemical characterization, and analysis of its expression profile. By solving the NAALADase L x-ray structure, we provide the first experimental evidence thatmore » it is a zinc-dependent metallopeptidase with a catalytic mechanism similar to that of glutamate carboxypeptidase II yet distinct substrate specificity. A proteome-based assay revealed that the NAALADL1 gene product possesses previously unrecognized aminopeptidase activity but no carboxy- or endopeptidase activity. These findings were corroborated by site-directed mutagenesis and identification of bestatin as a potent inhibitor of the enzyme. Analysis of NAALADL1 gene expression at both the mRNA and protein levels revealed the small intestine as the major site of protein expression and points toward extensive alternative splicing of the NAALADL1 gene transcript. Taken together, our data imply that the NAALADL1 gene product's primary physiological function is associated with the final stages of protein/peptide digestion and absorption in the human digestive system. Finally, based on these results, we suggest a new name for this enzyme: human ileal aminopeptidase (HILAP).« less

Iron limitation of microbial phosphorus acquisition in the tropical North Atlantic

NASA Astrophysics Data System (ADS)

Browning, Thomas; Achterberg, Eric; Yong, Jaw Chuen; Rapp, Insa; Utermann, Caroline; Engel, Anja; Moore, Mark

2017-04-01

Growth-limitation of marine phytoplankton by fixed nitrogen (N) has been demonstrated for most of the low-latitude oceans; however, in the (sub)tropical North Atlantic enhanced N2 fixation leads to secondary/(co-)limitation by phosphorus (P). The dissolved organic P pool is rarely fully depleted in the modern ocean and potentially represents a substantial additional P source. Microbes can use a variety of alkaline phosphatase enzymes to access P from a major fraction of this pool. In contrast to the relatively well studied PhoA family of alkaline phosphatases that utilize zinc (Zn) as a cofactor, the recent discovery of iron (Fe) as a cofactor in the more widespread PhoX[1] and PhoD[2] enzymes imply potential for a complex, biochemically-dependant interplay between oceanic Zn, Fe and P cycles. Here we demonstrate enhanced natural community alkaline phosphatase activity (APA) following Fe amendment within the low Zn and moderately low Fe western tropical North Atlantic. In contrast, beneath the Saharan dust plume in the Eastern Atlantic no APA response to trace metal addition was observed. This is the first demonstration of intermittent Fe limitation of microbial P acquisition, providing an additional facet in the argument for Fe control of the coupling between oceanic N and P cycles. 1. Yong, S. C. et al. A complex iron-calcium cofactor catalyzing phosphotransfer chemistry. Science 345, 1170-3 (2014). 2. Rodriguez, F. et al. Crystal structure of the Bacillus subtilis phosphodiesterase PhoD reveals an iron and calcium-containing active site. J. Biol. Chem. 289, 30889-30899 (2014).

A biomimetic colorimetric logic gate system based on multi-functional peptide-mediated gold nanoparticle assembly.

PubMed

Li, Yong; Li, Wang; He, Kai-Yu; Li, Pei; Huang, Yan; Nie, Zhou; Yao, Shou-Zhuo

2016-04-28

In natural biological systems, proteins exploit various functional peptide motifs to exert target response and activity switch, providing a functional and logic basis for complex cellular activities. Building biomimetic peptide-based bio-logic systems is highly intriguing but remains relatively unexplored due to limited logic recognition elements and complex signal outputs. In this proof-of-principle work, we attempted to address these problems by utilizing multi-functional peptide probes and the peptide-mediated nanoparticle assembly system. Here, the rationally designed peptide probes function as the dual-target responsive element specifically responsive to metal ions and enzymes as well as the mediator regulating the assembly of gold nanoparticles (AuNPs). Taking advantage of Zn2+ ions and chymotrypsin as the model inputs of metal ions and enzymes, respectively, we constructed the peptide logic system computed by the multi-functional peptide probes and outputted by the readable colour change of AuNPs. In this way, the representative binary basic logic gates (AND, OR, INHIBIT, NAND, IMPLICATION) have been achieved by delicately coding the peptide sequence, demonstrating the versatility of our logic system. Additionally, we demonstrated that the three-input combinational logic gate (INHIBIT-OR) could also be successfully integrated and applied as a multi-tasking biosensor for colorimetric detection of dual targets. This nanoparticle-based peptide logic system presents a valid strategy to illustrate peptide information processing and provides a practical platform for executing peptide computing or peptide-related multiplexing sensing, implying that the controllable nanomaterial assembly is a promising and potent methodology for the advancement of biomimetic bio-logic computation.

Anaerobic metabolism and thermal tolerance: The importance of opine pathways on survival of a gastropod after cardiac dysfunction.

PubMed

Han, Guodong; Zhang, Shu; Dong, Yunwei

2017-09-01

Organisms on rocky shores are frequently exposed to high temperatures, which cause impairment of cardiac function and retard cellular oxygen delivery. However, some gastropods can survive at several degrees Celsius higher than their Arrhenius break temperature of cardiac function (ABT), indicating the importance of anaerobic metabolism for their thermal tolerance. We measured the global molecular responses to heat stress in limpet Cellana toreuma using 454 GS-FLX to investigate the variations of genes involved in anaerobic metabolism at high temperatures. Next, the gene expression levels of 4 anaerobic enzymes and activity of alanopine dehydrogenase (AlDH), which is involved in opine pathway, were measured in response to elevated temperature. A total of 19 heat shock proteins (HSPs) were determined using real-time PCR at different temperatures. At high temperatures, the extensive upregulation of HSP genes was an effective but energetically expensive form of protection to prevent thermal damage. The upregulation of hypoxia-inducible factor 1 alpha mRNA indicated the condition of cellular hypoxia and the high gene expression and enzyme activity of AlDH suggested that opine pathway was the main anaerobic pathway. These results implied that anaerobic metabolism was enhanced to provide energy in the face of thermal stress. Our findings highlight the ecological significance of the anaerobic metabolism of gastropods to thermal adaptation. For predicting the ecological impact of global warming on the distribution of gastropods, the role of anaerobic pathways should be evaluated. © 2016 International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.

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.

Generation of thromboxane A2 and aorta-contracting activity from platelets stimulated with modified C-reactive protein.

PubMed Central

Simpson, R M; Prancan, A; Izzi, J M; Fiedel, B A

1982-01-01

The classical acute phase reactant, C-reactive protein (CRP), appears in markedly elevated concentration in the sera of individuals undergoing reactions of acute inflammation and tissue degradation. We previously demonstrated that like IgG, appropriately purified CRP could be thermally modified (H-CRP) such that it enhanced platelet activation in plasma and initiated platelet responses in isolated systems. We now report that this direct platelet activation by modified CRP results in the secretion of both platelet dense body and alpha-granule constituents, and is sensitive to non-steroidal anti-inflammatory drugs as well as the adenosine diphosphate (ADP)-removing enzyme system creatine phosphate/creatine phosphokinase. Thin-layer chromatographic (TLC) analysis of prostanoate endproducts following platelet activation with H-CRP revealed the formation of thromboxane B2 (the hydrated endproduct of thromboxane A2), an important endogenous platelet activator and contractor of vascular tissue; bioassay on rabbit aorta strips of supernatants obtained from platelets undergoing challenge with H-CRP supported the TLC analysis. Complexes formed between CRP and one major ligand, the polycation, were found to share certain platelet activating properties with H-CRP, as does latex-aggregated CRP. These data imply a potential agonist role for this acute phase reactant in platelet physiology and suggest that the interaction of modified forms of CRP with the platelet at sites of vascular damage could have pathological significance. PMID:7118160

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.

Organohalogens and their hydroxylated metabolites in the blood of pigs from an open waste dumping site in south India: association with hepatic cytochrome P450.

PubMed

Mizukawa, Hazuki; Nomiyama, Kei; Kunisue, Tatsuya; Watanabe, Michio X; Subramanian, Annamalai; Iwata, Hisato; Ishizuka, Mayumi; Tanabe, Shinsuke

2015-04-01

The concentrations of polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and their hydroxylated metabolites (OH-PCBs and OH-PBDEs) were measured in the blood of Eurasian wild pigs (Sus scrofa) from a municipal waste open dumping site (DS) and a reference site (RS) in South India. We showed that contamination with OH-PCBs was higher in female pigs from the DS than in all other adult pigs. The highest OH-PCB concentrations were found in piglets from the DS. Moreover, the hepatic expression levels of CYP1A and CYP2B were higher in piglets than in their dam, implying metabolism of PCBs by cytochrome P450 (CYP) enzymes. The OH-PCB congener profiles differed according to sex and collection sites, possibly because of variations in the expression levels of phase I and phase II enzymes among individual pigs, differences in the exposure sources, and maternal transfer of parent PCBs. The hepatic CYP1A expression levels were positively correlated with the blood concentrations of 4OH-CB107, 4OH-CB162, and 4OH-CB187, implying CYP1A-dependent formation of these OH-PCBs in the pig liver. We found no significant correlations between the blood concentrations of OH-PCBs and thyroid hormones (THs); however, the thyroxin (T4) levels were lower in pigs from the DS than in pigs from the RS. Our limited dataset suggest that induced CYP enzymes accelerate the metabolism of xenobiotics and endogenous molecules in pigs. Thus, besides parental compounds, the risk of hydroxylated metabolites entering wildlife and humans living in and around municipal open waste dumping sites should be considered. Copyright © 2015 Elsevier Inc. All rights reserved.

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

Metabolite concentrations, fluxes and free energies imply efficient enzyme usage

DOE PAGES

Park, Junyoung O.; Rubin, Sara A.; Xu, Yi -Fan; ...

2016-05-02

In metabolism, available free energy is limited and must be divided across pathway steps to maintain a negative Δ G throughout. For each reaction, Δ G is log proportional both to a concentration ratio (reaction quotient to equilibrium constant) and to a flux ratio (backward to forward flux). In this paper, we use isotope labeling to measure absolute metabolite concentrations and fluxes in Escherichia coli, yeast and a mammalian cell line. We then integrate this information to obtain a unified set of concentrations and Δ G for each organism. In glycolysis, we find that free energy is partitioned so asmore » to mitigate unproductive backward fluxes associated with Δ G near zero. Across metabolism, we observe that absolute metabolite concentrations and Δ G are substantially conserved and that most substrate (but not inhibitor) concentrations exceed the associated enzyme binding site dissociation constant ( K m or K i). Finally, the observed conservation of metabolite concentrations is consistent with an evolutionary drive to utilize enzymes efficiently given thermodynamic and osmotic constraints.« less

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

[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.

Immunomodulatory potencies of isolated compounds from Crataegus azarolus through their antioxidant activities.

PubMed

Mustapha, Nadia; Mokdad-Bzéouich, Imèn; Sassi, Aicha; Abed, Besma; Ghedira, Kamel; Hennebelle, Thierry; Chekir-Ghedira, Leila

2016-06-01

The search of natural immunomodulatory agents has become an area of great interest in order to reduce damage to the human body. In this study, the immunomodulatory potential of Crataegus azarolus and its isolated hyperoside on mouse lymphocytes and macrophages in vitro was assessed. The effect of C. azarolus natural compounds on splenocytes proliferation, natural killer (NK) and cytotoxic T lymphocytes (CTL) activities, and on macrophage-mediated cytotoxicity were assessed by MTT test. Phagocytic activity and inhibition of nitric oxide (NO) release by macrophages were also evaluated. The antioxidant capacity of these products was evaluated by determining their cellular antioxidant activity (CAA) in splenocytes and macrophages. Depending on the concentrations, both ethyl acetate (EA) extract and hyperoside (Hyp) from C. azarolus affect macrophage functions by modulating their lysosomal enzyme activity and nitric oxide release. Whereas, the above-mentioned products significantly promote LPS and lectin-stimulated splenocyte proliferation, implying a potential activation of lymphocytes B and T enhancing humoral and cellular immune responses. Moreover, EA extract and Hyp could enhance the activity of NK and T lymphocytes cells, as well as the macrophages-mediated cytotoxicity against B16F10 cells. The anti-inflammatory activity was concomitant with the cellular antioxidant effect of the tested compounds against macrophages and splenocytes. Collectively, C. azarolus and its isolated hyperoside exhibited an immunomodulatory effect through their antioxidant activity. These findings suggest that C. azarolus should be explored as a novel potential immunomodulatory agent for the treatment of inflammatory diseases.

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.

Role of hydrogen bonds in the reaction mechanism of chalcone isomerase.

PubMed

Jez, Joseph M; Bowman, Marianne E; Noel, Joseph P

2002-04-23

In flavonoid, isoflavonoid, and anthocyanin biosynthesis, chalcone isomerase (CHI) catalyzes the intramolecular cyclization of chalcones into (S)-flavanones with a second-order rate constant that approaches the diffusion-controlled limit. The three-dimensional structures of alfalfa CHI complexed with different flavanones indicate that two sets of hydrogen bonds may possess critical roles in catalysis. The first set of interactions includes two conserved amino acids (Thr48 and Tyr106) that mediate a hydrogen bond network with two active site water molecules. The second set of hydrogen bonds occurs between the flavanone 7-hydroxyl group and two active site residues (Asn113 and Thr190). Comparison of the steady-state kinetic parameters of wild-type and mutant CHIs demonstrates that efficient cyclization of various chalcones into their respective flavanones requires both sets of contacts. For example, the T48A, T48S, Y106F, N113A, and T190A mutants exhibit 1550-, 3-, 30-, 7-, and 6-fold reductions in k(cat) and 2-3-fold changes in K(m) with 4,2',4'-trihydroxychalcone as a substrate. Kinetic comparisons of the pH-dependence of the reactions catalyzed by wild-type and mutant enzymes indicate that the active site hydrogen bonds contributed by these four residues do not significantly alter the pK(a) of the intramolecular cyclization reaction. Determinations of solvent kinetic isotope and solvent viscosity effects for wild-type and mutant enzymes reveal a change from a diffusion-controlled reaction to one limited by chemistry in the T48A and Y106F mutants. The X-ray crystal structures of the T48A and Y106F mutants support the assertion that the observed kinetic effects result from the loss of key hydrogen bonds at the CHI active site. Our results are consistent with a reaction mechanism for CHI in which Thr48 polarizes the ketone of the substrate and Tyr106 stabilizes a key catalytic water molecule. Hydrogen bonds contributed by Asn113 and Thr190 provide additional stabilization in the transition state. Conservation of these residues in CHIs from other plant species implies a common reaction mechanism for enzyme-catalyzed flavanone formation in all plants.

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.

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.

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.

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.

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.

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.

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.

Expression level, cellular compartment and metabolic network position all influence the average selective constraint on mammalian enzymes

PubMed Central

2011-01-01

Background A gene's position in regulatory, protein interaction or metabolic networks can be predictive of the strength of purifying selection acting on it, but these relationships are neither universal nor invariably strong. Following work in bacteria, fungi and invertebrate animals, we explore the relationship between selective constraint and metabolic function in mammals. Results We measure the association between selective constraint, estimated by the ratio of nonsynonymous (Ka) to synonymous (Ks) substitutions, and several, primarily metabolic, measures of gene function. We find significant differences between the selective constraints acting on enzyme-coding genes from different cellular compartments, with the nucleus showing higher constraint than genes from either the cytoplasm or the mitochondria. Among metabolic genes, the centrality of an enzyme in the metabolic network is significantly correlated with Ka/Ks. In contrast to yeasts, gene expression magnitude does not appear to be the primary predictor of selective constraint in these organisms. Conclusions Our results imply that the relationship between selective constraint and enzyme centrality is complex: the strength of selective constraint acting on mammalian genes is quite variable and does not appear to exclusively follow patterns seen in other organisms. PMID:21470417

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.

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.

Anaerobic Cysteine Degradation and Potential Metabolic Coordination in Salmonella enterica and Escherichia coli

PubMed Central

Loddeke, Melissa; Schneider, Barbara; Oguri, Tamiko; Mehta, Iti; Xuan, Zhenyu

2017-01-01

ABSTRACT Salmonella enterica has two CyuR-activated enzymes that degrade cysteine, i.e., the aerobic CdsH and an unidentified anaerobic enzyme; Escherichia coli has only the latter. To identify the anaerobic enzyme, transcript profiling was performed for E. coli without cyuR and with overexpressed cyuR. Thirty-seven genes showed at least 5-fold changes in expression, and the cyuPA (formerly yhaOM) operon showed the greatest difference. Homology suggested that CyuP and CyuA represent a cysteine transporter and an iron-sulfur-containing cysteine desulfidase, respectively. E. coli and S. enterica ΔcyuA mutants grown with cysteine generated substantially less sulfide and had lower growth yields. Oxygen affected the CyuR-dependent genes reciprocally; cyuP-lacZ expression was greater anaerobically, whereas cdsH-lacZ expression was greater aerobically. In E. coli and S. enterica, anaerobic cyuP expression required cyuR and cysteine and was induced by l-cysteine, d-cysteine, and a few sulfur-containing compounds. Loss of either CyuA or RidA, both of which contribute to cysteine degradation to pyruvate, increased cyuP-lacZ expression, which suggests that CyuA modulates intracellular cysteine concentrations. Phylogenetic analysis showed that CyuA homologs are present in obligate and facultative anaerobes, confirming an anaerobic function, and in archaeal methanogens and bacterial acetogens, suggesting an ancient origin. Our results show that CyuA is the major anaerobic cysteine-catabolizing enzyme in both E. coli and S. enterica, and it is proposed that anaerobic cysteine catabolism can contribute to coordination of sulfur assimilation and amino acid synthesis. IMPORTANCE Sulfur-containing compounds such as cysteine and sulfide are essential and reactive metabolites. Exogenous sulfur-containing compounds can alter the thiol landscape and intracellular redox reactions and are known to affect several cellular processes, including swarming motility, antibiotic sensitivity, and biofilm formation. Cysteine inhibits several enzymes of amino acid synthesis; therefore, increasing cysteine concentrations could increase the levels of the inhibited enzymes. This inhibition implies that control of intracellular cysteine levels, which is the immediate product of sulfide assimilation, can affect several pathways and coordinate metabolism. For these and other reasons, cysteine and sulfide concentrations must be controlled, and this work shows that cysteine catabolism contributes to this control. PMID:28607157

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

Evidence That the [beta] Subunit of Chlamydia trachomatis Ribonucleotide Reductase Is Active with the Manganese Ion of Its Manganese(IV)/Iron(III) Cofactor in Site 1

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

Dassama, Laura M.K.; Boal, Amie K.; Krebs, Carsten

2014-10-02

The reaction of a class I ribonucleotide reductase (RNR) begins when a cofactor in the {beta} subunit oxidizes a cysteine residue {approx}35 {angstrom} away in the {alpha} subunit, generating a thiyl radical. In the class Ic enzyme from Chlamydia trachomatis (Ct), the cysteine oxidant is the Mn{sup IV} ion of a Mn{sup IV}/Fe{sup III} cluster, which assembles in a reaction between O{sub 2} and the Mn{sup II}/Fe{sup II} complex of {beta}. The heterodinuclear nature of the cofactor raises the question of which site, 1 or 2, contains the Mn{sup IV} ion. Because site 1 is closer to the conserved locationmore » of the cysteine-oxidizing tyrosyl radical of class Ia and Ib RNRs, we suggested that the Mn{sup IV} ion most likely resides in this site (i.e., {sup 1}Mn{sup IV}/{sup 2}Fe{sup III}), but a subsequent computational study favored its occupation of site 2 ({sup 1}Fe{sup III}/{sup 2}Mn{sup IV}). In this work, we have sought to resolve the location of the Mn{sup IV} ion in Ct RNR-{beta} by correlating X-ray crystallographic anomalous scattering intensities with catalytic activity for samples of the protein reconstituted in vitro by two different procedures. In samples containing primarily Mn{sup IV}/Fe{sup III} clusters, Mn preferentially occupies site 1, but some anomalous scattering from site 2 is observed, implying that both {sup 1}Mn{sup II}/{sup 2}Fe{sup II} and {sup 1}Fe{sup II}/{sup 2}Mn{sup II} complexes are competent to react with O{sub 2} to produce the corresponding oxidized states. However, with diminished Mn{sup II} loading in the reconstitution, there is no evidence for Mn occupancy of site 2, and the greater activity of these 'low-Mn' samples on a per-Mn basis implies that the {sup 1}Mn{sup IV}/{sup 2}Fe{sup III}-{beta} is at least the more active of the two oxidized forms and may be the only active form.« 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

Differential physiological effects of neonicotinoid insecticides on honey bees: A comparison between Apis mellifera and Apis cerana.

PubMed

Li, Zhiguo; Li, Meng; He, Jingfang; Zhao, Xiaomeng; Chaimanee, Veeranan; Huang, Wei-Fone; Nie, Hongyi; Zhao, Yazhou; Su, Songkun

2017-08-01

Acute toxicities (LD50s) of imidacloprid and clothianidin to Apis mellifera and A. cerana were investigated. Changing patterns of immune-related gene expressions and the activities of four enzymes between the two bee species were compared and analyzed after exposure to sublethal doses of insecticides. Results indicated that A. cerana was more sensitive to imidacloprid and clothianidin than A. mellifera. The acute oral LD50 values of imidacloprid and clothianidin for A. mellifera were 8.6 and 2.0ng/bee, respectively, whereas the corresponding values for A. cerana were 2.7 and 0.5ng/bee. The two bee species possessed distinct abilities to mount innate immune response against neonicotinoids. After 48h of imidacloprid treatment, carboxylesterase (CCE), prophenol oxidase (PPO), and acetylcholinesterase (AChE) activities were significantly downregulated in A. mellifera but were upregulated in A. cerana. Glutathione-S-transferase (GST) activity was significantly elevated in A. mellifera at 48h after exposure to imidacloprid, but no significant change was observed in A. cerana. AChE was downregulated in both bee species at three different time points during clothianidin exposure, and GST activities were upregulated in both species exposed to clothianidin. Different patterns of immune-related gene expression and enzymatic activities implied distinct detoxification and immune responses of A. cerana and A. mellifera to imidacloprid and clothianidin. Copyright © 2017 Elsevier Inc. All rights reserved.

Activation of cyclic electron flow by hydrogen peroxide in vivo

DOE PAGES

Strand, Deserah D.; Livingston, Aaron K.; Satoh-Cruz, Mio; ...

2015-04-13

Cyclic electron flow (CEF) around photosystem I is thought to balance the ATP/NADPH energy budget of photosynthesis, requiring that its rate be finely regulated. The mechanisms of this regulation are not well understood. We observed that mutants that exhibited constitutively high rates of CEF also showed elevated production of H 2O 2. We thus tested the hypothesis that CEF can be activated by H 2O 2 in vivo. CEF was strongly increased by H 2O 2 both by infiltration or in situ production by chloroplast-localized glycolate oxidase, implying that H 2O 2 can activate CEF either directly by redox modulationmore » of key enzymes, or indirectly by affecting other photosynthetic processes. CEF appeared with a half time of about 20 min after exposure to H 2O 2, suggesting activation of previously expressed CEF-related machinery. H 2O 2-dependent CEF was not sensitive to antimycin A or loss of PGR5, indicating that increased CEF probably does not involve the PGR5-PGRL1 associated pathway. In contrast, the rise in CEF was not observed in a mutant deficient in the chloroplast NADPH:PQ reductase (NDH), supporting the involvement of this complex in CEF activated by H 2O 2. In conclusion, we propose that H 2O 2 is a missing link between environmental stress, metabolism, and redox regulation of CEF in higher plants.« less

Improvement of catalytic performance of lignin peroxidase for the enhanced degradation of lignocellulose biomass based on the imbedded electron-relay in long-range electron transfer route.

PubMed

Pham, Le Thanh Mai; Kim, Su Jin; Kim, Yong Hwan

2016-01-01

Although lignin peroxidase is claimed as a key enzyme in enzyme-catalyzed lignin degradation, in vitro enzymatic degradation of lignin was not easily observed in lab-scale experiments. It implies that other factors may hinder the enzymatic degradation of lignin. Irreversible interaction between phenolic compound and lignin peroxidase was hypothesized when active enzyme could not be recovered after the reaction with degradation product (guaiacol) of lignin phenolic dimer. In the study of lignin peroxidase isozyme H8 from white-rot fungi Phanerochaete chrysosporium (LiPH8), W251 site was revealed to make the covalent coupling with one moiety of monolignolic radical (guaiacol radical) by LC-MS/MS analysis. Hypothetical electron-relay containing W251 residue was newly suggested based on the observation of repressed radical coupling and remarkably lower electron transfer rate for W215A mutant. Furthermore, the retardation of the suicidal radical coupling between the W251 residue and the monolignolic radical was attempted by supplementing the acidic microenvironment around the W251 residue to engineer radical-robust LiPH8. Among many mutants, mutant A242D showed exceptional catalytic performances by yielding 21.1- and 4.9-fold higher increases of k cat and k cat /K M values, respectively, in the oxidation of non-phenolic model lignin dimer. A mechanism-based suicide inhibition of LiPH8 by phenolic compounds was firstly revealed and investigated in this work. Radical-robust LiPH8 was also successfully engineered by manipulating the transient radical state of radical-susceptible electron-relay. Radical-robust LiPH8 will play an essential role in degradation of lignin, which will be consequently linked with improved production of sugars from lignocellulose biomass.

Mycobacterium tuberculosis thymidylate synthase gene thyX is essential and potentially bifunctional, while thyA deletion confers resistance to p-aminosalicylic acid

PubMed Central

Houghton, Joanna; Davis, Elaine O.

2012-01-01

Thymidylate synthase (TS) enzymes catalyse the biosynthesis of deoxythymidine monophosphate (dTMP or thymidylate), and so are important for DNA replication and repair. Two different types of TS proteins have been described (ThyA and ThyX), which have different enzymic mechanisms and unrelated structures. Mycobacteria are unusual as they encode both thyA and thyX, and the biological significance of this is not yet understood. Mycobacterium tuberculosis ThyX is thought to be essential and a potential drug target. We therefore analysed M. tuberculosis thyA and thyX expression levels, their essentiality and roles in pathogenesis. We show that both thyA and thyX are expressed in vitro, and that this expression significantly increased within murine macrophages. Under all conditions tested, thyA expression exceeded that of thyX. Mutational studies show that M. tuberculosis thyX is essential, confirming that the enzyme is a plausible drug target. The requirement for M. tuberculosis thyX in the presence of thyA implies that the essential function of ThyX is something other than dTMP synthase. We successfully deleted thyA from the M. tuberculosis genome, and this deletion conferred an in vitro growth defect that was not observed in vivo. Presumably ThyX performs TS activity within M. tuberculosis ΔthyA at a sufficient rate in vivo for normal growth, but the rate in vitro is less than optimal. We also demonstrate that thyA deletion confers M. tuberculosis p-aminosalicylic acid resistance, and show by complementation studies that ThyA T202A and V261G appear to be functional and non-functional, respectively. PMID:22034487

Mycobacterium tuberculosis thymidylate synthase gene thyX is essential and potentially bifunctional, while thyA deletion confers resistance to p-aminosalicylic acid.

PubMed

Fivian-Hughes, Amanda S; Houghton, Joanna; Davis, Elaine O

2012-02-01

Thymidylate synthase (TS) enzymes catalyse the biosynthesis of deoxythymidine monophosphate (dTMP or thymidylate), and so are important for DNA replication and repair. Two different types of TS proteins have been described (ThyA and ThyX), which have different enzymic mechanisms and unrelated structures. Mycobacteria are unusual as they encode both thyA and thyX, and the biological significance of this is not yet understood. Mycobacterium tuberculosis ThyX is thought to be essential and a potential drug target. We therefore analysed M. tuberculosis thyA and thyX expression levels, their essentiality and roles in pathogenesis. We show that both thyA and thyX are expressed in vitro, and that this expression significantly increased within murine macrophages. Under all conditions tested, thyA expression exceeded that of thyX. Mutational studies show that M. tuberculosis thyX is essential, confirming that the enzyme is a plausible drug target. The requirement for M. tuberculosis thyX in the presence of thyA implies that the essential function of ThyX is something other than dTM synthesis [corrected].We successfully deleted thyA from the M. tuberculosis genome, and this deletion conferred an in vitro growth defect that was not observed in vivo. Presumably ThyX performs TS activity within M. tuberculosis ΔthyA at a sufficient rate in vivo for normal growth, but the rate in vitro is less than optimal. We also demonstrate that thyA deletion confers M. tuberculosis p-aminosalicylic acid resistance, and show by complementation studies that ThyA T202A and V261G appear to be functional and non-functional, respectively.

Concomitant ingestion of lactic acid bacteria and black tea synergistically enhances flavonoid bioavailability and attenuates d-galactose-induced oxidative stress in mice via modulating glutathione antioxidant system.

PubMed

Zhao, Danyue; Shah, Nagendra P

2016-12-01

Black tea (BT) has been positively linked to improved redox status, while its efficacy is limited due to the low bioavailability of BT flavonoids. In addition to the direct antioxidant activity, flavonoids regulate redox balance via inducing endogenous antioxidants, particularly glutathione (GSH) and GSH-dependent antioxidant enzymes. This work first examined the effect of lactic acid bacteria (LAB) and BT alone or in combination on flavonoid bioavailability and metabolism; next, the effect of LAB-fermented BT diet in attenuating oxidative stress in mice and the underlying mechanisms were studied. Phenolic profiles of plasma, urine and feces from healthy mice consuming plain yogurt, BT milk (BTM) or BT yogurt (BTY) were acquired using LC-MS/MS. Plasma antioxidant capacity, lipid peroxidation level, content of nonprotein thiols and expression of GSH-related antioxidant enzymes and Nrf2 were examined in d-galactose-treated mice. Total flavonoid content in plasma following a single dose of BTY attained 0.657 μmol/l, increased by 50% compared with the BTM group. Increased excretion of phenolic metabolite and hippuric acid in urine and feces indicated enhanced metabolism of flavonoids in BTY-fed mice. In the second study, 8-week concomitant LAB-BT treatment of oxidatively stressed mice effectively restored plasma antioxidant capacity and GSH levels, and mitigated lipid peroxidation, which were associated with significant induction of GSH-dependent antioxidant enzymes and nuclear accumulation of Nrf2. Our results demonstrated the effect of LAB fermentation in enhancing BT flavonoid bioavailability in vivo. The synergistic antioxidant efficacy of LAB-BT diet implied its therapeutic potential in enhancing antioxidant defenses and protecting organisms from oxidative damage. Copyright © 2016. Published by Elsevier Inc.

Structural and Functional Adaptation of Vancomycin Resistance VanT Serine Racemases

PubMed Central

Meziane-Cherif, Djalal; Stogios, Peter J.; Evdokimova, Elena; Egorova, Olga

2015-01-01

ABSTRACT Vancomycin resistance in Gram-positive bacteria results from the replacement of the d-alanyl–d-alanine target of peptidoglycan precursors with d-alanyl–d-lactate or d-alanyl–d-serine (d-Ala-d-Ser), to which vancomycin has low binding affinity. VanT is one of the proteins required for the production of d-Ala-d-Ser-terminating precursors by converting l-Ser to d-Ser. VanT is composed of two domains, an N-terminal membrane-bound domain, likely involved in l-Ser uptake, and a C-terminal cytoplasmic catalytic domain which is related to bacterial alanine racemases. To gain insight into the molecular function of VanT, the crystal structure of the catalytic domain of VanTG from VanG-type resistant Enterococcus faecalis BM4518 was determined. The structure showed significant similarity to type III pyridoxal 5′-phosphate (PLP)-dependent alanine racemases, which are essential for peptidoglycan synthesis. Comparative structural analysis between VanTG and alanine racemases as well as site-directed mutagenesis identified three specific active site positions centered around Asn696 which are responsible for the l-amino acid specificity. This analysis also suggested that VanT racemases evolved from regular alanine racemases by acquiring additional selectivity toward serine while preserving that for alanine. The 4-fold-lower relative catalytic efficiency of VanTG against l-Ser versus l-Ala implied that this enzyme relies on its membrane-bound domain for l-Ser transport to increase the overall rate of d-Ser production. These findings illustrate how vancomycin pressure selected for molecular adaptation of a housekeeping enzyme to a bifunctional enzyme to allow for peptidoglycan remodeling, a strategy increasingly observed in antibiotic-resistant bacteria. PMID:26265719

Structural and functional adaptation of vancomycin resistance VanT serine racemases

DOE PAGES

Meziane-Cherif, Djalal; Stogios, Peter J.; Evdokimova, Elena; ...

2015-08-11

Vancomycin resistance in Gram-positive bacteria results from the replacement of the D-alanyl–D-alanine target of peptidoglycan precursors with D-alanyl–D-lactate or D-alanyl–D-serine (D-Ala-D-Ser), to which vancomycin has low binding affinity. VanT is one of the proteins required for the production of D-Ala-D-Ser-terminating precursors by converting L-Ser to D-Ser. VanT is composed of two domains, an N-terminal membrane-bound domain, likely involved in L-Ser uptake, and a C-terminal cytoplasmic catalytic domain which is related to bacterial alanine racemases. To gain insight into the molecular function of VanT, the crystal structure of the catalytic domain of VanT G from VanG-type resistant Enterococcus faecalis BM4518more » was determined. The structure showed significant similarity to type III pyridoxal 5'-phosphate (PLP)-dependent alanine racemases, which are essential for peptidoglycan synthesis. Comparative structural analysis between VanT G and alanine racemases as well as site-directed mutagenesis identified three specific active site positions centered around Asn 696 which are responsible for theL-amino acid specificity. This analysis also suggested that VanT racemases evolved from regular alanine racemases by acquiring additional selectivity toward serine while preserving that for alanine. The 4-fold-lower relative catalytic efficiency of VanT G against L-Ser versus L-Ala implied that this enzyme relies on its membrane-bound domain for L-Ser transport to increase the overall rate of D-Ser production. These findings illustrate how vancomycin pressure selected for molecular adaptation of a housekeeping enzyme to a bifunctional enzyme to allow for peptidoglycan remodeling, a strategy increasingly observed in antibiotic-resistant bacteria.« less

Evidence of carbon fixation pathway in a bacterium from candidate phylum SBR1093 revealed with genomic analysis.

PubMed

Wang, Zhiping; Guo, Feng; Liu, Lili; Zhang, Tong

2014-01-01

Autotrophic CO2 fixation is the most important biotransformation process in the biosphere. Research focusing on the diversity and distribution of relevant autotrophs is significant to our comprehension of the biosphere. In this study, a draft genome of a bacterium from candidate phylum SBR1093 was reconstructed with the metagenome of an industrial activated sludge. Based on comparative genomics, this autotrophy may occur via a newly discovered carbon fixation path, the hydroxypropionate-hydroxybutyrate (HPHB) cycle, which was demonstrated in a previous work to be uniquely possessed by some genera from Archaea. This bacterium possesses all of the thirteen enzymes required for the HPHB cycle; these enzymes share 30∼50% identity with those in the autotrophic species of Archaea that undergo the HPHB cycle and 30∼80% identity with the corresponding enzymes of the mixotrophic species within Bradyrhizobiaceae. Thus, this bacterium might have an autotrophic growth mode in certain conditions. A phylogenetic analysis based on the 16S rRNA gene reveals that the phylotypes within candidate phylum SBR1093 are primarily clustered into 5 clades with a shallow branching pattern. This bacterium is clustered with phylotypes from organically contaminated environments, implying a demand for organics in heterotrophic metabolism. Considering the types of regulators, such as FnR, Fur, and ArsR, this bacterium might be a facultative aerobic mixotroph with potential multi-antibiotic and heavy metal resistances. This is the first report on Bacteria that may perform potential carbon fixation via the HPHB cycle, thus may expand our knowledge of the distribution and importance of the HPHB cycle in the biosphere.

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.

A novel mechanism of programmed cell death in bacteria by toxin-antitoxin systems corrupts peptidoglycan synthesis.

PubMed

Mutschler, Hannes; Gebhardt, Maike; Shoeman, Robert L; Meinhart, Anton

2011-03-01

Most genomes of bacteria contain toxin-antitoxin (TA) systems. These gene systems encode a toxic protein and its cognate antitoxin. Upon antitoxin degradation, the toxin induces cell stasis or death. TA systems have been linked with numerous functions, including growth modulation, genome maintenance, and stress response. Members of the epsilon/zeta TA family are found throughout the genomes of pathogenic bacteria and were shown not only to stabilize resistance plasmids but also to promote virulence. The broad distribution of epsilon/zeta systems implies that zeta toxins utilize a ubiquitous bacteriotoxic mechanism. However, whereas all other TA families known to date poison macromolecules involved in translation or replication, the target of zeta toxins remained inscrutable. We used in vivo techniques such as microscropy and permeability assays to show that pneumococcal zeta toxin PezT impairs cell wall synthesis and triggers autolysis in Escherichia coli. Subsequently, we demonstrated in vitro that zeta toxins in general phosphorylate the ubiquitous peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) and that this activity is counteracted by binding of antitoxin. After identification of the product we verified the kinase activity in vivo by analyzing metabolite extracts of cells poisoned by PezT using high pressure liquid chromatograpy (HPLC). We further show that phosphorylated UNAG inhibitis MurA, the enzyme catalyzing the initial step in bacterial peptidoglycan biosynthesis. Additionally, we provide what is to our knowledge the first crystal structure of a zeta toxin bound to its substrate. We show that zeta toxins are novel kinases that poison bacteria through global inhibition of peptidoglycan synthesis. This provides a fundamental understanding of how epsilon/zeta TA systems stabilize mobile genetic elements. Additionally, our results imply a mechanism that connects activity of zeta toxin PezT to virulence of pneumococcal infections. Finally, we discuss how phosphorylated UNAG likely poisons additional pathways of bacterial cell wall synthesis, making it an attractive lead compound for development of new antibiotics.

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.

An Alkaline Phosphatase Paradox in a Shelf Sea

NASA Astrophysics Data System (ADS)

Davis, C. E.; Mahaffey, C.

2016-02-01

Alkaline phosphatase (AP) is an ubiquitous hydrolytic phosphoenzyme that hydrolyses phosphomonester bonds. In the open ocean, the generally accepted paradigm is that when phosphate concentrations are sufficiently depleted (less than 50 nM), AP is produced by organisms to enable utilisation of dissolved organic phosphorus to meet the phosphorus demands of biological processes such as growth and carbon fixation. At higher phosphate concentrations (greater than 100 nM), AP is repressed implying that the excess product competes for active sites at enzyme surfaces. However, our ongoing work on phosphorus cycling in the Celtic Sea, a temperate shelf sea, has challenged this paradigm. We find elevated rates of AP below the thermocline where phosphate concentrations are greater than 700 nM, and a significant correlation between AP and total dissolved phosphorus. Using enzyme labelled fluorescence (ELF) and particle concentrate bioassays, we show that the AP is associated with large detrital and sinking particulate matter, suggesting that rather than AP being induced by the lack of phosphate, it plays an important role in organic matter cycling in this nitrogen limited environment. At the shelf edge, AP was found to be associated with diatoms, which have been found in culture studies to express AP under silica limitation. Our study highlights the need to consider the environmental conditions under which AP is induced or repressed and presents an opportunity to use AP as an indicator of organic phosphorus recycling in high phosphate environments.

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.

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.

The Biogeochemistry of Sulfur in Hydrothermal Systems

NASA Technical Reports Server (NTRS)

Schulte, Mitchell; Rogers, K. L.; DeVincenzi, Donald L. (Technical Monitor)

2001-01-01

The incorporation of sulfur into many biomolecules likely dates back to the development of the earliest metabolic strategies. Sulfur is common in enzymes and co-enzymes and is an indispensable structural component in many peptides and proteins. Early metabolism may have been heavily influenced by the abundance of sulfide minerals in hydrothermal systems. The incorporation of sulfur into many biomolecules likely dates back to the development of the earliest metabolic strategies. Sulfur is common in enzymes and co-enzymes and is an indispensable structural component in many peptides and proteins. Early metabolism may have been heavily influenced by the abundance of sulfide minerals in hydrothermal systems. Understanding how sulfur became prevalent in biochemical processes and many biomolecules requires knowledge of the reaction properties of sulfur-bearing compounds. We have previously estimated thermodynamic data for thiols, the simplest organic sulfur compounds, at elevated temperatures and pressures. If life began in hydrothermal environments, it is especially important to understand reactions at elevated temperatures among sulfur-bearing compounds and other organic molecules essential for the origin and persistence of life. Here we examine reactions that may have formed amino acids with thiols as reaction intermediates in hypothetical early Earth hydrothermal environments. (There are two amino acids, cysteine and methionine, that contain sulfur.) Our calculations suggest that significant amounts of some amino acids were produced in early Earth hydrothermal fluids, given reasonable concentrations H2, NH3, H2S and CO. For example, preliminary results indicate that glycine activities as high as 1 mmol can be reached in these systems at 100 C. Alanine formation from propanethiol is also a favorable reaction. On the other hand, the calculated equilibrium log activities of cysteine and serine from propanethiol are -21 and -19, respectively, at 100 C. These results indicate that while amino acid formation with thiols as intermediates is favored in some cases, other mechanisms may have been necessary to produce significant amounts of other amino acids. Coupled with our previous results for thiols, these studies imply that sulfur may have been easily incorporated into the organic geochemistry of early Earth hydrothermal systems, leading to its widespread use in biomolecules. Formation of more complex biomolecules in hydrothermal systems may have required sulfur-bearing organic compounds as reaction intermediates.

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

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.

An appraisal of the enzyme stability-activity trade-off.

PubMed

Miller, Scott R

2017-07-01

A longstanding idea in evolutionary physiology is that an enzyme cannot jointly optimize performance at both high and low temperatures due to a trade-off between stability and activity. Although a stability-activity trade-off has been observed for well-characterized examples, such a trade-off is not imposed by any physical chemical constraint. To better understand the pervasiveness of this trade-off, I investigated the stability-activity relationship for comparative biochemical studies of purified orthologous enzymes identified by a literature search. The nature of this relationship varied greatly among studies. Notably, studies of enzymes with low mean synonymous nucleotide sequence divergence were less likely to exhibit the predicted negative correlation between stability and activity. Similarly, a survey of directed evolution investigations of the stability-activity relationship indicated that these traits are often uncoupled among nearly identical yet phenotypically divergent enzymes. This suggests that the presumptive trade-off often reported for investigations of enzymes with high mean sequence divergence may in some cases instead be a consequence of the degeneration over time of enzyme function in unselected environments, rather than a direct effect of thermal adaptation. The results caution against the general assertion of a stability-activity trade-off during enzyme adaptation. © 2017 The Author(s). Evolution © 2017 The Society for the Study of Evolution.

Primordial-like enzymes from bacteria with reduced genomes.

PubMed

Ferla, Matteo P; Brewster, Jodi L; Hall, Kelsi R; Evans, Gary B; Patrick, Wayne M

2017-08-01

The first cells probably possessed rudimentary metabolic networks, built using a handful of multifunctional enzymes. The promiscuous activities of modern enzymes are often assumed to be relics of this primordial era; however, by definition these activities are no longer physiological. There are many fewer examples of enzymes using a single active site to catalyze multiple physiologically-relevant reactions. Previously, we characterized the promiscuous alanine racemase (ALR) activity of Escherichia coli cystathionine β-lyase (CBL). Now we have discovered that several bacteria with reduced genomes lack alr, but contain metC (encoding CBL). We characterized the CBL enzymes from three of these: Pelagibacter ubique, the Wolbachia endosymbiont of Drosophila melanogaster (wMel) and Thermotoga maritima. Each is a multifunctional CBL/ALR. However, we also show that CBL activity is no longer required in these bacteria. Instead, the wMel and T. maritima enzymes are physiologically bi-functional alanine/glutamate racemases. They are not highly active, but they are clearly sufficient. Given the abundance of the microorganisms using them, we suggest that much of the planet's biochemistry is carried out by enzymes that are quite different from the highly-active exemplars usually found in textbooks. Instead, primordial-like enzymes may be an essential part of the adaptive strategy associated with streamlining. © 2017 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.

Modeling nitrous oxide production and reduction in soil through explicit representation of denitrification enzyme kinetics.

PubMed

Zheng, Jianqiu; Doskey, Paul V

2015-02-17

An enzyme-explicit denitrification model with representations for pre- and de novo synthesized enzymes was developed to improve predictions of nitrous oxide (N2O) accumulations in soil and emissions from the surface. The metabolic model of denitrification is based on dual-substrate utilization and Monod growth kinetics. Enzyme synthesis/activation was incorporated into each sequential reduction step of denitrification to regulate dynamics of the denitrifier population and the active enzyme pool, which controlled the rate function. Parameterizations were developed from observations of the dynamics of N2O production and reduction in soil incubation experiments. The model successfully reproduced the dynamics of N2O and N2 accumulation in the incubations and revealed an important regulatory effect of denitrification enzyme kinetics on the accumulation of denitrification products. Pre-synthesized denitrification enzymes contributed 20, 13, 43, and 62% of N2O that accumulated in 48 h incubations of soil collected from depths of 0-5, 5-10, 10-15, and 15-25 cm, respectively. An enzyme activity function (E) was defined to estimate the relative concentration of active enzymes and variation in response to environmental conditions. The value of E allows for activities of pre-synthesized denitrification enzymes to be differentiated from de novo synthesized enzymes. Incorporating explicit representations of denitrification enzyme kinetics into biogeochemical models is a promising approach for accurately simulating dynamics of the production and reduction of N2O in soils.

Development of Activity-based Cost Functions for Cellulase, Invertase, and Other Enzymes

NASA Astrophysics Data System (ADS)

Stowers, Chris C.; Ferguson, Elizabeth M.; Tanner, Robert D.

As enzyme chemistry plays an increasingly important role in the chemical industry, cost analysis of these enzymes becomes a necessity. In this paper, we examine the aspects that affect the cost of enzymes based upon enzyme activity. The basis for this study stems from a previously developed objective function that quantifies the tradeoffs in enzyme purification via the foam fractionation process (Cherry et al., Braz J Chem Eng 17:233-238, 2000). A generalized cost function is developed from our results that could be used to aid in both industrial and lab scale chemical processing. The generalized cost function shows several nonobvious results that could lead to significant savings. Additionally, the parameters involved in the operation and scaling up of enzyme processing could be optimized to minimize costs. We show that there are typically three regimes in the enzyme cost analysis function: the low activity prelinear region, the moderate activity linear region, and high activity power-law region. The overall form of the cost analysis function appears to robustly fit the power law form.

Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

PubMed

Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

2014-12-16

Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

Activation of Phenylalanine Hydroxylase by Phenylalanine Does Not Require Binding in the Active Site

PubMed Central

2015-01-01

Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein’s regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The kcat/Kphe value is down 104 for the mutant enzyme, and the Km value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain. PMID:25453233

Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

DOEpatents

Jarvis, E.E.; Roessler, P.G.

1999-07-27

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. 8 figs.

Ectomycorrhizal Fungal Communities and Enzymatic Activities Vary across an Ecotone between a Forest and Field

PubMed Central

Rúa, Megan A.; Moore, Becky; Hergott, Nicole; Van, Lily; Jackson, Colin R.; Hoeksema, Jason D.

2015-01-01

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. PMID:29376908

Redox Switch for the Inhibited State of Yeast Glycogen Synthase Mimics Regulation by Phosphorylation

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

Mahalingan, Krishna K.; Baskaran, Sulochanadevi; DePaoli-Roach, Anna A.

Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS is negatively regulated by covalent phosphorylation and allosterically activated by glucose-6-phosphate (G-6-P). To gain structural insights into the inhibited state of the enzyme, we solved the crystal structure of yGsy2-R589A/R592A to a resolution of 3.3 Å. The double mutant has an activity ratio similar to the phosphorylated enzyme and also retains the ability to be activated by G-6-P. When compared to the 2.88 Å structure of the wild-type G-6-P activated enzyme, the crystal structure of the low-activity mutant showed that the N-terminal domain of themore » inhibited state is tightly held against the dimer-related interface thereby hindering acceptor access to the catalytic cleft. On the basis of these two structural observations, we developed a reversible redox regulatory feature in yeast GS by substituting cysteine residues for two highly conserved arginine residues. When oxidized, the cysteine mutant enzyme exhibits activity levels similar to the phosphorylated enzyme but cannot be activated by G-6-P. Upon reduction, the cysteine mutant enzyme regains normal activity levels and regulatory response to G-6-P activation.« less

Dramatic enhancement of enzymatic activity in organic solvents by lyoprotectants

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

Dabulis, K.; Klibanov, A.M.

1993-03-05

When seven different hydrolytic enzymes (four proteases and three lipases) were lyophilized from aqueous solution containing a ligand, N-Ac-L-Phe-NH[sub 2], their catalytic activity in anhydrous solvents was far greater (one to two orders of magnitude) than that of the enzymes lyophilized without the ligand. This ligand-induced activation was expressed regardless of whether the substrate employed in organic solvents structurally resembled the ligand. Furthermore, nonligand lyoprotectants [sorbitol, other sugars, and poly(ethylene glycol)] also dramatically enhanced enzymatic activity in anhydrous solvents when present in enzyme aqueous solution prior to lyophilization. The effects of the ligand and of the lyoprotectants were nonadditive, suggestingmore » the same mechanism of action. Excipient-activated and nonactivated enzymes exhibited identical activities in water. Also, addition of the excipients directly to suspensions of nonactivated enzymes in organic solvents had no appreciable effect on catalytic activity. These observations indicate that the mechanism of the excipient-induced activation is based on the ability of the excipients to alleviate reversible denaturation of enzymes upon lyophilization. Activity enhancement induced by the excipients is displayed even after their removal by washing enzymes with anhydrous solvents. Subtilisin Carlsberg, lyophilized with sorbitol, was found to be a much more efficient practical catalyst than its regular' counterpart.« less

Sphingolipid Long-Chain Base Synthesis in Plants (Characterization of Serine Palmitoyltransferase Activity in Squash Fruit Microsomes).

PubMed

Lynch, D. V.; Fairfield, S. R.

1993-12-01

The activity of serine palmitoyltransferase (palmitoyl-coenzyme A [CoA]:L-serine [Ser]-C-palmitoyltransferase [decarboxylating], EC 2.3.1.50), the enzyme catalyzing the first step in the synthesis of the long-chain base required for sphingolipid assembly, has been characterized in a plant system. Enzyme activity in a microsomal membrane fraction from summer squash fruit (Cucurbita pepo L. cv Early Prolific Straightneck) was assayed by monitoring the incorporation of L-[3H]Ser into the chloroform-soluble product, 3-ketosphinganine. Addition of NADPH to the assay system resulted in the conversion of 3-ketosphinganine to sphinganine. The apparent Km for Ser was approximately 1.8 mM. The enzyme exhibited a strong preference for palmitoyl-CoA, with optimal activity at a substrate concentration of 200 [mu]M. Pyridoxal 5[prime]-phosphate was required as a coenzyme. The pH optimum was 7.6, and the temperature optimum was 36 to 40[deg]C. Enzyme activity was greatest in the microsomal fraction obtained by differential centrifugation and was localized to the endoplasmic reticulum using marker enzymes. Two known mechanism-based inhibitors of the mammalian enzyme, L-cycloserine and [beta]-chloro-L-alanine, were effective inhibitors of enzyme activity in squash microsomes. Changes in enzyme activity with size (age) of squash fruit were observed. The results from this study suggest that the properties and catalytic mechanism of Ser palmitoyltransferase from squash are similar to those of the animal, fungal, and bacterial enzyme in most respects. The specific activity of the enzyme in squash microsomes ranged from 0.57 to 0.84 nmol min-1 mg-1 of protein, values 2- to 20-fold higher than those previously reported for preparations from animal tissues.

Variation in Its C-Terminal Amino Acids Determines Whether Endo-β-Mannanase Is Active or Inactive in Ripening Tomato Fruits of Different Cultivars1

PubMed Central

Bourgault, Richard; Bewley, J. Derek

2002-01-01

Endo-β-mannanase cDNAs were cloned and characterized from ripening tomato (Lycopersicon esculentum Mill. cv Trust) fruit, which produces an active enzyme, and from the tomato cv Walter, which produces an inactive enzyme. There is a two-nucleotide deletion in the gene from tomato cv Walter, which results in a frame shift and the deletion of four amino acids at the C terminus of the full-length protein. Other cultivars that produce either active or inactive enzyme show the same absence or presence of the two-nucleotide deletion. The endo-β-mannanase enzyme protein was purified and characterized from ripe fruit to ensure that cDNA codes for the enzyme from fruit. Immunoblot analysis demonstrated that non-ripening mutants, which also fail to exhibit endo-β-mannanase activity, do so because they fail to express the protein. In a two-way genetic cross between tomato cvs Walter and Trust, all F1 progeny from both crosses produced fruit with active enzyme, suggesting that this form is dominant and homozygous in tomato cv Trust. Self-pollination of a plant from the heterozygous F1 generation yielded F2 plants that bear fruit with and without active enzyme at a ratio appropriate to Mendelian genetic segregation of alleles. Heterologous expression of the two endo-β-mannanase genes in Escherichia coli resulted in active enzyme being produced from cultures containing the tomato cv Trust gene and inactive enzyme being produced from those containing the tomato cv Walter gene. Site-directed mutagenesis was used to establish key elements in the C terminus of the endo-β-mannanase protein that are essential for full enzyme activity. PMID:12427992

Enzymes immobilized in mesoporous silica: a physical-chemical perspective.

PubMed

Carlsson, Nils; Gustafsson, Hanna; Thörn, Christian; Olsson, Lisbeth; Holmberg, Krister; Åkerman, Björn

2014-03-01

Mesoporous materials as support for immobilized enzymes have been explored extensively during the last two decades, primarily not only for biocatalysis applications, but also for biosensing, biofuels and enzyme-controlled drug delivery. The activity of the immobilized enzymes inside the pores is often different compared to that of the free enzymes, and an important challenge is to understand how the immobilization affects the enzymes in order to design immobilization conditions that lead to optimal enzyme activity. This review summarizes methods that can be used to understand how material properties can be linked to changes in enzyme activity. Real-time monitoring of the immobilization process and techniques that demonstrate that the enzymes are located inside the pores is discussed by contrasting them to the common practice of indirectly measuring the depletion of the protein concentration or enzyme activity in the surrounding bulk phase. We propose that pore filling (pore volume fraction occupied by proteins) is the best standard for comparing the amount of immobilized enzymes at the molecular level, and present equations to calculate pore filling from the more commonly reported immobilized mass. Methods to detect changes in enzyme structure upon immobilization and to study the microenvironment inside the pores are discussed in detail. Combining the knowledge generated from these methodologies should aid in rationally designing biocatalyst based on enzymes immobilized in mesoporous materials. © 2013 Elsevier B.V. All rights reserved.

Molecular dynamics simulations of glycosyltransferase LgtC.

PubMed

Snajdrová, Lenka; Kulhánek, Petr; Imberty, Anne; Koca, Jaroslav

2004-04-02

Molecular dynamics simulations have been performed on fully solvated alpha-(1-->4)-galactosyltransferase LgtC from Neisseria meningitidis with and without the donor substrate UDP-Gal and in the presence of the manganese ion. The analysis of the trajectories revealed a limited movement in the loop X (residues 75-80) and a larger conformational change in the loop Y (residues 246-251) in the simulation, when UDP-Gal was not present. In this case, the loops X and Y open by almost 10A, exposing the active site to the solvent. The 'hinge region' responsible for the opening is composed of residues 246-247. We have also analyzed the behavior of the manganese ion in the simulations. The coordination number is 6 when UDP-Gal is present and it increases to 7 when it is absent. In the latter case, three water molecules become coordinated to the ion. In both cases, the coordination is very stable implying that the manganese ion is tightly bound in the active site of the enzyme even if UDP-Gal is not present. Further analysis of the structural water molecules location confirmed that the mobility of water molecules in the active site and the accessibility of this site for solvent are higher in the absence of the substrate.

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

Immobilization of Chloroperoxidase on Aminopropyl-Glass

PubMed Central

Kadima, Tenshuk A.; Pickard, Michael A.

1990-01-01

Chloroperoxidase (CPO) purified from Caldariomyces fumago CMI 89362 was covalently bound to aminopropyl-glass by using a modification of an established method. Acid-washed glass was derivatized by using aminopropyltriethoxysilane, and the enzyme was ionically bound at low ionic strength. Further treatment with glutaraldehyde covalently linked the enzyme to the glass beads in an active form. No elution of bound activity from glass beads could be detected with a variety of washings. The loading of enzyme protein to the glass beads was highest, 100 mg of CPO per g of glass, at high reaction ratios of CPO to glass, but the specific activity of the immobilized enzyme was highest, 36% of theoretical, at low enzyme-to-carrier ratios. No differences in the properties of the soluble and immobilized enzymes could be detected by a number of criteria: their pH-activity and pH-stability profiles were similar, as were their thermal stabilities. After five uses, the immobilized enzyme retained full activity between pH 6.0 and 6.7. PMID:16348352

Enzyme Prodrug Therapy Engineered into Electrospun Fibers with Embedded Liposomes for Controlled, Localized Synthesis of Therapeutics.

PubMed

Chandrawati, Rona; Olesen, Morten T J; Marini, Thatiane C C; Bisra, Gurpal; Guex, Anne Géraldine; de Oliveira, Marcelo G; Zelikin, Alexander N; Stevens, Molly M

2017-09-01

Enzyme prodrug therapy (EPT) enables localized conversion of inert prodrugs to active drugs by enzymes. Performance of EPT necessitates that the enzyme remains active throughout the time frame of the envisioned therapeutic application. β-glucuronidase is an enzyme with historically validated performance in EPT, however it retains its activity in biomaterials for an insufficiently long period of time, typically not exceeding 7 d. Herein, the encapsulation of β-glucuronidase in liposomal subcompartments within poly(vinyl alcohol) electrospun fibers is reported, leading to the assembly of biocatalytically active materials with activity of the enzyme sustained over at least seven weeks. It is further shown that liposomes provide the highly beneficial stabilization of the enzyme when incubated in cell culture media. The assembled biocatalytic materials successfully produce antiproliferative drugs (SN-38) using externally administered prodrugs (SN-38-glucuronide) and effectively suppress cell proliferation, with envisioned utility in the design of cardiovascular grafts. © 2017 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Loss in photosynthesis during senescence is accompanied by an increase in the activity of β-galactosidase in leaves of Arabidopsis thaliana: modulation of the enzyme activity by water stress.

PubMed

Pandey, Jitendra Kumar; Dash, Sidhartha Kumar; Biswal, Basanti

2017-07-01

The precise nature of the developmental modulation of the activity of cell wall hydrolases that breakdown the wall polysaccharides to maintain cellular sugar homeostasis under sugar starvation environment still remains unclear. In this work, the activity of β-galactosidase (EC 3.2.1.23), a cell-wall-bound enzyme known to degrade the wall polysaccharides, has been demonstrated to remarkably enhance during senescence-induced loss in photosynthesis in Arabidopsis thaliana. The enhancement in the enzyme activity reaches a peak at the terminal phase of senescence when the rate of photosynthesis is at its minimum. Although the precise nature of chemistry of the interface between the decline in photosynthesis and enhancement in the activity of the enzyme could not be fully resolved, the enhancement in its activity in dark and its suppression in light or with exogenous sugars may indicate the involvement of loss of photosynthetic production of sugars as a key factor that initiates and stimulates the activity of the enzyme. The hydrolase possibly participates in the catabolic network of cell wall polysaccharides to produce sugars for execution of energy-dependant senescence program in the background of loss of photosynthesis. Drought stress experienced by the senescing leaves accelerates the decline in photosynthesis with further stimulation in the activity of the enzyme. The stress recovery of photosynthesis and suppression of the enzyme activity on withdrawal of stress support the proposition of photosynthetic modulation of the cell-wall-bound enzyme activity.

Purification and characterization of endo-beta-1,4 mannanase from Aspergillus niger gr for application in food processing industry.

PubMed

Naganagouda, K; Salimath, P V; Mulimani, V H

2009-10-01

A thermostable extracellular beta-mannanase from the culture supernatant of a fungus Aspergillus niger gr was purified to homogeneity. SDS-PAGE of the purified enzyme showed a single protein band of molecular mass 66 kDa. The beta- mannanase exhibited optimum catalytic activity at pH 5.5 and 55 degrees C. It was thermostable at 55 degrees C, and retained 50% activity after 6 h at 55 degrees C. The enzyme was stable at a pH range of 3.0 to 7.0. The metal ions Hg(2+), Cu(2+), and Ag(2+) inhibited complete enzyme activity. The inhibitors tested, EDTA, PMSF, and 1,10-phenanthroline, did not inhibit the enzyme activity. N-Bromosuccinimide completely inhibited enzyme activity. The relative substrate specificity of enzyme towards the various mannans is in the order of locust bean gum>guar gum>copra mannan, with K(m) of 0.11, 0.28, and 0.33 mg/ml, respectively. Since the enzyme is active over a wide range of pH and temperature, it could find potential use in the food-processing industry.

Remarkable Reproducibility of Enzyme Activity Profiles in Tomato Fruits Grown under Contrasting Environments Provides a Roadmap for Studies of Fruit Metabolism1[W][OPEN

PubMed Central

Biais, Benoît; Bénard, Camille; Beauvoit, Bertrand; Colombié, Sophie; Prodhomme, Duyên; Ménard, Guillaume; Bernillon, Stéphane; Gehl, Bernadette; Gautier, Hélène; Ballias, Patricia; Mazat, Jean-Pierre; Sweetlove, Lee; Génard, Michel; Gibon, Yves

2014-01-01

To assess the influence of the environment on fruit metabolism, tomato (Solanum lycopersicum ‘Moneymaker’) plants were grown under contrasting conditions (optimal for commercial, water limited, or shaded production) and locations. Samples were harvested at nine stages of development, and 36 enzyme activities of central metabolism were measured as well as protein, starch, and major metabolites, such as hexoses, sucrose, organic acids, and amino acids. The most remarkable result was the high reproducibility of enzyme activities throughout development, irrespective of conditions or location. Hierarchical clustering of enzyme activities also revealed tight relationships between metabolic pathways and phases of development. Thus, cell division was characterized by high activities of fructokinase, glucokinase, pyruvate kinase, and tricarboxylic acid cycle enzymes, indicating ATP production as a priority, whereas cell expansion was characterized by enzymes involved in the lower part of glycolysis, suggesting a metabolic reprogramming to anaplerosis. As expected, enzymes involved in the accumulation of sugars, citrate, and glutamate were strongly increased during ripening. However, a group of enzymes involved in ATP production, which is probably fueled by starch degradation, was also increased. Metabolites levels seemed more sensitive than enzymes to the environment, although such differences tended to decrease at ripening. The integration of enzyme and metabolite data obtained under contrasting growth conditions using principal component analysis suggests that, with the exceptions of alanine amino transferase and glutamate and malate dehydrogenase and malate, there are no links between single enzyme activities and metabolite time courses or levels. PMID:24474652

Simultaneously and separately immobilizing incompatible dual-enzymes on polymer substrate via visible light induced graft polymerization

NASA Astrophysics Data System (ADS)

Zhu, Xing; He, Bin; Zhao, Changwen; Ma, Yuhong; Yang, Wantai

2018-04-01

Developing facile and mild strategy to construct multi-enzymes immobilization system has attracted considerable attentions in recent years. Here a simple immobilization strategy called visible light induced graft polymerization that can simultaneously and separately encapsulate two kinds of enzymes on one polymer film was proposed. Two incompatible enzymes, trypsin and transglutaminase (TGase) were selected as model dual-enzymes system and simultaneously immobilized on two sides of low-density polyethylene (LDPE) film. After immobilization, it was found that more than 90% of the enzymes can be embedded into dual-enzymes loaded film without leakage. And the activities of both separately immobilized enzymes were higher than the activities of mixed co-immobilized enzymes or the sequential immobilized ones. This dual-enzymes loaded film (DEL film) showed excellent recyclability and can retain >87% activities of both enzymes after 4 cycles of utilization. As an example, this DEL film was used to conjugate a prodrug of cytarabine with a target peptide. The successful preparation of expected product demonstrated that the separately immobilized two enzymes can worked well together to catalyze a two-step reaction.

Kinetic study of Candida antarctica lipase B immobilization using poly(methyl methacrylate) nanoparticles obtained by miniemulsion polymerization as support.

PubMed

Valério, Alexsandra; Nicoletti, Gabrieli; Cipolatti, Eliane P; Ninow, Jorge L; Araújo, Pedro H H; Sayer, Cláudia; de Oliveira, Débora

2015-03-01

With the objective to obtain immobilized Candida antarctica lipase B (CalB) with good activity and improved utilization rate, this study evaluated the influence of enzyme and crodamol concentrations and initiator type on the CalB enzyme immobilization in nanoparticles consisting of poly(methyl methacrylate) (PMMA) obtained by miniemulsion polymerization. The kinetic study of immobilized CalB enzyme in PMMA nanoparticles was evaluated in terms of monomer conversion, particle size, zeta potential, and relative activity. The optimum immobilization condition for CalB was compared with free enzyme in the p-NPL hydrolysis activity measurement. Results showed a higher CalB enzyme stability after 20 hydrolysis cycles compared with free CalB enzyme; in particular, the relative immobilized enzyme activity was maintained up to 40%. In conclusion, PMMA nanoparticles proved to be a good support for the CalB enzyme immobilization and may be used as a feasible alternative catalyst in industrial processes.

Function and biotechnology of extremophilic enzymes in low water activity

PubMed Central

2012-01-01

Enzymes from extremophilic microorganisms usually catalyze chemical reactions in non-standard conditions. Such conditions promote aggregation, precipitation, and denaturation, reducing the activity of most non-extremophilic enzymes, frequently due to the absence of sufficient hydration. Some extremophilic enzymes maintain a tight hydration shell and remain active in solution even when liquid water is limiting, e.g. in the presence of high ionic concentrations, or at cold temperature when water is close to the freezing point. Extremophilic enzymes are able to compete for hydration via alterations especially to their surface through greater surface charges and increased molecular motion. These properties have enabled some extremophilic enzymes to function in the presence of non-aqueous organic solvents, with potential for design of useful catalysts. In this review, we summarize the current state of knowledge of extremophilic enzymes functioning in high salinity and cold temperatures, focusing on their strategy for function at low water activity. We discuss how the understanding of extremophilic enzyme function is leading to the design of a new generation of enzyme catalysts and their applications to biotechnology. PMID:22480329

Deletion of a non-catalytic region increases the enzymatic activity of a β-agarase from Flammeovirga sp. MY04

NASA Astrophysics Data System (ADS)

Han, Wenjun; Gu, Jingyan; Liu, Huihui; Li, Fuchuan; Wu, Zhihong; Li, Yuezhong

2015-10-01

A Glycoside hydrolase (GH) typically contains one catalytic module and varied non-catalytic regions (NCRs). However, effects of the NCRs to the catalytic modules remain mostly unclear except the carbohydrate-binding modules (CBMs). AgaG4 is a GH16 endo- β-agarase of the agarolytic marine bacterium Flammeovirga sp. MY04. The enzyme consists of an extra sugar-binding peptide within the catalytic module, with no predictable CBMs but function-unknown sequences in the NCR, which is a new characteristic of agarase sequences. In this study, we deleted the NCR sequence, a 140-amino acid peptide at the C-terminus and expressed the truncated gene, agaG4-T140, in Escherichia coli. After purification and refolding, the truncated agarase rAgaG4-T140 retained the same catalytic temperature and pH value as rAgaG4. Using combined fluorescent labeling, HPLC and MS/MS techniques, we identified the end-products of agarose degradation by rAgaG4-T140 as neoagarotetraose and neoagarohexaose, with a final molar ratio of 1.53:1 and a conversion ratio of approximately 70%, which were similar to those of rAgaG4. However, the truncated agarase rAgaG4-T140 markedly decreased in protein solubility by 15 times and increased in enzymatic activities by 35 times. The oligosaccharide production of rAgaG4-T140 was approximately 25 times the weight of that produced by equimolar rAgaG4. This study provides some insights into the influences of NCR on the biochemical characteristics of agarase AgaG4 and implies some new strategies to improve the properties of a GH enzyme.

Genetic construction and functional analysis of hybrid polyketide synthases containing heterologous acyl carrier proteins.

PubMed Central

Khosla, C; McDaniel, R; Ebert-Khosla, S; Torres, R; Sherman, D H; Bibb, M J; Hopwood, D A

1993-01-01

The gene that encodes the acyl carrier protein (ACP) of the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2) was replaced with homologs from the granaticin, oxytetracycline, tetracenomycin, and putative frenolicin polyketide synthase gene clusters. All of the replacements led to expression of functional synthases, and the recombinants synthesized aromatic polyketides similar in chromatographic properties to actinorhodin or to shunt products produced by mutants defective in the actinorhodin pathway. Some regions within the ACP were also shown to be interchangeable and allow production of a functional hybrid ACP. Structural analysis of the most abundant polyketide product of one of the recombinants by electrospray mass spectrometry suggested that it is identical to mutactin, a previously characterized shunt product of an actVII mutant (deficient in cyclase and dehydrase activities). Quantitative differences in the product profiles of strains that express the various hybrid synthases were observed. These can be explained, at least in part, by differences in ribosome-binding sites upstream of each ACP gene, implying either that the ACP concentration in some strains is rate limiting to overall PKS activity or that the level of ACP expression also influences the expression of another enzyme(s) encoded by a downstream gene(s) in the same operon as the actinorhodin ACP gene. These results reaffirm the idea that construction of hybrid polyketide synthases will be a useful approach for dissecting the molecular basis of the specificity of PKS-catalyzed reactions. However, they also point to the need for reducing the chemical complexity of the approach by minimizing the diversity of polyketide products synthesized in strains that produce recombinant polyketide synthases. Images PMID:8468280

The Role of α-Glucosidase in Germinating Barley Grains1[W][OA

PubMed Central

Stanley, Duncan; Rejzek, Martin; Naested, Henrik; Smedley, Mark; Otero, Sofía; Fahy, Brendan; Thorpe, Frazer; Nash, Robert J.; Harwood, Wendy; Svensson, Birte; Denyer, Kay; Field, Robert A.; Smith, Alison M.

2011-01-01

The importance of α-glucosidase in the endosperm starch metabolism of barley (Hordeum vulgare) seedlings is poorly understood. The enzyme converts maltose to glucose (Glc), but in vitro studies indicate that it can also attack starch granules. To discover its role in vivo, we took complementary chemical-genetic and reverse-genetic approaches. We identified iminosugar inhibitors of a recombinant form of an α-glucosidase previously discovered in barley endosperm (ALPHA-GLUCOSIDASE97 [HvAGL97]), and applied four of them to germinating grains. All four decreased the Glc-to-maltose ratio in the endosperm 10 d after imbibition, implying inhibition of maltase activity. Three of the four inhibitors also reduced starch degradation and seedling growth, but the fourth did not affect these parameters. Inhibition of starch degradation was apparently not due to inhibition of amylases. Inhibition of seedling growth was primarily a direct effect of the inhibitors on roots and coleoptiles rather than an indirect effect of the inhibition of endosperm metabolism. It may reflect inhibition of glycoprotein-processing glucosidases in these organs. In transgenic seedlings carrying an RNA interference silencing cassette for HvAgl97, α-glucosidase activity was reduced by up to 50%. There was a large decrease in the Glc-to-maltose ratio in these lines but no effect on starch degradation or seedling growth. Our results suggest that the α-glucosidase HvAGL97 is the major endosperm enzyme catalyzing the conversion of maltose to Glc but is not required for starch degradation. However, the effects of three glucosidase inhibitors on starch degradation in the endosperm indicate the existence of unidentified glucosidase(s) required for this process. PMID:21098673

Identification of a nicotinamide/nicotinate mononucleotide adenylyltransferase in Giardia lamblia (GlNMNAT).

PubMed

Forero-Baena, Nicolás; Sánchez-Lancheros, Diana; Buitrago, July Constanza; Bustos, Victor; Ramírez-Hernández, María Helena

2015-01-01

Giardia lamblia is an intestinal protozoan parasite that causes giardiasis, a disease of high prevalence in Latin America, Asia and Africa. Giardiasis leads to poor absorption of nutrients, severe electrolyte loss and growth retardation. In addition to its clinical importance, this parasite is of special biological interest due to its basal evolutionary position and simplified metabolism, which has not been studied thoroughly. One of the most important and conserved metabolic pathways is the biosynthesis of nicotinamide adenine dinucleotide (NAD). This molecule is widely known as a coenzyme in multiple redox reactions and as a substrate in cellular processes such as synthesis of Ca 2+ mobilizing agents, DNA repair and gene expression regulation. There are two pathways for NAD biosynthesis, which converge at the step catalyzed by nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1/18). Using bioinformatics tools, we found two NMNAT sequences in Giardia lamblia ( glnmnat-a and glnmnat-b ). We first verified the identity of the sequences in silico . Subsequently, glnmnat-a was cloned into an expression vector. The recombinant protein (His-GlNMNAT) was purified by nickel-affinity binding and was used in direct in vitro enzyme assays assessed by C18-HPLC, verifying adenylyltransferase activity with both nicotinamide (NMN) and nicotinic acid (NAMN) mononucleotides. Optimal reaction pH and temperature were 7.3 and 26 °C. Michaelis-Menten kinetics were observed for NMN and ATP, but saturation was not accomplished with NAMN, implying low affinity yet detectable activity with this substrate. Double-reciprocal plots showed no cooperativity for this enzyme. This represents an advance in the study of NAD metabolism in Giardia spp.

[Hepatic allopurinol oxidizing enzyme in mice].

PubMed

Huh, K; Iwata, H; Yamamoto, I

1975-03-01

The relationship between allopurinol oxidizing enzyme and aldehyde oxidase was investaged in mice. The oxidation of both N-methylnicotinamide and allopurinol appears to be catalized by a single enzyme, aldehyde oxidase (aldehyde-oxygen oxidoreductase EC, 1.2.3.1.). This conclusion is based on the following evidence; The postnatal changes of allopurinol and N-methylnicotinamide oxidizing activities were similar during growth and the levels of both activities increased in a parallel fashion upon the attainment of sexual maturity. The rates of loss of the activities of both enzymes by heat denaturation as well as dexamethasone administration were similar. The inhibitors of allopurinol oxidizing enzyme also suppressed N-methylnicotinamide oxidation. Competition of N-methylnicotineamide and allopurinol for oxidation was demonstrated. The rate of increase of the activities in both enzymes was almost parallel during each step of the purification from mouse liver supernatant. It was ascertained that xanthine oxidase in the enzyme preparation does not influence allopurinol oxidation.

Kinase Activity Studied in Living Cells Using an Immunoassay

ERIC Educational Resources Information Center

Bavec, Aljos?a

2014-01-01

This laboratory exercise demonstrates the use of an immunoassay for studying kinase enzyme activity in living cells. The advantage over the classical method, in which students have to isolate the enzyme from cell material and measure its activity in vitro, is that enzyme activity is modulated and measured in living cells, providing a more…

Elevated stearoyl-CoA desaturase-1 expression in skeletal muscle contributes to abnormal fatty acid partitioning in obese humans

PubMed Central

Hulver, Matthew W.; Berggren, Jason R.; Carper, Michael J.; Miyazaki, Makoto; Ntambi, James M.; Hoffman, Eric P.; Thyfault, John P.; Stevens, Robert; Dohm, G. Lynis; Houmard, Joseph A.; Muoio, Deborah M.

2014-01-01

Summary Obesity and type 2 diabetes are strongly associated with abnormal lipid metabolism and accumulation of intramyocellular triacylglycerol, but the underlying cause of these perturbations are yet unknown. Herein, we show that the lipogenic gene, stearoyl-CoA desaturase 1 (SCD1), is robustly up-regulated in skeletal muscle from extremely obese humans. High expression and activity of SCD1, an enzyme that catalyzes the synthesis of monounsaturated fatty acids, corresponded with low rates of fatty acid oxidation, increased triacylglycerol synthesis and increased monounsaturation of muscle lipids. Elevated SCD1 expression and abnormal lipid partitioning were retained in primary skeletal myocytes derived from obese compared to lean donors, implying that these traits might be driven by epigenetic and/or heritable mechanisms. Overexpression of human SCD1 in myotubes from lean subjects was sufficient to mimic the obese phenotype. These results suggest that elevated expression of SCD1 in skeletal muscle contributes to abnormal lipid metabolism and progression of obesity. PMID:16213227

Elevated stearoyl-CoA desaturase-1 expression in skeletal muscle contributes to abnormal fatty acid partitioning in obese humans.

PubMed

Hulver, Matthew W; Berggren, Jason R; Carper, Michael J; Miyazaki, Makoto; Ntambi, James M; Hoffman, Eric P; Thyfault, John P; Stevens, Robert; Dohm, G Lynis; Houmard, Joseph A; Muoio, Deborah M

2005-10-01

Obesity and type 2 diabetes are strongly associated with abnormal lipid metabolism and accumulation of intramyocellular triacylglycerol, but the underlying cause of these perturbations are yet unknown. Herein, we show that the lipogenic gene, stearoyl-CoA desaturase 1 (SCD1), is robustly up-regulated in skeletal muscle from extremely obese humans. High expression and activity of SCD1, an enzyme that catalyzes the synthesis of monounsaturated fatty acids, corresponded with low rates of fatty acid oxidation, increased triacylglycerol synthesis and increased monounsaturation of muscle lipids. Elevated SCD1 expression and abnormal lipid partitioning were retained in primary skeletal myocytes derived from obese compared to lean donors, implying that these traits might be driven by epigenetic and/or heritable mechanisms. Overexpression of human SCD1 in myotubes from lean subjects was sufficient to mimic the obese phenotype. These results suggest that elevated expression of SCD1 in skeletal muscle contributes to abnormal lipid metabolism and progression of obesity.

Effects of dietary lead acetate on hepatic detoxication enzyme activity

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

Wagstaff, D.J.

1979-12-01

Lead-containing compounds usually inhibit enzymic and metabolic processes. This inhibition is presumed to be the mechanism of intoxication by these compounds. Inhibition of detoxication activities of liver microsomal enzymes could be particularly detrimental because the toxicity of many different substances would be increased. Exposure of experimental animals to lead compounds in several studies has been associated with depressed activity of hepatic microsomal enzymes, reduced levels of hepatic cytochrome P-450, reduced levels of hepatic microsomal protein, and prolonged hexobarbital sleep times. The present report contains observations that under certain experimental conditions there is stimulated hepatic meicrosomal enzyme activity in rats fedmore » lead acetate.« less

The kinetics of the oxidation of cytochrome c by Paracoccus cytochrome c peroxidase.

PubMed

Gilmour, R; Goodhew, C F; Pettigrew, G W; Prazeres, S; Moura, J J; Moura, I

1994-06-15

In work that is complementary to our investigation of the spectroscopic features of the cytochrome c peroxidase from Paracoccus denitrificans [Gilmour, Goodhew, Pettigrew, Prazeres, Moura and Moura (1993) Biochem. J. 294, 745-752], we have studied the kinetics of oxidation of cytochrome c by this enzyme. The enzyme, as isolated, is in the fully oxidized form and is relatively inactive. Reduction of the high-potential haem at pH 6 with ascorbate results in partial activation of the enzyme. Full activation is achieved by addition of 1 mM CaCl2. Enzyme activation is associated with formation of a high-spin state at the oxidized low-potential haem. EGTA treatment of the oxidized enzyme prevents activation after reduction with ascorbate, while treatment with EGTA of the reduced, partially activated, form abolishes the activity. We conclude that the active enzyme is a mixed-valence form with the low-potential haem in a high-spin state that is stabilized by Ca2+. Dilution of the enzyme results in a progressive loss of activity, the extent of which depends on the degree of dilution. Most of the activity lost upon dilution can be recovered after reconcentration. The M(r) of the enzyme on molecular-exclusion chromatography is concentration-dependent, with a shift to lower values at lower concentrations. Values of M(r) obtained are intermediate between those of a monomer (39,565) and a dimer. We propose that the active form of the enzyme is a dimer which dissociates at high dilution to give inactive monomers. From the activity of the enzyme at different dilutions, a KD of 0.8 microM can be calculated for the monomerdimer equilibrium. The cytochrome c peroxidase oxidizes horse ferrocytochrome c with first-order kinetics, even at high ferrocytochrome c concentrations. The maximal catalytic-centre activity ('turnover number') under the assay conditions used is 62,000 min-1, with a half-saturating ferrocytochrome c concentration of 3.3 microM. The corresponding values for the Paracoccus cytochrome c-550 (presumed to be the physiological substrate) are 85,000 min-1 and 13 microM. However, in this case, the kinetics deviate from first-order progress curves at all ferrocytochrome c concentrations. Consideration of the periplasmic environment in Paracoccus denitrificans leads us to propose that the enzyme will be present as the fully active dimer supplied with saturating ferrocytochrome c-550.

Effect of vitamin A deprivation on the cholesterol side-chain cleavage enzyme activity of testes and ovaries of rats (Short Communication)

PubMed Central

Jayaram, M.; Murthy, S. K.; Ganguly, J.

1973-01-01

The cholesterol side-chain cleavage enzyme activity is decreased considerably at the mild stage of vitamin A deficiency in rat testes and ovaries and the decrease in activity becomes more pronounced with progress of deficiency. Supplementation of the deficient rats with retinyl acetate, but not retinoic acid, restores the enzyme activity to normal values. The cholesterol side-chain cleavage enzyme of adrenals is not affected by any of the above treatments. PMID:4772624

iPhone-imaged and cell-powered electrophoresis titration chip for the alkaline phosphatase assay in serum by the moving reaction boundary.

PubMed

Cao, Xin-Yu; Kong, Fan-Zhi; Zhang, Qiang; Liu, Wei-Wen; Liu, Xiao-Ping; Li, Guo-Qing; Zhong, Ran; Fan, Liu-Yin; Xiao, Hua; Cao, Cheng-Xi

2018-05-21

As a vital enzyme, alkaline phosphatase (ALP) has great clinical significance in diagnoses of bone or liver cancer, bone metastases, rickets, and extrahepatic biliary obstruction. However, there is still no really portable chip for the ALP assay in blood. Herein, a simple electrophoresis titration (ET) model was developed for ALP detection via a moving reaction boundary (MRB). In the model, ALP catalyzed the dephosphorylation of a 4-methylumbelliferyl phosphate disodium salt (4-MUP) substrate in the cathode well to 4-methylumbelliferone ([4-MU]-) with a negative charge and blue fluorescence under UV excitation. After the catalysis, an electric field was used between the cathode and the anode. Under the electric field, [4-MU]- moved into the channel and neutralized the acidic Tris-HCl buffer, resulting in the quenching of [4-MU]- and creating a MRB. The ET system just had an ET chip, a lithium cell, a UV LED and an iPhone used as a recorder, having no traditional expensive power supply and fluorescence detector. The relevant method was developed, and a series of experiments were conducted via the ET chip. The experiments showed: (i) a MRB could be formed between the [4-MU]- base and the acidic buffer, and the MRB motion had a linear relationship with the ALP activity, validating the ET model; (ii) the ET run was not impacted by many interferences, implying good selectivity; and (iii) the ET chip could be used for portable detection within 10 min, implying an on-site and rapid analysis. In addition, the ET method had a relatively good sensitivity (0.1 U L-1), linearity (V = 0.033A + 3.87, R2 = 0.9980), stability (RSD 2.4-6.8%) and recoveries (101-105%). Finally, the ET method was successfully used for ALP assays in real serum samples. All the results implied that the developed method was simple, rapid and low-cost, and had potential for POCT clinical ALP assays.

Methods for determining enzymatic activity comprising heating and agitation of closed volumes

DOEpatents

Thompson, David Neil; Henriksen, Emily DeCrescenzo; Reed, David William; Jensen, Jill Renee

2016-03-15

Methods for determining thermophilic enzymatic activity include heating a substrate solution in a plurality of closed volumes to a predetermined reaction temperature. Without opening the closed volumes, at least one enzyme is added, substantially simultaneously, to the closed volumes. At the predetermined reaction temperature, the closed volumes are agitated and then the activity of the at least one enzyme is determined. The methods are conducive for characterizing enzymes of high-temperature reactions, with insoluble substrates, with substrates and enzymes that do not readily intermix, and with low volumes of substrate and enzyme. Systems for characterizing the enzymes are also disclosed.

A biochemical and physicochemical comparison of two recombinant enzymes used for enzyme replacement therapies of hunter syndrome.

PubMed

Chung, Yo Kyung; Sohn, Young Bae; Sohn, Jong Mun; Lee, Jieun; Chang, Mi Sun; Kwun, Younghee; Kim, Chi Hwa; Lee, Jin Young; Yook, Yeon Joo; Ko, Ah-Ra; Jin, Dong-Kyu

2014-05-01

Mucopolysaccharidosis II (MPS II, Hunter syndrome; OMIM 309900) is an X-linked lysosomal storage disease caused by a deficiency in the enzyme iduronate-2-sulfatase (IDS), leading to accumulation of glycosaminoglycans (GAGs). For enzyme replacement therapy (ERT) of Hunter syndrome, two recombinant enzymes, idursulfase (Elaprase(®), Shire Human Genetic Therapies, Lexington, MA) and idursulfase beta (Hunterase(®), Green Cross Corporation, Yongin, Korea), are currently available in Korea. To compare the biochemical and physicochemical differences between idursulfase and idursulfase beta, we examined the formylglycine (FGly) content, specific enzyme activity, mannose-6-phosphate (M6P) content, sialic acid content, and in vitro cell uptake activity of normal human fibroblasts of these two enzymes.The FGly content, which determines the enzyme activity, of idursulfase beta was significantly higher than that of idursulfase (79.4 ± 0.9 vs. 68.1 ± 2.2 %, P < 0.001). In accordance with the FGly content, the specific enzyme activity of idursulfase beta was significantly higher than that of idursulfase (42.6 ± 1.1 vs. 27.8 ± 0.9 nmol/min/μg protein, P < 0.001). The levels of M6P and sialic acid were not significantly different (2.4 ± 0.1 vs 2.4 ± 0.3 mol/mol protein for M6P and 12.3 ± 0.7 vs. 12.4 ± 0.4 mol/mol protein for sialic acid). However, the cellular uptake activity of the normal human fibroblasts in vitro showed a significant difference (Kuptake, 5.09 ± 0.96 vs. 6.50 ± 1.28 nM protein, P = 0.017).In conclusion, idursulfase beta exhibited significantly higher specific enzyme activity than idursulfase, resulting from higher FGly content. These biochemical differences may be partly attributed to clinical efficacy. However, long-term clinical evaluations of Hunter syndrome patients treated with these two enzymes will be needed to demonstrate the clinical implications of significant difference of the enzyme activity and the FGly content.

Determining Enzyme Activity by Radial Diffusion

ERIC Educational Resources Information Center

Davis, Bill D.

1977-01-01

Discusses advantages of radial diffusion assay in determining presence of enzyme and/or rough approximation of amount of enzyme activities. Procedures are included for the preparation of starch-agar plates, and the application and determination of enzyme. Techniques using plant materials (homogenates, tissues, ungerminated embryos, and seedlings)…

Towards Understanding the Catalytic Mechanism of Human Paraoxonase 1: Experimental and In Silico Mutagenesis Studies.

PubMed

Tripathy, Rajan K; Aggarwal, Geetika; Bajaj, Priyanka; Kathuria, Deepika; Bharatam, Prasad V; Pande, Abhay H

2017-08-01

Human paraoxonase 1 (h-PON1) is a ~45-kDa serum enzyme that can hydrolyze a variety of substrates, including organophosphate (OP) compounds. It is a potential candidate for the development of antidote against OP poisoning in humans. However, insufficient OP-hydrolyzing activity of native enzyme affirms the urgent need to develop improved variant(s) having enhanced OP-hydrolyzing activity. The crystal structure of h-PON1 remains unsolved, and the molecular details of how the enzyme catalyses hydrolysis of different types of substrates are also not clear. Understanding the molecular details of the catalytic mechanism of h-PON1 is essential to engineer better variant(s) of enzyme. In this study, we have used a random mutagenesis approach to increase the OP-hydrolyzing activity of recombinant h-PON1. The mutants not only showed a 10-340-fold increased OP-hydrolyzing activity against different OP substrates but also exhibited differential lactonase and arylesterase activities. In order to investigate the mechanistic details of the effect of observed mutations on the hydrolytic activities of enzyme, molecular docking studies were performed with selected mutants. The results suggested that the observed mutations permit differential binding of substrate/inhibitor into the enzyme's active site. This may explain differential hydrolytic activities of the enzyme towards different substrates.

Microbial Enzyme Activity and Carbon Cycling in Grassland Soil Fractions

NASA Astrophysics Data System (ADS)

Allison, S. D.; Jastrow, J. D.

2004-12-01

Extracellular enzymes are necessary to degrade complex organic compounds present in soils. Using physical fractionation procedures, we tested whether old soil carbon is spatially isolated from degradative enzymes across a prairie restoration chronosequence in Illinois, USA. We found that carbon-degrading enzymes were abundant in all soil fractions, including macroaggregates, microaggregates, and the clay fraction, which contains carbon with a mean residence time of ~200 years. The activities of two cellulose-degrading enzymes and a chitin-degrading enzyme were 2-10 times greater in organic matter fractions than in bulk soil, consistent with the rapid turnover of these fractions. Polyphenol oxidase activity was 3 times greater in the clay fraction than in the bulk soil, despite very slow carbon turnover in this fraction. Changes in enzyme activity across the restoration chronosequence were small once adjusted for increases in soil carbon concentration, although polyphenol oxidase activity per unit carbon declined by 50% in native prairie versus cultivated soil. These results are consistent with a `two-pool' model of enzyme and carbon turnover in grassland soils. In light organic matter fractions, enzyme production and carbon turnover both occur rapidly. However, in mineral-dominated fractions, both enzymes and their carbon substrates are immobilized on mineral surfaces, leading to slow turnover. Soil carbon accumulation in the clay fraction and across the prairie restoration chronosequence probably reflects increasing physical isolation of enzymes and substrates on the molecular scale, rather than the micron to millimeter scale.

Partial purification of saccharifying and cell wall-hydrolyzing enzymes from malt in waste from beer fermentation broth.

PubMed

Khattak, Waleed Ahmad; Kang, Minkyung; Ul-Islam, Mazhar; Park, Joong Kon

2013-06-01

A number of hydrolyzing enzymes that are secreted from malt during brewing, including cell wall-hydrolyzing, saccharide-hydrolyzing, protein-degrading, lipid-hydrolyzing, and polyphenol and thiol-hydrolyzing enzymes, are expected to exist in an active form in waste from beer fermentation broth (WBFB). In this study, the existence of these enzymes was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, after which enzyme extract was partially purified through a series of purification steps. The hydrolyzing enzyme activity was then measured under various conditions at each purification step using carboxymethyl cellulose as a substrate. The best hydrolyzing activities of partially purified enzymes were found at pH 4.5 and 50 °C in a citrate buffer system. The enzymes showed highest thermal stability at 30 °C when exposed for prolonged time. As the temperature increased gradually from 25 to 70 °C, yeast cells in the chemically defined medium with enzyme extract lost their cell wall and viability earlier than those without enzyme extract. Cell wall degradation and the release of cell matrix into the culture media at elevated temperature (45-70 °C) in the presence of enzyme extract were monitored through microscopic pictures. Saccharification enzymes from malt were relatively more active in the original WBFB than supernatant and diluted sediments. The presence of hydrolyzing enzymes from malt in WBFB is expected to play a role in bioethanol production using simultaneous saccharification and fermentation without the need for additional enzymes, nutrients, or microbial cells via a cell-free enzyme system.

Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis

DOE PAGES

Weyman, Philip D.; Beeri, Karen; Lefebvre, Stephane C.; ...

2014-10-10

Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow onmore » urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle.« less

Influence of cadmium on antioxidant capacity and four microelement concentrations in tomato seedlings (Lycopersicon esculentum).

PubMed

Dong, Jing; Wu, Feibo; Zhang, Guoping

2006-09-01

Tomato (Lycopersicon esculentum) seedlings were grown in four cadmium (Cd) levels of 0-10 microM in a hydroponic system to analyze the antioxidative enzymes, Cd concentration in the plants, and the interaction between Cd and four microelements. The results showed that there was a significant increase in malondialdehyde (MDA) concentration, and superoxide dismutase (SOD) and peroxidase (POD) activities in the plants subjected to 1-10 microM Cd. This indicates that Cd stress induces an oxidative stress response in tomato plants, characterized by an accumulation of MDA and increase in activities of SOD and POD. Root, stem and leaf Cd concentrations increased with its exposure Cd level, and the highest Cd concentration occurred in roots, followed by leaves and stems. A concentration- and tissue-dependent response was found in the four microelement concentrations to Cd stress in the tomato leaves, stems and roots. Regression analysis showed that there was a significantly negative correlation between Cd and Mn, implying the antagonistic effect of Cd on Mn absorption and translocation. The correlation between Cd and Zn, Cu and Fe were inconsistent among leaves, stems and roots.

Utilization of 2,6-diaminopurine by Salmonella typhimurium.

PubMed Central

Garber, B B; Gots, J S

1980-01-01

The pathway for the utilization of 2,6-diaminopurine (DAP) as an exogenous purine source in Salmonella typhimurium was examined. In strains able to use DAP as a purine source, mutant derivatives lacking either purine nucleoside phosphorylase or adenosine deaminase activity lost the ability to do so. The implied pathway of DAP utilization was via its conversion to DAP ribonucleoside by purine nucleoside phosphorylase, followed by deamination to guanosine by adenosine deaminase. Guanosine can then enter the established purine salvage pathways. In the course of defining this pathway, purine auxotrophs able to utilize DAP as sole purine source were isolated and partially characterized. These mutants fell into several classes, including (i) strains that only required an exogenous source of guanine nucleotides (e.g., guaA and guaB strains); (ii) strains that had a purF genetic lesion (i.e., were defective in alpha-5-phosphoribosyl 1-pyrophosphate amidotransferase activity); and (iii) strains that had constitutive levels of purine nucleoside phosphorylase. Selection among purine auxotrophs blocked in the de novo synthesis of inosine 5'-monophosphate, for efficient growth on DAP as sole source of purine nucleotides, readily yielded mutants which were defective in the regulation of their deoxyribonucleoside-catabolizing enzymes (e.g., deoR mutants). PMID:6782081

Should we test TPMT enzyme levels before starting azathioprine?

PubMed

Richard, Vijay Samuel; Al-Ismail, Deana; Salamat, Ahmed

2007-08-01

Thiopurine methyltransferase (TPMT) is the main enzyme responsible for inactivating toxic products of azathioprine (AZA) metabolism. Patients with homozygous deficiency of this enzyme have no enzyme activity and ideally should not be given AZA. Patients with heterozygous deficiency have 50% of enzyme activity and have been shown to respond well and tolerate half a standard dose. We describe a patient with homozygous deficiency of TPMT who developed life threatening neutropenic sepsis, and advocate that all patients should be tested for TPMT activity prior to starting AZA therapy.

Redox-active quinones and ascorbate: an innovative cancer therapy that exploits the vulnerability of cancer cells to oxidative stress.

PubMed

Verrax, J; Beck, R; Dejeans, N; Glorieux, C; Sid, B; Pedrosa, R Curi; Benites, J; Vásquez, D; Valderrama, J A; Calderon, P Buc

2011-02-01

Cancer cells are particularly vulnerable to treatments impairing redox homeostasis. Reactive oxygen species (ROS) can indeed play an important role in the initiation and progression of cancer, and advanced stage tumors frequently exhibit high basal levels of ROS that stimulate cell proliferation and promote genetic instability. In addition, an inverse correlation between histological grade and antioxidant enzyme activities is frequently observed in human tumors, further supporting the existence of a redox dysregulation in cancer cells. This biochemical property can be exploited by using redox-modulating compounds, which represent an interesting approach to induce cancer cell death. Thus, we have developed a new strategy based on the use of pharmacologic concentrations of ascorbate and redox-active quinones. Ascorbate-driven quinone redox cycling leads to ROS formation and provoke an oxidative stress that preferentially kill cancer cells and spare healthy tissues. Cancer cell death occurs through necrosis and the underlying mechanism implies an energetic impairment (ATP depletion) that is likely due to glycolysis inhibition. Additional mechanisms that participate to cell death include calcium equilibrium impairment and oxidative cleavage of protein chaperone Hsp90. Given the low systemic toxicity of ascorbate and the impairment of crucial survival pathways when associated with redox-active quinones, these combinations could represent an original approach that could be combined to standard cancer therapy.

Toxic effect of nonylphenol on the marine macroalgae Gracilaria lemaneiformis (Gracilariales, Rhodophyta): antioxidant system and antitumor activity.

PubMed

Zhong, Mingqin; Yin, Pinghe; Zhao, Ling

2017-04-01

The objective of the present work was to evaluate the toxic effect of nonylphenol (NP) on the antioxidant response and antitumor activity of Gracilaria lemaneiformis. An obvious oxidative damage was observed in this study. The thallus exposed to NP showed 1.2-2.0-fold increase in lipid peroxide and displayed a maximum level of 16.58 μmol g -1 Fw on 0.6 mg L -1 for 15-day exposure. The activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) enhanced significantly by 1.1-3.2-fold and subsequently diminished at the high concentrations and prolonged exposure. The results of DNA damage in comet assay also supported that NP was obviously toxic on G. lemaneiformis with increasing the percentage of tail DNA in a dose-dependent manner. Furthermore, the ethanol extract of G. lemaneiformis (EEGL) did exhibit antitumor potential against HepG-2 cells. While decreased in cell inhibition, ROS generation, apoptosis, and caspase-3 in HepG-2 cells treated with the EEGL were observed when G. lemaneiformis was exposed to NP for 15 days, and which were related to exposure concentration of NP. These suggested that NP has strongly toxic effect on the antitumor activity of G. lemaneiformis. The results revealed in this study imply that macroalgae can be useful biomarkers to evaluate marine pollutions.

Protective effect of saponins from Panax notoginseng against doxorubicin-induced cardiotoxicity in mice.

PubMed

Liu, Li; Shi, Run; Shi, Qiang; Cheng, Yiyu; Huo, Yang

2008-02-01

The dried rhizome of Panax notoginseng is a traditional Chinese herb extensively used for treatment of cardiovascular diseases and other ailments. Panax notoginseng saponins (PNS) are known as the major pharmacologically active constituents. The purpose of this study was to investigate the cardioprotective effects of PNS against doxorubicin-induced cardiotoxicity and its possible influence on the anti-tumor activity of doxorubicin. Five groups of ICR mice were treated with saline (control group), doxorubicin alone (20 mg/kg I. P.), PNS alone, doxorubicin pretreated with PNS (100 mg/kg I. G. for 5 consecutive days) or amifostine (single dose of 200 mg/kg I. V., used as positive control). After 72 h of doxorubicin treatment, cardiac function, serum levels of lactate dehydrogenase (LDH), creatine kinase (CK) and creatine kinase isoenzyme (CK-MB) and activities of antioxidant enzymes in heart tissue were measured. Pretreatment with PNS significantly protected the mice from DOX-induced cardiotoxicity as evidenced from improved ventricular contractile function, lower levels of serum LDH, CK and CK-MB, minimal morphological changes in hearts, and normalization of myocardial superoxide dismutase, glutathione peroxidase and catalase activities. Additionally, IN VITRO cytotoxic studies demonstrated that PNS did not compromise the inhibitory effect of doxorubicin on the proliferation of cancer cells. These results imply the potentially clinical application of PNS to overcome the negative side effects of doxorubicin.

Application of carbohydrate arrays coupled with mass spectrometry to detect activity of plant-polysaccharide degradative enzymes from the fungus Aspergillus niger

PubMed Central

van Munster, Jolanda M.; Thomas, Baptiste; Riese, Michel; Davis, Adrienne L.; Gray, Christopher J.; Archer, David B.; Flitsch, Sabine L.

2017-01-01

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. PMID:28220903

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

Velletri, P.A.; Aquilano, D.R.; Bruckwick, E.

Hypophysectomy of prepubescent (3-week-old) rats prevented the pubertal development of testicular, but not pulmonary, angiotensin-converting enzyme (EC 3.4.15.1). Additionally, hypophysectomy resulted in a loss of testicular converting enzyme activity in 10-week-old rats that had achieved puberty and had developed enzyme activity. Hormone regimens consisting of FSH/LH (7.5 U/rat X day), hCG (10 U/rat X day), or testosterone (1 mg/rat X day) were employed to ascertain their ability to maintain activity in hypophysectomized rats. All three of the above hormone regimens, if initiated on the first day after hypophysectomy of 10-week-old rats, were capable of maintaining testicular converting enzyme activity. Centrifugalmore » elutriation of dispersed testicular cells indicated that the majority of enzyme activity in mature rats was associated with the germinal cells, a result consistent with the data accumulated from the hormonal studies. Lastly, (/sup 3/H)captopril bound specifically to cellular fractions enriched in germinal cells. The above studies suggest that the pituitary gland is required for the development and maintenance of testicular angiotensin-converting enzyme in the rat by stimulating steroidogenesis in the testes. Furthermore, the sensitivity of converting enzyme activity to androgen coupled with the centrifugal elutriation and (/sup 3/H) captopril binding studies strongly support the notion that testicular converting enzyme is associated with germinal cells.« less

Expression, purification and immobilization of tannase from Staphylococcus lugdunensis MTCC 3614.

PubMed

Chaitanyakumar, Amballa; Anbalagan, M

2016-12-01

Enzymes find their applications in various industries, due to their error free conversion of substrate into product. Tannase is an enzyme used by various industries for degradation of tannin. Biochemical characterization of a specific enzyme from one organism to other is one of the ways to search for enzymes with better traits for industrial applications. Here, tannase encoding gene from Staphylococcus lugdunensis was cloned and suitability of the enzyme in various conditions was analysed to find its application in various industry. The recombinant protein was expressed with 6× His tag and purified using nickel affinity beads. The enzyme was purified up to homogeneity, with approximate molecular weight of 66 kDa. Purified tannase exhibited specific activity of about 716 U/mg. Optimum enzyme activity was found to be 40 °C at pH 7.0. Biochemical characterization revealed; metal ions such as Zn 2+ , Fe 2+ , Fe 3+ and Mn 2+ inhibited tannase activity, and SDS at lower concentration, increased tannase activity. Non polar organic solvents increased the tannase activity and polar solvents inhibited the tannase activity. Tannase immobilization studies show protection of the enzyme under wide range of pH and temperature. Also in this study we report a method for recovery and repeated use of the tannase.

A Chaperone Enhances Blood α-Glucosidase Activity in Pompe Disease Patients Treated With Enzyme Replacement Therapy

PubMed Central

Parenti, Giancarlo; Fecarotta, Simona; la Marca, Giancarlo; Rossi, Barbara; Ascione, Serena; Donati, Maria Alice; Morandi, Lucia Ovidia; Ravaglia, Sabrina; Pichiecchio, Anna; Ombrone, Daniela; Sacchini, Michele; Pasanisi, Maria Barbara; De Filippi, Paola; Danesino, Cesare; Della Casa, Roberto; Romano, Alfonso; Mollica, Carmine; Rosa, Margherita; Agovino, Teresa; Nusco, Edoardo; Porto, Caterina; Andria, Generoso

2014-01-01

Enzyme replacement therapy is currently the only approved treatment for Pompe disease, due to acid α-glucosidase deficiency. Clinical efficacy of this approach is variable, and more effective therapies are needed. We showed in preclinical studies that chaperones stabilize the recombinant enzyme used for enzyme replacement therapy. Here, we evaluated the effects of a combination of enzyme therapy and a chaperone on α-glucosidase activity in Pompe disease patients. α-Glucosidase activity was analyzed by tandem-mass spectrometry in dried blood spots from patients treated with enzyme replacement therapy, either alone or in combination with the chaperone N-butyldeoxynojirimycin given at the time of the enzyme infusion. Thirteen patients with different presentations (3 infantile-onset, 10 late-onset) were enrolled. In 11 patients, the combination treatment resulted in α-glucosidase activities greater than 1.85-fold the activities with enzyme replacement therapy alone. In the whole patient population, α-glucosidase activity was significantly increased at 12 hours (2.19-fold, P = 0.002), 24 hours (6.07-fold, P = 0.001), and 36 hours (3.95-fold, P = 0.003). The areas under the curve were also significantly increased (6.78-fold, P = 0.002). These results suggest improved stability of recombinant α-glucosidase in blood in the presence of the chaperone. PMID:25052852

Assessing the Contributions of Motor Enzymes and Microtubule Dynamics to Mitotic Chromosome Motions.

PubMed

McIntosh, J Richard

2017-10-06

During my graduate work with Keith Porter, I became fascinated by the mitotic spindle, an interest that has motivated much of my scientific work ever since. I began spindle studies by using electron microscopes, instruments that have made significant contributions to our understanding of spindle organization. Such instruments have helped to elucidate the distributions of spindle microtubules, the interactions among them, their molecular polarity, and their associations with both kinetochores and spindle poles. Our lab has also investigated some processes of spindle physiology: microtubule dynamics, the actions of microtubule-associated proteins (including motor enzymes), the character of forces generated by specific spindle components, and factors that control mitotic progression. Here, I give a personal perspective on some of this intellectual history and on what recent discoveries imply about the mechanisms of chromosome motion.

Dihydroquercetin Does Not Affect Age-Dependent Increase in Blood Pressure and Angiotensin-Converting Enzyme Activity in the Aorta of Hypertensive Rats.

PubMed

Slashcheva, G A; Rykov, V A; Lobanov, A V; Murashev, A N; Kim, Yu A; Arutyunyan, T V; Korystova, A F; Kublik, L N; Levitman, M Kh; Shaposhnikona, V V; Korystov, Yu N

2016-09-01

We analyzed changes in angiotensin-converting enzyme activity in the aorta of hypertensive SHR rats against the background of age-related BP increase (from week 7 to 14) and the effect of dihydroquercetin on BP rise and angiotensin-converting enzyme activity. Normotensive WKY rats of the same age were used as the control. BP and activity of angiotensin-converting enzyme in the aorta of SHR rats increased with age. Dihydroquercetin in doses of 100 and 300 μg/kg per day had no effect on the increase of these parameters; dihydroquercetin administered to 14-week-old WKY rats in a dose of 300 μg/kg reduced activity of the angiotensin-converting enzyme. Thus, the early (7-14 weeks) increase in BP and angiotensin-converting enzyme activity in the aorta of SHR rats was not modified by flavonoids (dihydroquercetin) in contrast to other rat strains and humans, which is indicative of specificity of hypertension mechanism in SHR rats.

Enhanced stability and catalytic activity of immobilized α-amylase on modified Fe3O4 nanoparticles for potential application in food industries

NASA Astrophysics Data System (ADS)

Hosseinipour, Seyyedeh Leila; Khiabani, Mahmoud Sowti; Hamishehkar, Hamed; Salehi, Roya

2015-09-01

Enzymes play an essential role in catalyzing various reactions. However, their instability upon repetitive/prolonged use, elevated temperature, acidic or alkaline pH remains an area of concern. α-Amylase, a widely used enzyme in food industries for starch hydrolysis, was covalently immobilized on the surface of two developed matrices, amino-functionalized silica-coated magnetite nanoparticles (AFSMNPs) alone and covered with chitosan. The synthesis steps and characterizations of NPs were examined by FT-IR, VSM, and SEM. Modified nanoparticles with average diameters of 20-80 nm were obtained. Enzyme immobilization efficiencies of 89 and 74 were obtained for AFSMNPs and chitosan-coated AFSMNPs, respectively. The optimum pH obtained was 6.5 and 8.0 for the enzyme immobilized on AFSMNPs and chitosan-coated AFSMNPs, respectively. Optimum temperature for the immobilized enzyme shifted toward higher temperatures. Considerable enhancements in thermal stabilities were observed for the immobilized enzyme at elevated temperatures up to 80 °C. A frequent use experiment demonstrated that the immobilized enzyme retained 74 and 85 % of its original activity even after 20 times of repeated use in AFSMNPs and chitosan-coated AFSMNPs, respectively. Storage stability demonstrated that free enzyme lost its activity completely within 30 days. But, immobilized enzyme on AFSMNPs and chitosan-coated AFSMNPs preserved 65.73 and 78.63 % of its initial activity, respectively, after 80 days of incubation. In conclusion, a substantial improvement in the performance of the immobilized enzyme with reference to the free enzyme was obtained. Furthermore, the relative activities of immobilized enzyme are superior than free enzyme over the broader pH and temperature ranges.

Comparison of Free and Immobilized L-asparaginase Synthesized by Gamma-Irradiated Penicillium cyclopium.

PubMed

El-Refai, Heba A; Shafei, Mona S; Mostafa, Hanan; El-Refai, Abdel-Monem H; Araby, Eman M; El-Beih, Fawkia M; Easa, Saadia M; Gomaa, Sanaa K

2016-01-01

Gamma irradiation is used on Penicillium cyclopium in order to obtain mutant cells of high L-asparaginase productivity. Using gamma irradiation dose of 4 KGy, P. cyclopium cells yielded L-asparaginase with extracellular enzyme activity of 210.8 ± 3 U/ml, and specific activity of 752.5 ± 1.5 U/mg protein, which are 1.75 and 1.53 times, respectively, the activity of the wild strain. The enzyme was partially purified by 40-60% acetone precipitation. L-asparaginase was immobilized onto Amberlite IR-120 by ionic binding. Both free and immobilized enzymes exhibited maximum activity at pH 8 and 40 degrees C. The immobilization process improved the enzyme thermal stability significantly. The immobilized enzyme remained 100% active at temperatures up to 60 degrees C, while the free asparaginase was less tolerant to high temperatures. The immobilized enzyme was more stable at pH 9.0 for 50 min, retaining 70% of its relative activity. The maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) of the free form were significantly changed after immobilization. The K(m) value for immobilized L-asparaginase was about 1.3 times higher than that of free enzyme. The ions K+, Ba2+ and Na+ showed stimulatory effect on enzyme activity with percentages of 110%, 109% and 106% respectively.

Catalytic power of enzymes decreases with temperature: New insights for understanding soil C cycling and microbial ecology under warming.

PubMed

Alvarez, Gaël; Shahzad, Tanvir; Andanson, Laurence; Bahn, Michael; Wallenstein, Matthew D; Fontaine, Sébastien

2018-04-23

Most current models of soil C dynamics predict that climate warming will accelerate soil C mineralization, resulting in a long-term CO 2 release and positive feedback to global warming. However, ecosystem warming experiments show that CO 2 loss from warmed soils declines to control levels within a few years. Here, we explore the temperature dependence of enzymatic conversion of polymerized soil organic C (SOC) into assimilable compounds, which is presumed the rate-limiting step of SOC mineralization. Combining literature review, modelling and enzyme assays, we studied the effect of temperature on activity of enzymes considering their thermal inactivation and catalytic activity. We defined the catalytic power of enzymes (E power ) as the cumulative amount of degraded substrate by one unit of enzyme until its complete inactivation. We show a universal pattern of enzyme's thermodynamic properties: activation energy of catalytic activity (EA cat ) < activation energy of thermal inactivation (EA inact ). By investing in stable enzymes (high EA inact ) having high catalytic activity (low EA cat ), microorganisms may maximize the E power of their enzymes. The counterpart of such EAs' hierarchical pattern is the higher relative temperature sensitivity of enzyme inactivation than catalysis, resulting in a reduction in E power under warming. Our findings could explain the decrease with temperature in soil enzyme pools, microbial biomass (MB) and carbon use efficiency (CUE) reported in some warming experiments and studies monitoring the seasonal variation in soil enzymes. They also suggest that a decrease in soil enzyme pools due to their faster inactivation under warming contributes to the observed attenuation of warming effect on soil C mineralization. This testable theory predicts that the ultimate response of SOC degradation to warming can be positive or negative depending on the relative temperature response of E power and microbial production of enzymes. © 2018 John Wiley & Sons Ltd.

A Simulation Game for the Study of Enzyme Kinetics and Inhibition.

ERIC Educational Resources Information Center

Chayoth, Reuben; Cohen, Annette

1996-01-01

Presents a simulation game that facilitates understanding of the concepts of enzyme kinetics and inhibition. The first part of the game deals with the relationship between enzyme activity and substrate concentration while the second part deals with characterization of competitive and noncompetitive inhibition of enzyme activity. (JRH)

[Effects of snow pack on soil nitrogen transformation enzyme activities in a subalpine Abies faxioniana forest of western Sichuan, China].

PubMed

Xiong, Li; Xu, Zhen-Feng; Wu, Fu-Zhong; Yang, Wan-Qin; Yin, Rui; Li, Zhi-Ping; Gou, Xiao-Lin; Tang, Shi-Shan

2014-05-01

This study characterized the dynamics of the activities of urease, nitrate reductase and nitrite reductase in both soil organic layer and mineral soil layer under three depths of snow pack (deep snowpack, moderate snowpack and shallow snowpack) over the three critical periods (snow formed period, snow stable period, and snow melt period) in the subalpine Abies faxoniana forest of western Sichuan in the winter of 2012 and 2013. Throughout the winter, soil temperature under deep snowpack increased by 46.2% and 26.2%, respectively in comparison with moderate snowpack and shallow snowpack. In general, the three nitrogen-related soil enzyme activities under shallow snowpack were 0.8 to 3.9 times of those under deep snowpack during the winter. In the beginning and thawing periods of seasonal snow pack, shallow snowpack significantly increased the activities of urease, nitrate and nitrite reductase enzyme in both soil organic layer and mineral soil layer. Although the activities of the studied enzymes in soil organic layer and mineral soil layer were observed to be higher than those under deep- and moderate snowpacks in deep winter, no significant difference was found under the three snow packs. Meanwhile, the effects of snowpack on the activities of the measured enzymes were related with season, soil layer and enzyme type. Significant variations of the activities of nitrogen-related enzymes were found in three critical periods over the winter, and the three measured soil enzymes were significantly higher in organic layer than in mineral layer. In addition, the activities of the three measured soil enzymes were closely related with temperature and moisture in soils. In conclusion, the decrease of snow pack induced by winter warming might increase the activities of soil enzymes related with nitrogen transformation and further stimulate the process of wintertime nitrogen transformation in soils of the subalpine forest.

Enzyme activity in dialkyl phosphate ionic liquids

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

Thomas, M.F.; Dunn, J.; Li, L.-L.

2011-12-01

The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariellavolvacea were studied in the following ionic liquids, 1,3-dimethylimidazolium dimethyl phosphate, [mmim][dmp], 1-ethyl-3-methylimidazolium dimethyl phosphate, [emim][dmp], 1-ethyl-3-methylimidazolium diethyl phosphate, [emim][dep] and 1-ethyl-3-methylimidazolium acetate, [emim][OAc]. Activity was determined by analyzing the hydrolysis of para-nitrobenzene carbohydrate derivatives. In general, the enzymes were most active in the dimethyl phosphate ionic liquids, followed by acetate. Generally speaking, activity decreased sharply for concentrations of [emim][dep] above 10% v/v, while the other ionic liquids showed less impact on activity up to 20% v/v.

Some enzymes of carbohydrate metabolism in Mesocestoides corti and Heterakis spumosa.

PubMed

Dubinský, P; Ruscinová, B; Hetmanski, S L; Arme, C; Turceková, L; Rybos, M

1991-09-01

The activities of selected enzymes of carbohydrate metabolism were measured in tetrathyridia of Mesocestoides corti and in adult females and males of Heterakis spumosa. When the species were compared, only lactate dehydrogenase and phosphoenolpyruvate carboxykinase activities were considerably higher in M. corti. Activities of other enzymes were higher in H. spumosa, with malate dehydrogenase activity being considerably so. In H. spumosa, enzyme activity was higher, and succinate dehydrogenase markedly so in males, when compared with females. Tetrathyridia aged 170 and 210 days show relatively stable malate and lactate dehydrogenase activities, and mice of ICR and BALB/c strains are suitable for the maintenance of tetrathyridia.

In-vitro engineering of novel bioactivity in the natural enzymes

NASA Astrophysics Data System (ADS)

Tiwari, Vishvanath

2016-10-01

Enzymes catalyze various biochemical functions with high efficiency and specificity. In-vitro design of the enzyme leads to novel bioactivity in this natural biomolecule that give answers of some vital questions like crucial residues in binding with substrate, molecular evolution, cofactor specificity etc. Enzyme engineering technology involves directed evolution, rational designing, semi-rational designing and structure-based designing using chemical modifications. Similarly, combined computational and in-vitro evolution approaches together help in artificial designing of novel bioactivity in the natural enzyme. DNA shuffling, error prone PCR and staggered extension process are used to artificially redesign active site of enzyme, which can alter its efficiency and specificity. Modifications of the enzyme can lead to the discovery of new path of molecular evolution, designing of efficient enzymes, locating active sites and crucial residues, shift in substrate and cofactor specificity. The methods and thermodynamics of in-vitro designing of the enzyme are also discussed. Similarly, engineered thermophilic and psychrophilic enzymes attain substrate specificity and activity of mesophilic enzymes that may also be beneficial for industry and therapeutics.

Dried blood spots for the enzymatic diagnosis of lysosomal storage diseases in dogs and cats.

PubMed

Sewell, Adrian C; Haskins, Mark E; Giger, Urs

2012-12-01

In people, lysosomal storage diseases (LSD) can be diagnosed by assaying enzyme activities in dried blood spots (DBS). The aim of this study was to evaluate the feasibility of using DBS samples from dogs and cats to measure lysosomal enzymatic activities and diagnose LSD. Drops of fresh whole blood collected in EDTA from dogs and cats with known or suspected LSD and from clinically healthy dogs and cats were placed on neonatal screening cards, dried, and mailed to the Metabolic Laboratory, University Children's Hospital, Frankfurt, Germany. Activities of selected lysosomal enzymes were measured using fluorescent substrates in a 2-mm diameter disk (~2.6 μL blood) punched from the DBS. Results were expressed as nmol substrate hydrolyzed per mL of blood per minute or hour. Reference values were established for several lysosomal enzyme activities in DBS from dogs and cats; for most enzymes, activities were higher than those published for human samples. Activities of β-glucuronidase, N-acetylglucosamine-4-sulfatase (arylsulfatase B), α-mannosidase, α-galactosidase, α-fucosidase, and hexosaminidase A were measureable in DBS from healthy cats and dogs; α-iduronidase activity was measureable only in cats. In samples from animals with LSD, markedly reduced activity of a specific enzyme was found. In contrast, in samples from cats affected with mucolipidosis II, activities of lysosomal enzymes were markedly increased. Measurement of lysosomal enzyme activities in DBS provides an inexpensive, simple, and convenient method to screen animals for suspected LSD and requires only a small sample volume. For diseases in which the relevant enzyme activity can be measured in DBS, a specific diagnosis can be made. © 2012 American Society for Veterinary Clinical Pathology.

Activities of five enzymes following soil disturbance and weed control in a Missouri forest

Treesearch

Felix, Jr. Ponder; Frieda Eivazi

2008-01-01

Forest disturbances associated with harvesting activities can affect soil properties including enzyme activity and overall soil quality. The activities of five enzymes (acid and alkaline phosphatases, betaglucosidase, aryl-sulfatase, and beta-glucosominidase) were measured after 8 years in soil from clearcut and uncut control plots of a Missouri oak-hickory (...

Purification and properties of a novel ferricyanide-linked xanthine dehydrogenase from Pseudomonas putida 40.

PubMed Central

Woolfolk, C A

1985-01-01

The isolation of a xanthine dehydrogenase from Pseudomonas putida 40 which utilizes ferricyanide as an electron acceptor at high efficiency is presented. The new activity is separate from the NAD+ and oxygen-utilizing activities of the same organism but displays a broad pattern for reducing substrates typical of those of previously studied xanthine-oxidizing enzymes. Unlike the previously studied enzymes, the new enzyme appears to lack flavin but possess heme and is resistant to cyanide treatment. However, sensitivity of the purified enzyme to methanol and the selective elimination of the activity when tungstate is added to certain growth media suggest a role for molybdenum. The enzyme is subject to a selective proteolytic action during processing which is not accompanied by denaturation or loss of activity and which is minimized by the continuous exposure of the activity to EDTA and phenylmethylsulfonyl fluoride. Electrophoresis of the denatured enzyme in the presence of sodium dodecyl sulfate suggests that the enzyme is constructed of subunits with a molecular weight of approximately 72,000. Electrophoresis under native conditions of a purified enzyme previously exposed to magnesium ion reveals a series of major and minor activity bands which display some selectivity toward both electron donors and acceptors. An analysis of the effect of gel concentration on this pattern suggests that the enzyme forms a series of charge and size isomers with a pair of trimeric forms predominating. Comparison of the rate of sedimentation of the enzyme in sucrose gradients with its elution profile from standardized Sepharose 6B columns suggests a molecular weight of 255,000 for the major form of the native enzyme. Images PMID:3860496

Gene expression and activity of digestive enzymes of Daphnia pulex in response to food quality differences.

PubMed

Schwarzenberger, Anke; Fink, Patrick

2018-04-01

Food quality is an important factor influencing organisms' well-being. In freshwater ecosystems, food quality has been studied extensively for the keystone herbivore genus Daphnia, as they form the critical trophic link between primary producers and higher order consumers such as fish. For Daphnia, the edible fraction of phytoplankton in lakes (consisting mostly of unicellular algae and cyanobacteria) is extraordinarily diverse. To be able to digest different food particles, Daphnia possess a set of digestive enzymes that metabolize carbohydrates, lipids and proteins. Recent studies have found a connection between gene expression and activity of single digestive enzyme types of Daphnia, i.e. lipases and proteases, and transcriptome studies have shown that a variety of genes coding for gut enzymes are differentially expressed in response to different food algae. However, never before has a set of digestive enzymes been studied simultaneously both on the gene expression and the enzyme activity level in Daphnia. Here, we investigated several digestive enzymes of Daphnia pulex in a comparison between a high-quality (green algal) and a low-quality (cyanobacterial) diet. Diet significantly affected the expression of all investigated digestive enzyme genes and enzyme activity was altered between treatments. Furthermore, we found that gene expression and enzyme activity were significantly correlated in cellulase, triacylglycerol lipase and β-glucosidase when switched from high to low-quality food. We conclude that one of the factors causing the often observed low biomass and energy transfer efficiency from cyanobacteria to Daphnia is probably the switch to a cost-effective overall increase of gene expression and activity of digestive enzymes of this herbivore. Copyright © 2018 Elsevier Inc. All rights reserved.

Activity loss by H46A mutation in Mycobacterium tuberculosis isocitrate lyase is due to decrease in structural plasticity and collective motions of the active site.

PubMed

Shukla, Rohit; Shukla, Harish; Tripathi, Timir

2018-01-01

Mycobacterium tuberculosis isocitrate lyase (MtbICL) is a crucial enzyme of the glyoxylate cycle and is a validated anti-tuberculosis drug target. Structurally distant, non-active site mutation (H46A) in MtbICL has been found to cause loss of enzyme activity. The aim of the present work was to explore the structural alterations induced by H46A mutation that caused the loss of enzyme activity. The structural and dynamic consequences of H46A mutation were studied using multiple computational methods such as docking, molecular dynamics simulation and residue interaction network analysis (RIN). Principal component analysis and cross correlation analysis revealed the difference in conformational flexibility and collective modes of motions between the wild-type and mutant enzyme, particularly in the active site region. RIN analysis revealed that the active site geometry was disturbed in the mutant enzyme. Thus, the dynamic perturbation of the active site led to enzyme transition from its active form to inactive form upon mutation. The computational analyses elucidated the mutant-specific conformational alterations, differential dominant motions, and anomalous residue level interactions that contributed to the abrogated function of mutant MtbICL. An understanding of interactions of mutant enzymes may help in modifying the existing drugs and designing improved drugs for successful control of tuberculosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ecotoxicological effects of copper and selenium combined pollution on soil enzyme activities in planted and unplanted soils.

PubMed

Hu, Bin; Liang, Dongli; Liu, Juanjuan; Xie, Junyu

2013-04-01

The present study explored the joint effects of Cu and Se pollution mechanisms on soil enzymes to provide references for the phytoremediation of contaminated areas and agricultural environmental protection. Pot experiments and laboratory analyses were carried out to study the individual and combined influences of Cu and Se on soil enzyme activities. The activities of four soil enzymes (urease, catalase, alkaline phosphatase, and nitrate reductase) were chosen. All soil enzyme activities tested were inhibited by Cu and Se pollution, either individually or combined, in varying degrees, following the order nitrate reductase>urease>catalase>alkaline phosphatase. Growing plants stimulated soil enzyme activity in a similar trend compared with treatments without plants. The joint effects of Cu and Se on catalase activity showed synergism at low concentrations and antagonism at high concentrations, whereas the opposite was observed for urease activity. However, nitrate reductase activity showed synergism both with and without plant treatments. The half maximal effective concentration (EC50) of exchangeable fractions had a similar trend with the EC50 of total content and was lower than that of total content. The EC50 values of nitrate reductase and urease activities were significantly lower for both Se and Cu (p<0.05), which indicated that they were more sensitive than the other two enzymes. Copyright © 2013 SETAC.

Comparative kinetic studies of Mn2+-activated and fructose-1,6-P-modified Mg2+-activated pyruvate kinase from Concholepas concholepas.

PubMed

Carvajal, N; González, R; Morán, A; Oyarce, A M

1985-01-01

Initial velocity and product inhibition studies of Mn2+-activated and FDP-modified Mg2+-activated pyruvate kinase from Concholepas concholepas, were performed. Evidence is presented to show that the Mn2+-enzyme catalyzes an ordered sequential mechanism, with ADP being the first substrate and pyruvate the last product. The results presented are consistent with a random combination of reactants with the FDP-modified Mg2+-activated enzyme and the formation of the dead-end complexes enzyme ADP-ATP and enzyme-PEP-ATP.

The Molecular Basis of Dominance

PubMed Central

Kacser, Henrik; Burns, James A.

1981-01-01

The best known genes of microbes, mice and men are those that specify enzymes. Wild type, mutant and heterozygote for variants of such genes differ in the catalytic activity at the step in the enzyme network specified by the gene in question. The effect on the respective phenotypes of such changes in catalytic activity, however, is not defined by the enzyme change as estimated by in vitro determination of the activities obtained from the extracts of the three types. In vivo enzymes do not act in isolation, but are kinetically linked to other enzymes via their substrates and products. These interactions modify the effect of enzyme variation on the phenotype, depending on the nature and quantity of the other enzymes present. An output of such a system, say a flux, is therefore a systemic property, and its response to variation at one locus must be measured in the whole system. This response is best described by the sensitivity coefficient, Z, which is defined by the fractional change in flux over the fractional change in enzyme activity.(see PDF)Its magnitude determines the extent to which a particular enzyme "controls" a particular flux or phenotype and, implicitly, determines the values that the three phenotypes will have. There are as many sensitivity coefficients for a given flux as there are enzymes in the system. It can be shown that the sum of all such coefficients equals unity.(see PDF)Since n, the number of enzymes, is large, this summation property results in the individual coefficients being small. The effect of making a large change in enzyme activity therefore usually results in only a negligible change in flux. A reduction to 50% activity in the heterozygote, a common feature for many mutants, is therefore not expected to be detectable in the phenotype. The mutant would therefore be described as "recessive". The widespread occurrence of recessive mutants is thus seen to be the inevitable consequence of the kinetic structure of enzyme networks. The ad hoc hypothesis of "modifiers" selected to maximize the fitness of the heterozygote, as proposed by Fisher, is therefore unnecessary. It is based on the false general expectation of an intermediate phenotype in the heterozygote. Wright's analysis, substantially sound in its approach, proposed selection of a "safety factor" in enzyme activity. The derivation of the summation property explains why such safety factors are automatically present in almost all enzymes without selection. PMID:7297851

Microbial dynamics and enzyme activities in tropical Andosols depending on land use and nutrient inputs

NASA Astrophysics Data System (ADS)

Mganga, Kevin; Razavi, Bahar; Kuzyakov, Yakov

2015-04-01

Microbial decomposition of soil organic matter is mediated by enzymes and is a key source of terrestrial CO2 emissions. Microbial and enzyme activities are necessary to understand soil biochemical functioning and identify changes in soil quality. However, little is known about land use and nutrients availability effects on enzyme activities and microbial processes, especially in tropical soils of Africa. This study was conducted to examine how microbial and enzyme activities differ between different land uses and nutrient availability. As Andosols of Mt. Kilimanjaro are limited by nutrient concentrations, we hypothesize that N and P additions will stimulate enzyme activity. N and P were added to soil samples (0-20 cm) representing common land use types in East Africa: (1) savannah, (2) maize fields, (3) lower montane forest, (4) coffee plantation, (5) grasslands and (6) traditional Chagga homegardens. Total CO2 efflux from soil, microbial biomass and activities of β-glucosidase, cellobiohydrolase, chitinase and phosphatase involved in C, N and P cycling, respectively was monitored for 60 days. Total CO2 production, microbial biomass and enzyme activities varied in the order forest soils > grassland soils > arable soils. Increased β-glucosidase and cellobiohydrolase activities after N addition of grassland soils suggest that microorganisms increased N uptake and utilization to produce C-acquiring enzymes. Low N concentration in all soils inhibited chitinase activity. Depending on land use, N and P addition had an inhibitory or neutral effect on phosphatase activity. We attribute this to the high P retention of Andosols and low impact of N and P on the labile P fractions. Enhanced CO2 production after P addition suggests that increased P availability could stimulate soil organic matter biodegradation in Andosols. In conclusion, land use and nutrients influenced soil enzyme activities and microbial dynamics and demonstrated the decline in soil quality after landuse change. Key words: Andosols, β-glucosidase, Cellobiohydrolase, Chitinase, Phosphatase, Mt. Kilimanjaro

Enzyme Activity Experiments Using a Simple Spectrophotometer

ERIC Educational Resources Information Center

Hurlbut, Jeffrey A.; And Others

1977-01-01

Experimental procedures for studying enzyme activity using a Spectronic 20 spectrophotometer are described. The experiments demonstrate the effect of pH, temperature, and inhibitors on enzyme activity and allow the determination of Km, Vmax, and Kcat. These procedures are designed for teaching large lower-level biochemistry classes. (MR)

[Effect of space flight on the Kosmos-1129 biosatellite on enzyme activity of the rat liver].

PubMed

Nemeth, S; Tigranian, R A

1983-01-01

After the 18.5 day Cosmos-1129 flight the activity of 7 glucocorticoid-stimulated enzymes of the rat liver was measured. Immediately postflight the activity of tyrosine aminotransferase, tryptophan pyrolase and serine dehydrogenase increased. These enzymes rapidly (within several hours) react to increased glucocorticoids. The activity of aspartate and alanine aminotransferases also increased. These enzymes require many days of a continuous effect of glucocorticoids. The glycogen concentration in the rat liver also grew. At R + 6 the activity of tryptophan pyrolase and serine dehydrogenase decreased and that of the other enzymes returned to normal. The immobilization stress applied postflight led to an increased activity of tyrosine aminotransferase and tryptophan pyrolase. This study gives evidence that after space flight rats are in an acute stress state, evidently, produced by the biosatellite recovery.

Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium

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

Desjardins, Morgan; Mak, Wai Shun; O’Brien, Terrence E.

Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs showsmore » that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. Finally, these results are directly applicable to rational enzyme design and engineering.« less

Small heat shock protein AgsA: an effective stabilizer of enzyme activities.

PubMed

Tomoyasu, Toshifumi; Tabata, Atsushi; Ishikawa, Yoko; Whiley, Robert A; Nagamune, Hideaki

2013-01-01

A small heat shock protein, AgsA, possesses chaperone activity that can reduce the amount of heat-aggregated protein in vivo, and suppress the aggregation of chemical- and heat-denatured proteins in vitro. Therefore, we examined the ability of AgsA to stabilize the activity of several enzymes by using this chaperone activity. We observed that AgsA can stabilize the enzymatic activities of Renilla (Renilla reniformis) luciferase, firefly (Photinus pyralis) luciferase, and β-galactosidase, and showed comparable or greater stabilization of these enzymes than bovine serum albumin (BSA), a well-known stabilizer of enzyme activities. In particular, AgsA revealed better stabilization of Renilla luciferase and β-galactosidase than BSA under disulfide bond-reducing conditions with dithiothreitol. In addition, AgsA also increased the enzymatic performance of β-galactosidase and various restriction enzymes to a comparable or greater extent than BSA. These data indicate that AgsA may be useful as a general stabilizer of enzyme activities. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium

DOE PAGES

Desjardins, Morgan; Mak, Wai Shun; O’Brien, Terrence E.; ...

2017-07-07

Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs showsmore » that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. Finally, these results are directly applicable to rational enzyme design and engineering.« less

Enzyme-polymer composites with high biocatalytic activity and stability

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

Kim, Jungbae; Kosto, Timothy J.; Manimala, Joseph C.

2004-08-22

We have applied vacuum-spraying and electrospinning to incorporate an enzyme into a polymer matrix, creating a novel and highly active biocatalytic composite. As a unique technical approach, enzymes were co-dissolved in toluene with polymers, and the solvent was then rapidly removed by injecting the mixture into a vacuum chamber or by electrospinning. Subsequent crosslinking of the enzyme with glutaraldehyde resulted in stable entrapped enzyme within the polymeric matrices. For example, an amorphous composite of alpha-chymotrypsin and polyethylene showed no significant loss of enzymatic activity in aqueous buffer for one month. Nanofibers of alpha-chymotrypsin and polystyrene also showed no decrease inmore » activity for more than two weeks. The normalized activity of amorphous composite in organic solvents was 3-13 times higher than that of native alpha-chymotrypsin. The activity of nanofibers was 5-7 times higher than that of amorphous composite in aqueous buffer solution. The composites of alpha-chymotrypsin and polymers demonstrate the feasibility of obtaining a wide variety of active and stable biocatalytic materials with many combinations of enzymes and polymers.« less

In vitro antibody-enzyme conjugates with specific bactericidal activity.

PubMed

Knowles, D M; Sulivan, T J; Parker, C W; Williams, R C

1973-06-01

IgG with antibacterial antibody opsonic activity was isolated from rabbit antisera produced by intravenous hyperimmunization with several test strains of pneumococci, Group A beta-hemolytic streptococci, Staphylococcus aureus, Proteus mirabilis, Pseudomonas aeruginosa, and Escherichia coli. Antibody-enzyme conjugates were prepared, using diethylmalonimidate to couple glucose oxidase to IgG antibacterial antibody preparations. Opsonic human IgG obtained from serum of patients with subacute bacterial endocarditis was also conjugated to glucose oxidase. Antibody-enzyme conjugates retained combining specificity for test bacteria as demonstrated by indirect immunofluorescence. In vitro test for bactericidal activity of antibody-enzyme conjugates utilized potassium iodide, lactoperoxidase, and glucose as cofactors. Under these conditions glucose oxidase conjugated to antibody generates hydrogen peroxide, and lactoperoxidase enzyme catalyzes the reduction of hydrogen peroxide with simultaneous oxidation of I(-) and halogenation and killing of test bacteria. Potent in vitro bactericidal activity of this system was repeatedly demonstrated for antibody-enzyme conjugates against pneumococci, streptococci, S. aureus, P. mirabilis, and E. coli. However, no bactericidal effect was demonstrable with antibody-enzyme conjugates and two test strains of P. aeruginosa. Bactericidal activity of antibody-enzyme conjugates appeared to parallel original opsonic potency of unconjugated IgG preparations. Antibody-enzyme conjugates at concentrations as low as 0.01 mg/ml were capable of intense bactericidal activity producing substantial drops in surviving bacterial counts within 30-60 min after initiation of assay. These in vitro bactericidal systems indicate that the concept of antibacterial antibody-enzyme conjugates may possibly be adaptable as a mechanism for treatment of patients with leukocyte dysfunction or fulminant bacteremia.

Enzymes in Commercial Cellulase Preparations Bind Differently to Dioxane Extracted Lignins

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

Yarbrough, John M.; Mittal, Ashutosh; Katahira, Rui

Commercial fungal cellulases used in biomass-to-biofuels processes can be grouped into three general classes: native, augmented, and engineered. To evaluate lignin binding affinities of different enzyme activities in various commercial cellulase formulations in order to determine if enzyme losses due to lignin binding can be modulated by using different enzymes of the same activity We used water:dioxane (1:9) to extract lignin from pretreated corn stover. Commercial cellulases were incubated with lignin and the unbound supernatants were evaluated for individual enzyme loss by SDS=PAGE and these were correlated with activity loss using various pNP-sugar substrates. Colorimetric assays for general glycosyl hydrolasemore » activities showed distinct differences in enzyme binding to lignin for each enzyme activity. Native systems demonstrated low binding of endo- and exo-cellulases, high binding of xylanase, and moderate ..beta..-glucosidase binding. Engineered cellulase mixtures exhibited low binding of exo-cellulases, very strong binding of endocellulases and ..beta..- glucosidase, and mixed binding of xylanase activity. The augmented cellulase had low binding of exocellulase, high binding of endocellulase and xylanase, and moderate binding of ..beta..-glucosidase activities. Bound and unbound activities were correlated with general molecular weight ranges of proteins as measured by loss of proteins bands in bound fractions on SDS-PAGE gels. Lignin-bound high molecular weight bands correlated with binding of ..beta..-glucosidase activity. While ..beta..-glucosidases demonstrated high binding in many cases, they have been shown to remain active. Bound low molecular weight bands correlated with xylanase activity binding. Contrary to other literature, exocellulase activity did not show strong lignin binding. The variation in enzyme activity binding between the three classes of cellulases preparations indicate that it is certainly possible to alter the binding of specific glycosyl hydrolase activities. It remains unclear whether loss of endocellulase activity to lignin binding is problematic for biomass conversion.« less

Functional and structural characterization of the pentapeptide insertion of Theileria annulata lactate dehydrogenase by site-directed mutagenesis, comparative modeling and molecular dynamics simulations.

PubMed

Erdemir, Aysegul; Mutlu, Ozal

2017-06-01

Lactate dehydrogenase (LDH) is an important metabolic enzyme in glycolysis and it has been considered as the main energy source in many organisms including apicomplexan parasites. Differences at the active site loop of the host and parasite LDH's makes this enzyme an attractive target for drug inhibitors. In this study, five amino acid insertions in the active site pocket of Theileria annulata LDH (TaLDH) were deleted by PCR-based site-directed mutagenesis, expression and activity analysis of mutant and wild type TaLDH enzymes were performed. Removal of the insertion at the active site loop caused production of an inactive enzyme. Furthermore, structures of wild and mutant enzymes were predicted by comparative modeling and the importance of the insertions at the active site loop were also assigned by molecular docking and dynamics simulations in order to evaluate essential role of this loop for the enzymatic activity. Pentapeptide insertion removal resulted in loss of LDH activity due to deletion of Trp96 and conformational change of Arg98 because of loop instability. Analysis of wild type and mutant enzymes with comparative molecular dynamics simulations showed that the fluctuations of the loop residues increase in mutant enzyme. Together with in silico studies, in vitro results revealed that active site loop has a vital role in the enzyme activity and our findings promise hope for the further drug design studies against theileriosis and other apicomplexan parasite diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Heterologous Expression of Secreted Bacterial BPP and HAP Phytases in Plants Stimulates Arabidopsis thaliana Growth on Phytate.

PubMed

Valeeva, Lia R; Nyamsuren, Chuluuntsetseg; Sharipova, Margarita R; Shakirov, Eugene V

2018-01-01

Phytases are specialized phosphatases capable of releasing inorganic phosphate from myo -inositol hexakisphosphate (phytate), which is highly abundant in many soils. As inorganic phosphorus reserves decrease over time in many agricultural soils, genetic manipulation of plants to enable secretion of potent phytases into the rhizosphere has been proposed as a promising approach to improve plant phosphorus nutrition. Several families of biotechnologically important phytases have been discovered and characterized, but little data are available on which phytase families can offer the most benefits toward improving plant phosphorus intake. We have developed transgenic Arabidopsis thaliana plants expressing bacterial phytases PaPhyC (HAP family of phytases) and 168phyA (BPP family) under the control of root-specific inducible promoter Pht1;2 . The effects of each phytase expression on growth, morphology and inorganic phosphorus accumulation in plants grown on phytate hydroponically or in perlite as the only source of phosphorus were investigated. The most enzymatic activity for both phytases was detected in cell wall-bound fractions of roots, indicating that these enzymes were efficiently secreted. Expression of both bacterial phytases in roots improved plant growth on phytate and resulted in larger rosette leaf area and diameter, higher phosphorus content and increased shoot dry weight, implying that these plants were indeed capable of utilizing phytate as the source of phosphorus for growth and development. When grown on phytate the HAP-type phytase outperformed its BPP-type counterpart for plant biomass production, though this effect was only observed in hydroponic conditions and not in perlite. Furthermore, we found no evidence of adverse side effects of microbial phytase expression in A. thaliana on plant physiology and seed germination. Our data highlight important functional differences between these members of bacterial phytase families and indicate that future crop biotechnologies involving such enzymes will require a very careful evaluation of phytase source and activity. Overall, our data suggest feasibility of using bacterial phytases to improve plant growth in conditions of phosphorus deficiency and demonstrate that inducible expression of recombinant enzymes should be investigated further as a viable approach to plant biotechnology.

Heterologous Expression of Secreted Bacterial BPP and HAP Phytases in Plants Stimulates Arabidopsis thaliana Growth on Phytate

PubMed Central

Valeeva, Lia R.; Nyamsuren, Chuluuntsetseg; Sharipova, Margarita R.; Shakirov, Eugene V.

2018-01-01

Phytases are specialized phosphatases capable of releasing inorganic phosphate from myo-inositol hexakisphosphate (phytate), which is highly abundant in many soils. As inorganic phosphorus reserves decrease over time in many agricultural soils, genetic manipulation of plants to enable secretion of potent phytases into the rhizosphere has been proposed as a promising approach to improve plant phosphorus nutrition. Several families of biotechnologically important phytases have been discovered and characterized, but little data are available on which phytase families can offer the most benefits toward improving plant phosphorus intake. We have developed transgenic Arabidopsis thaliana plants expressing bacterial phytases PaPhyC (HAP family of phytases) and 168phyA (BPP family) under the control of root-specific inducible promoter Pht1;2. The effects of each phytase expression on growth, morphology and inorganic phosphorus accumulation in plants grown on phytate hydroponically or in perlite as the only source of phosphorus were investigated. The most enzymatic activity for both phytases was detected in cell wall-bound fractions of roots, indicating that these enzymes were efficiently secreted. Expression of both bacterial phytases in roots improved plant growth on phytate and resulted in larger rosette leaf area and diameter, higher phosphorus content and increased shoot dry weight, implying that these plants were indeed capable of utilizing phytate as the source of phosphorus for growth and development. When grown on phytate the HAP-type phytase outperformed its BPP-type counterpart for plant biomass production, though this effect was only observed in hydroponic conditions and not in perlite. Furthermore, we found no evidence of adverse side effects of microbial phytase expression in A. thaliana on plant physiology and seed germination. Our data highlight important functional differences between these members of bacterial phytase families and indicate that future crop biotechnologies involving such enzymes will require a very careful evaluation of phytase source and activity. Overall, our data suggest feasibility of using bacterial phytases to improve plant growth in conditions of phosphorus deficiency and demonstrate that inducible expression of recombinant enzymes should be investigated further as a viable approach to plant biotechnology. PMID:29515604

Emerging roles of the nucleolus in regulating the DNA damage response: the noncanonical DNA repair enzyme APE1/Ref-1 as a paradigmatical example.

PubMed

Antoniali, Giulia; Lirussi, Lisa; Poletto, Mattia; Tell, Gianluca

2014-02-01

An emerging concept in DNA repair mechanisms is the evidence that some key enzymes, besides their role in the maintenance of genome stability, display also unexpected noncanonical functions associated with RNA metabolism in specific subcellular districts (e.g., nucleoli). During the evolution of these key enzymes, the acquisition of unfolded domains significantly amplified the possibility to interact with different partners and substrates, possibly explaining their phylogenetic gain of functions. After nucleolar stress or DNA damage, many DNA repair proteins can freely relocalize from nucleoli to the nucleoplasm. This process may represent a surveillance mechanism to monitor the synthesis and correct assembly of ribosomal units affecting cell cycle progression or inducing p53-mediated apoptosis or senescence. A paradigm for this kind of regulation is represented by some enzymes of the DNA base excision repair (BER) pathway, such as apurinic/apyrimidinic endonuclease 1 (APE1). In this review, the role of the nucleolus and the noncanonical functions of the APE1 protein are discussed in light of their possible implications in human pathologies. A productive cross-talk between DNA repair enzymes and proteins involved in RNA metabolism seems reasonable as the nucleolus is emerging as a dynamic functional hub that coordinates cell growth arrest and DNA repair mechanisms. These findings will drive further analyses on other BER proteins and might imply that nucleic acid processing enzymes are more versatile than originally thought having evolved DNA-targeted functions after a previous life in the early RNA world.

Improved production of an enzyme that hydrolyses raw yam starch by Penicillium sp. S-22 using fed-batch fermentation.

PubMed

Sun, Hai-Yan; Ge, Xiang-Yang; Zhang, Wei-Guo

2006-11-01

A newly isolated strain, Penicillium sp. S-22, was used to produce an enzyme that hydrolyses raw yam starch [raw yam starch digesting enzyme (RYSDE)]. The enzyme activity and overall enzyme productivity were respectively 16 U/ml and 0.19 U/ml h in the batch culture. The enzyme activity increased to 85 U/ml by feeding of partially hydrolyzed raw yam starch. When a mixture containing partially hydrolyzed raw yam starch and peptone was fed by a pH-stat strategy, the enzyme activity reached 366 U/ml, 23-fold of that obtained in the batch culture, and the overall productivity reached 3.4 U/ml h, which was 18-fold of that in the batch culture.

Effect of monensin on the levels of tachykinins and their processing enzyme activity in rat dorsal root ganglia.

PubMed

Chikuma, Toshiyuki; Inomata, Yuji; Tsuchida, Ken; Hojo, Hiroshi; Kato, Takeshi

2002-06-28

Th effect of monensin, which inhibits trans-Golgi function, on the levels of tachykinins and their processing enzyme activity was examined in organ-cultured rat dorsal root ganglia (DRG). Using an enzyme immunoassay method, we measured neurokinin A and substance P immunoreactivity in the DRG cultured for 72 h with and without 0.1 microM monensin. Both tachykinins were reduced in the DRG treated with monensin. Treatment with monensin also reduced the activity of carboxypeptidase E, which is one of the proteolytic processing enzymes of neuropeptides. These data suggest that proteolytic processing enzymes may in part modulate the biological activity of neuropeptides within a trans-Golgi apparatus.

The effect of preparation, storage and shipping of dried blood spots on the activity of five lysosomal enzymes.

PubMed

Elbin, Carole S; Olivova, Petra; Marashio, Carla A; Cooper, Samantha K; Cullen, Emmaline; Keutzer, Joan M; Zhang, X Kate

2011-06-11

Fluorometric and tandem mass spectrometry assays can be used to measure lysosomal enzyme activities in dried blood spots (DBS). The effect of DBS preparation, storage and shipping was evaluated on the activities of acid α-glucosidase, acid α-galactosidase, acid β-glucocerebrosidase, acid sphingomyelinase, and galactocerebrosidase. Whole blood from normal donors was used to prepare DBS following Clinical and Laboratory Standards Institute guidelines and by several deviations. Some DBS were subjected to various treatments, storage and shipping conditions. The activity of 5 lysosomal enzymes (GAA, GLA, GBA, ASM, and GALC) was measured using tandem mass spectrometric and fluorometric (GAA only) assays with 2 distinct and commonly used synthetic substrates. Enzyme activities were strongly affected by the way DBS were prepared and stored. Exposure of DBS to elevated heat and humidity can destroy enzyme functions rapidly. DBS prepared from poorly mixed blood caused significant variation on enzyme activities. EDTA, but not heparin, as an anti-coagulant gave more precise results. The study confirmed the importance of proper and consistent DBS preparation and storage when screening for deficiencies of lysosomal enzymes. Copyright © 2011 Elsevier B.V. All rights reserved.

Arylsulfatase Activity in Salt Marsh Soils †

PubMed Central

Oshrain, R. L.; Wiebe, W. J.

1979-01-01

The presence of arylsulfatase(s) was confirmed in salt marsh soils. The temperatures of maximum activity and inactivation, the pH range over which the enzyme was active, and the Km values were similar to those of soil enzymes. Unlike soil arylsulfatases, however, the salt marsh enzymes do not appear to be repressed by sulfate. It is postulated that these enzymes may be necessary for the initiation of arylsulfate ester metabolism. PMID:16345425

Cobalamin-Independent Methionine Synthase (MetE): A Face-to-Face Double Barrel that Evolved by Gene Duplication

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

Pejcha, Robert; Ludwig, Martha L.

2010-03-08

Cobalamin-independent methionine synthase (MetE) catalyzes the transfer of a methyl group from methyltetrahydrofolate to L-homocysteine (Hcy) without using an intermediate methyl carrier. Although MetE displays no detectable sequence homology with cobalamin-dependent methionine synthase (MetH), both enzymes require zinc for activation and binding of Hcy. Crystallographic analyses of MetE from T. maritima reveal an unusual dual-barrel structure in which the active site lies between the tops of the two ({beta}{alpha}){sub 8} barrels. The fold of the N-terminal barrel confirms that it has evolved from the C-terminal polypeptide by gene duplication; comparisons of the barrels provide an intriguing example of homologous domainmore » evolution in which binding sites are obliterated. The C-terminal barrel incorporates the zinc ion that binds and activates Hcy. The zinc-binding site in MetE is distinguished from the (Cys){sub 3}Zn site in the related enzymes, MetH and betaine-homocysteine methyltransferase, by its position in the barrel and by the metal ligands, which are histidine, cysteine, glutamate, and cysteine in the resting form of MetE. Hcy associates at the face of the metal opposite glutamate, which moves away from the zinc in the binary E {center_dot} Hcy complex. The folate substrate is not intimately associated with the N-terminal barrel; instead, elements from both barrels contribute binding determinants in a binary complex in which the folate substrate is incorrectly oriented for methyl transfer. Atypical locations of the Hcy and folate sites in the C-terminal barrel presumably permit direct interaction of the substrates in a ternary complex. Structures of the binary substrate complexes imply that rearrangement of folate, perhaps accompanied by domain rearrangement, must occur before formation of a ternary complex that is competent for methyl transfer.« less

Perfusion chromatography separation of the tomato fruit-specific pectin methylesterase from a semipurified commercial enzyme preparation.

PubMed

Savary, B J

2001-08-01

A rapid and simple method was developed, using perfusion chromatography media, to separate the fruit-specific pectin methylesterase (PME) isoform from the depolymerizing enzyme polygalacturonase (PG) and other contaminating pectinases present in a commercial tomato enzyme preparation. Pectinase activities were adsorbed onto a Poros HS (a strong cation exchanger) column in 20 M HEPES buffer at pH 7.5. The fruit-specific PME was eluted from the column with 80 mM NaCl, followed by a step to 300 mM NaCl to elute PG activity. Rechromatography of the PME activity peak with a linear gradient further resolved two PME isoenzymes and removed residual traces of PG activity. The PG activity peak was further treated with lectin affinity chromatography to provide purified PG enzyme, which was separated from a salt-dependent PME (tentatively identified as a "ubiquitous-type" isoform), and a pectin acetylesterase. The later enzyme has not been reported previously in tomato. This method provides monocomponent enzymes that will be useful for studying enzyme mechanisms and for modifying pectin structure and functional properties.

Celluloytic enzymes, nucleic acids encoding them and methods for making and using them

DOEpatents

Gray, Kevin A; Zhao, Lishan; Cayouette, Michelle H

2015-11-04

The invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

Celluloytic enzymes, nucleic acids encoding them and methods for making and using them

DOEpatents

Gray, Kevin A.; Zhao, Lishan; Cayouette, Michelle H.

2015-09-08

The invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

L-asparaginase activity in Aeromonas sp. isolated from freshwater mussel.

PubMed

Pattnaik, S; Kabi, R; Janaki Ram, K; Bhanot, K K

2000-11-01

Aeromonas sp. from Lamellidens marginalis produced L-asparaginase when grown at 37 degrees C. The optimum enzyme activity was at pH 9 when temperature was 45 degrees C. Half-life of partially purified enzyme at 50 degrees C and 55 degrees C was 35 and 20 min, respectively. Activation and deactivation energies of partially purified enzyme were 17.48 and 24.86 kcal mol-1 respectively. The enzyme exhibited a Km (L-asparagine) value of 4.9 x 10(-6) mol l-1 and a Vmax of 9.803 IU ml-1. Three metal ions inhibited the enzyme activity at 10-20 mumol l-1 concentrations. Catalytic activity was also inhibited by EDTA, iodoacetic acid, parachloromercuribenzoic acid and phenylmethylsulphonyl fluoride at 0.1 mumol l-1.

Sodium and Potassium Ions in Proteins and Enzyme Catalysis.

PubMed

Vašák, Milan; Schnabl, Joachim

2016-01-01

The group I alkali metal ions Na(+) and K(+) are ubiquitous components of biological fluids that surround biological macromolecules. They play important roles other than being nonspecific ionic buffering agents or mediators of solute exchange and transport. Molecular evolution and regulated high intracellular and extracellular M(+) concentrations led to incorporation of selective Na(+) and K(+) binding sites into enzymes to stabilize catalytic intermediates or to provide optimal positioning of substrates. The mechanism of M(+) activation, as derived from kinetic studies along with structural analysis, has led to the classification of cofactor-like (type I) or allosteric effector (type II) activated enzymes. In the type I mechanism substrate anchoring to the enzyme active site is mediated by M(+), often acting in tandem with a divalent cation like Mg(2+), Mn(2+) or Zn(2+). In the allosteric type II mechanism, M(+) binding enhances enzyme activity through conformational transitions triggered upon binding to a distant site. In this chapter, following the discussion of the coordination chemistry of Na(+) and K(+) ions and the structural features responsible for the metal binding site selectivity in M(+)-activated enzymes, well-defined examples of M(+)-activated enzymes are used to illustrate the structural basis for type I and type II activation by Na(+) and K(+).

Standardized Assay Medium To Measure Lactococcus lactis Enzyme Activities while Mimicking Intracellular Conditions

PubMed Central

Goel, Anisha; Santos, Filipe; de Vos, Willem M.; Teusink, Bas

2012-01-01

Knowledge of how the activity of enzymes is affected under in vivo conditions is essential for analyzing their regulation and constructing models that yield an integrated understanding of cell behavior. Current kinetic parameters for Lactococcus lactis are scattered through different studies and performed under different assay conditions. Furthermore, assay conditions often diverge from conditions prevailing in the intracellular environment. To establish uniform assay conditions that resemble intracellular conditions, we analyzed the intracellular composition of anaerobic glucose-limited chemostat cultures of L. lactis subsp. cremoris MG 1363. Based on this, we designed a new assay medium for enzyme activity measurements of growing cells of L. lactis, mimicking as closely as practically possible its intracellular environment. Procedures were optimized to be carried out in 96-well plates, and the reproducibility and dynamic range were checked for all enzyme activity measurements. The effects of freezing and the carryover of ammonium sulfate from the addition of coupling enzymes were also established. Activities of all 10 glycolytic and 4 fermentative enzymes were measured. Remarkably, most in vivo-like activities were lower than previously published data. Yet, the ratios of Vmax over measured in vivo fluxes were above 1. With this work, we have developed and extensively validated standard protocols for enzyme activity measurements for L. lactis. PMID:22020503

Positron emitter labeled enzyme inhibitors

DOEpatents

Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.

1987-05-22

This invention involved a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide in activators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography. 2 figs.

Positron emitter labeled enzyme inhibitors

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

Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.

This invention involved a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide in activators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgylinemore » and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography. 2 figs.« less

Multifunctional enzymes from reduced genomes - model proteins for simple primordial metabolism?

PubMed

Seelig, Burckhard

2017-08-01

Billions of years of evolution have yielded today's complex metabolic networks driven by efficient and highly specialized enzymes. In contrast, the metabolism of the earliest cellular life forms was likely much simpler with only a few enzymes of comparatively low activity. It has been speculated that these early enzymes had low specificities and in turn were able to perform multiple functions. In this issue of Molecular Microbiology, Ferla et al. describe examples of enzymes that catalyze chemically distinct reactions while using the same active site. Most importantly, the authors demonstrated that the comparatively weak activities of these multifunctional enzymes are each physiologically relevant. These findings contrast with simply promiscuous enzyme activities, which have been described numerous times but are not physiologically relevant. Ferla et al. elegantly combined initial bioinformatics searches for enzyme candidates with sound kinetic measurements, evolutionary considerations and even structural discussions. The phenomenon of multifunctionality appears to be a mechanism for bacteria with reduced genomes to compensate for their lack of certain enzymes. In the broader context of evolution, these organisms could be considered living model systems to study features of long-extinct early cellular life. © 2017 John Wiley & Sons Ltd.

Immobilization of fungal beta-glucosidase on silica gel and kaolin carriers.

PubMed

Karagulyan, Hakob K; Gasparyan, Vardan K; Decker, Stephen R

2008-03-01

Beta-glucosidase is a key enzyme in the hydrolysis of cellulose for producing feedstock glucose for various industrial processes. Reuse of enzyme through immobilization can significantly improve the economic characteristics of the process. Immobilization of the fungal beta-glucosidase by covalent binding and physical adsorption on silica gel and kaolin was conducted for consequent application of these procedures in large-scale industrial processes. Different immobilization parameters (incubation time, ionic strength, pH, enzyme/support ratio, glutaric aldehyde concentration, etc.) were evaluated for their effect on the thermal stability of the immobilized enzyme. It was shown that the immobilized enzyme activity is stable at 50 degrees C over 8 days. It has also been shown that in the case of immobilization on kaolin, approximately 95% of the initial enzyme was immobilized onto support, and loss of activity was not observed. However, covalent binding of the enzyme to silica gel brings significant loss of enzyme activity, and only 35% of activity was preserved. In the case of physical adsorption on kaolin, gradual desorption of enzyme takes place. To prevent this process, we have carried out chemical modification of the protein. As a result, after repeated washings, enzyme desorption from kaolin has been reduced from 75 to 20-25% loss.

The Holo-Transcriptome of the Zoantharian Protopalythoa variabilis (Cnidaria: Anthozoa): A Plentiful Source of Enzymes for Potential Application in Green Chemistry, Industrial and Pharmaceutical Biotechnology.

PubMed

R L Morlighem, Jean-Étienne; Huang, Chen; Liao, Qiwen; Braga Gomes, Paula; Daniel Pérez, Carlos; de Brandão Prieto-da-Silva, Álvaro Rossan; Ming-Yuen Lee, Simon; Rádis-Baptista, Gandhi

2018-06-13

Marine invertebrates, such as sponges, tunicates and cnidarians (zoantharians and scleractinian corals), form functional assemblages, known as holobionts, with numerous microbes. This type of species-specific symbiotic association can be a repository of myriad valuable low molecular weight organic compounds, bioactive peptides and enzymes. The zoantharian Protopalythoa variabilis (Cnidaria: Anthozoa) is one such example of a marine holobiont that inhabits the coastal reefs of the tropical Atlantic coast and is an interesting source of secondary metabolites and biologically active polypeptides. In the present study, we analyzed the entire holo-transcriptome of P. variabilis , looking for enzyme precursors expressed in the zoantharian-microbiota assemblage that are potentially useful as industrial biocatalysts and biopharmaceuticals. In addition to hundreds of predicted enzymes that fit into the classes of hydrolases, oxidoreductases and transferases that were found, novel enzyme precursors with multiple activities in single structures and enzymes with incomplete Enzyme Commission numbers were revealed. Our results indicated the predictive expression of thirteen multifunctional enzymes and 694 enzyme sequences with partially characterized activities, distributed in 23 sub-subclasses. These predicted enzyme structures and activities can prospectively be harnessed for applications in diverse areas of industrial and pharmaceutical biotechnology.

Aβ-degrading enzymes: potential for treatment of Alzheimer disease.

PubMed

Miners, James Scott; Barua, Neil; Kehoe, Patrick Gavin; Gill, Steven; Love, Seth

2011-11-01

There is increasing evidence that deficient clearance of β-amyloid (Aβ) contributes to its accumulation in late-onset Alzheimer disease (AD). Several Aβ-degrading enzymes, including neprilysin (NEP), insulin-degrading enzyme, and endothelin-converting enzyme reduce Aβ levels and protect against cognitive impairment in mouse models of AD. The activity of several Aβ-degrading enzymes rises with age and increases still further in AD, perhaps as a physiological response to minimize the buildup of Aβ. The age- and disease-related changes in expression of more recently recognized Aβ-degrading enzymes (e.g. NEP-2 and cathepsin B) remain to be investigated, and there is strong evidence that reduced NEP activity contributes to the development of cerebral amyloid angiopathy. Regardless of the role of Aβ-degrading enzymes in the development of AD, experimental data indicate that increasing the activity of these enzymes (NEP in particular) has therapeutic potential in AD, although targeting their delivery to the brain remains a major challenge. The most promising current approaches include the peripheral administration of agents that enhance the activity of Aβ-degrading enzymes and the direct intracerebral delivery of NEP by convection-enhanced delivery. In the longer term, genetic approaches to increasing the intracerebral expression of NEP or other Aβ-degrading enzymes may offer advantages.

[Importance of the 11β-hydroxysteroid dehydrogenase enzyme in clinical disorders].

PubMed

Feldman, Karolina; Likó, István; Nagy, Zsolt; Szappanos, Agnes; Grolmusz, Vince Kornél; Tóth, Miklós; Rácz, Károly; Patócs, Attila

2013-02-24

Glucocorticoids play an important role in the regulation of carbohydrate and amino acid metabolism, they modulate the function of the immune system, and contribute to stress response. Increased and decreased production of glucocorticoids causes specific diseases. In addition to systemic hypo- or hypercortisolism, alteration of local synthesis and metabolism of cortisol may result in tissue-specific hypo- or hypercortisolism. One of the key enzymes participating in the local synthesis and metabolism of cortisol is the 11β-hydroxysteroid dehydrogenase enzyme. Two isoforms, type 1 and type 2 enzymes are located in the endoplasmic reticulum and catalyze the interconversion of hormonally active cortisol and inactive cortisone. The type 1 enzyme mainly works as an activator, and it is responsible for the generation of cortisol from cortisone in liver, adipose tissue, brain and bone. The gene encoding this enzyme is located on chromosome 1. The authors review the physiological and pathophysiological processes related to the function of the type 1 11β-hydroxysteroid dehydrogenase enzyme. They summarize the potential significance of polymorphic variants of the enzyme in clinical diseases as well as knowledge related to inhibitors of enzyme activity. Although further studies are still needed, inhibition of the enzyme activity may prove to be an effective tool for the treatment of several diseases such as obesity, osteoporosis and type 2 diabetes.

Immobilization of Fungal β-Glucosidase on Silica Gel and Kaolin Carriers

NASA Astrophysics Data System (ADS)

Karagulyan, Hakob K.; Gasparyan, Vardan K.; Decker, Stephen R.

β-Glucosidase is a key enzyme in the hydrolysis of cellulose for producing feedstock glucose for various industrial processes. Reuse of enzyme through immobilization can significantly improve the economic characteristics of the process. Immobilization of the fungal β-glucosidase by covalent binding and physical adsorption on silica gel and kaolin was conducted for consequent application of these procedures in large-scale industrial processes. Different immobilization parameters (incubation time, ionic strength, pH, enzyme/support ratio, glutaric aldehyde concentration, etc.) were evaluated for their effect on the thermal stability of the immobilized enzyme. It was shown that the immobilized enzyme activity is stable at 50 °C over 8 days. It has also been shown that in the case of immobilization on kaolin, approximately 95% of the initial enzyme was immobilized onto support, and loss of activity was not observed. However, covalent binding of the enzyme to silica gel brings significant loss of enzyme activity, and only 35% of activity was preserved. In the case of physical adsorption on kaolin, gradual desorption of enzyme takes place. To prevent this process, we have carried out chemical modification of the protein. As a result, after repeated washings, enzyme desorption from kaolin has been reduced from 75 to 20-25% loss.

Stability of the anti-oxidative enzymes in aqueous and detergent solution.

PubMed

Mailer, K; Del Maestro, R F

1991-09-18

Activities of the anti-oxidative enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase were studied in rat tissues to determine the ability of detergents both to solubilize the enzymes and also to stabilize enzyme activity. Rat brain, heart and liver were homogenized in 0.1M KCl, 0.1% sodium dodecyl sulfate, 0.1% lubrol, or 0.1% cetyl-trimethylammonium bromide. In general lubrol was more effective than the other solutions in solubilizing GPx and catalase. Lubrol and 0.1M KCl were equally effective in solubilizing SOD. The highest enzyme activities were (1) SOD: 2484 ng/mg (brain), 2501 ng/mg (heart), and 5586 ng/mg (liver); (2) GPx: 224 mU/mg (brain), 1870 mU/mg (heart), and 7332 mU/mg (liver); (3) catalase: 2.8 mU/mg (brain), 10.6 mU/mg (heart), and 309 mU/mg (liver). While cetyl trimethylammonium bromide is marginally better than sodium dodecyl sulfate in solubilizing active enzyme, neither ionic detergent has any advantage over lubrol or 0.1M KCl. For catalase and GPx, enzyme activity loss with time is biphasic. After initial, rapid activity loss (1-5 days for GPx and 7-10 days for catalase) the differences noted among the homogenizing solutions disappear and very little if any activity loss is noted over the next 2-3 weeks. For catalase and GPx, only baseline enzyme activity from t = 0-3 weeks is found in the most chaotropic solution, 0.1% sodium dodecyl sulfate while biphasic activity loss is most pronounced in 0.1% lubrol. These results may indicate active GPx and catalase species stabilized by a lipid-like environment. Correlating in vitro catalase or GPx measurements with in vivo anti-oxidative protection may underestimate tissue defences.

Partial purification and some properties of a latent CO2 reductase from green potato tuber chloroplasts.

PubMed

Arora, S; Ramaswamy, N K; Nair, P M

1985-12-16

We have partially purified the CO2 reductase, present in green potato tuber chloroplasts, as a latent form. Illumination of the chloroplasts in the absence of substrate, bicarbonate, activated the enzyme, which could then be obtained in soluble forms. Purification of the enzyme was achieved by (NH4)2SO4 fractionation (0-30%) and adsorption and elution from a DEAE-Sephadex A-50 column. The final preparation showed 15-fold purification and 50% recovery of the activity. The pH optimum for CO2 reductase was 8.0. Hepes and Tricine buffers showed maximum activity whereas Tris/phosphate or borate failed to show any activity. The enzyme reaction was sensitive to the presence of metal ions like Fe3+, Hg2+, Cu2+, Mo6+ and Zn2+, however, a threefold activation was observed with Fe2+. The metal requirement for CO2 reductase was evident from the observed inhibition by metal chelators like o-phenanthroline, alpha, alpha'-dipyridyl, bathocuproine, 8-hydroxyquinoline etc. Out of these o-phenanthroline was the strongest inhibitor and its concentration for 50% inhibition was 40 microM. The presence of Fe2+ ions in the reaction mixture protected the enzyme from heat denaturation upto 50 degrees C. Maximum enzyme activity was observed at 15 degrees C. The enzyme activity showed a 30-s lag period and the maximum was reached in 90 s. Supplementation of sodium dithionite in the reaction activated enzyme activity threefold, suggesting involvement of dithiol groups in the catalytic activity. There was strong inhibition by -SH inhibitors like 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide and -SH reagents like dithiothreitol, 2-mercaptoethanol and cysteine. Various nucleotide coenzyme tried inhibited the enzyme strongly.

Impact of heavy metal on activity of some microbial enzymes in the riverbed sediments: Ecotoxicological implications in the Ganga River (India).

PubMed

Jaiswal, Deepa; Pandey, Jitendra

2018-04-15

We studied the extracellular enzyme activity (EEA) in the riverbed sediment along a 518km gradient of the Ganga River receiving carbon and nutrient load from varied human sources. Also, we tested, together with substrate-driven stimulation, if the heavy metal accumulated in the sediment inhibits enzyme activities. Because pristine values are not available, we considered Dev Prayag, a least polluted site located 624km upstream to main study stretch, as a reference site. There were distinct increases in enzyme activities in the sediment along the study gradient from Dev Prayag, however, between-site differences were in concordance with sediment carbon(C), nitrogen (N) and phosphorus (P). Fluorescein diacetate hydrolysis (FDAase), β-glucosidase (Glu) and protease activities showed positive correlation with C, N and P while alkaline phosphatase was found negatively correlated with P. Enzyme activities were found negatively correlated with heavy metal, although ecological risk index (E R i ) varied with site and metal species. Dynamic fit curves showed significant positive correlation between heavy metal and microbial metabolic quotient (qCO 2 ) indicating a decrease in microbial activity in response to increasing heavy metal concentrations. This study forms the first report linking microbial enzyme activities to regional scale sediment heavy metal accumulation in the Ganga River, suggests that the microbial enzyme activities in the riverbed sediment were well associated with the proportion of C, N and P and appeared to be a sensitive indicator of C, N and P accumulation in the river. Heavy metal accumulated in the sediment inhibits enzyme activities, although C rich sediment showed relatively low toxicity due probably to reduced bioavailability of the metal. The study has relevance from ecotoxicological as well as from biomonitoring perspectives. Copyright © 2017 Elsevier Inc. All rights reserved.

Enzymatic hydrolysis of steam-pretreated lignocellulosic materials with Trichoderma atroviride enzymes produced in-house

PubMed Central

Kovacs, Krisztina; Macrelli, Stefano; Szakacs, George; Zacchi, Guido

2009-01-01

Background Improvement of the process of cellulase production and development of more efficient lignocellulose-degrading enzymes are necessary in order to reduce the cost of enzymes required in the biomass-to-bioethanol process. Results Lignocellulolytic enzyme complexes were produced by the mutant Trichoderma atroviride TUB F-1663 on three different steam-pretreated lignocellulosic substrates, namely spruce, wheat straw and sugarcane bagasse. Filter paper activities of the enzymes produced on the three materials were very similar, while β-glucosidase and hemicellulase activities were more dependent on the nature of the substrate. Hydrolysis of the enzyme preparations investigated produced similar glucose yields. However, the enzymes produced in-house proved to degrade the xylan and the xylose oligomers less efficiently than a commercial mixture of cellulase and β-glucosidase. Furthermore, accumulation of xylose oligomers was observed when the TUB F-1663 supernatants were applied to xylan-containing substrates, probably due to the low β-xylosidase activity of the enzymes. The efficiency of the enzymes produced in-house was enhanced by supplementation with extra commercial β-glucosidase and β-xylosidase. When the hydrolytic capacities of various mixtures of a commercial cellulase and a T. atroviride supernatant produced in the lab were investigated at the same enzyme loading, the glucose yield appeared to be correlated with the β-glucosidase activity, while the xylose yield seemed to be correlated with the β-xylosidase level in the mixtures. Conclusion Enzyme supernatants produced by the mutant T. atroviride TUB F-1663 on various pretreated lignocellulosic substrates have good filter paper activity values combined with high levels of β-glucosidase activities, leading to cellulose conversion in the enzymatic hydrolysis that is as efficient as with a commercial cellulase mixture. On the other hand, in order to achieve good xylan conversion, the supernatants produced by the mutant have to be supplemented with additional β-xylosidase activity. PMID:19580644

Assembling high activity phosphotriesterase composites using hybrid nanoparticle peptide-DNA scaffolded architectures

NASA Astrophysics Data System (ADS)

Breger, Joyce C.; Buckhout-White, Susan; Walper, Scott A.; Oh, Eunkeu; Susumu, Kimihiro; Ancona, Mario G.; Medintz, Igor L.

2017-06-01

Nanoparticle (NP) display potentially offers a new way to both stabilize and, in many cases, enhance enzyme activity over that seen for native protein in solution. However, the large, globular and sometimes multimeric nature of many enzymes limits their ability to attach directly to the surface of NPs, especially when the latter are colloidally stabilized with bulky PEGylated ligands. Engineering extended protein linkers into the enzymes to achieve direct attachment through the PEG surface often detrimentally alters the enzymes catalytic ability. Here, we demonstrate an alternate, hybrid biomaterials-based approach to achieving directed enzyme assembly on PEGylated NPs. We self-assemble a unique architecture consisting of a central semiconductor quantum dot (QD) scaffold displaying controlled ratios of extended peptide-DNA linkers which penetrate through the PEG surface to directly couple enzymes to the QD surface. As a test case, we utilize phosphotriesterase (PTE), an enzyme of bio-defense interest due to its ability to hydrolyze organophosphate nerve agents. Moreover, this unique approach still allows PTE to maintain enhanced activity while also suggesting the ability of DNA to enhance enzyme activity in and of itself.

Mineralogical impact on long-term patterns of soil nitrogen and phosphorus enzyme activities

NASA Astrophysics Data System (ADS)

Mikutta, Robert; Turner, Stephanie; Meyer-Stüve, Sandra; Guggenberger, Georg; Dohrmann, Reiner; Schippers, Axel

2014-05-01

Soil chronosequences provide a unique opportunity to study microbial activity over time in mineralogical diverse soils of different ages. The main objective of this study was to test the effect of mineralogical properties, nutrient and organic matter availability over whole soil pro-files on the abundance and activity of the microbial communities. We focused on microbio-logical processes involved in nitrogen and phosphorus cycling at the 120,000-year Franz Josef soil chronosequence. Microbial abundances (microbial biomass and total cell counts) and enzyme activities (protease, urease, aminopeptidase, and phosphatase) were determined and related to nutrient contents and mineralogical soil properties. Both, microbial abundances and enzyme activities decreased with soil depth at all sites. In the organic layers, microbial biomass and the activities of N-hydrolyzing enzymes showed their maximum at the intermediate-aged sites, corresponding to a high aboveground biomass. In contrast, the phosphatase activity increased with site age. The activities of N-hydrolyzing enzymes were positively correlated with total carbon and nitrogen contents, whereas the phosphatase activity was negatively correlated with the phosphorus content. In the mineral soil, the enzyme activities were generally low, thus reflecting the presence of strongly sorbing minerals. Sub-strate-normalized enzyme activities correlated negatively to clay content as well as poorly crystalline Al and Fe oxyhydroxides, supporting the view that the evolution of reactive sec-ondary mineral phases alters the activity of the microbial communities by constraining sub-strate availability. Our data suggest a strong mineralogical influence on nutrient cycling par-ticularly in subsoil environments.

A Modular Approach for Interlocking Enzymes in Whatman Paper.

PubMed

Riccardi, Caterina; Kumar, Challa; Kasi, Rajeswari; McCormick, Shelby

2018-06-13

We report a potentially universal approach for enzyme attachment to cellulose that significantly enhances enzyme stability while retaining high activity, and involves no chemical functionalization of cellulose. In our design, bovine serum albumin (BSA) was interlocked in cellulose to form a protein-friendly surface (named BSA-Paper), while also providing COOH and NH2 groups for subsequent attachment of enzymes. The desired enzyme is then mixed with additional BSA and interlocked on BSA-Paper. The 2nd layer dilutes and crosslinks the enzyme for improved stability. Laccase was tested as a model enzyme for interlocking on BSA-Paper, and was found to retain over 100% activity and was 240 times more stable at 25 °C (half life = 180 d) than laccase. This new approach was also tested with a few other enzymes with encouraging results, thus providing a potentially universal method for stabilization of enzymes on cellulose with retention of high activities. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lipophilic flavonoids from Orthosiphon spicatus prevent oxidative inactivation of 15-lipoxygenase.

PubMed

Lyckander, I M; Malterud, K E

1996-04-01

15-Lipoxygenase from soybeans is inactivated by bubbling air through enzyme solutions. This inactivation is prevented by flavonoids from the East Asian medicinal plant Orthosiphon spicatus, previously found to be inhibitors of the enzyme. 5,7,4'-Trimethylapigenin, eupatorin and 5,7,3',4'-tetramethylluteolin show the strongest enzyme-stabilizing effects, decreasing loss of activity by 50% at concentrations of 2.0 +/- 0.04, 2.4 +/- 0.3 and 4.3 +/- 1.1 microM, respectively. There is no significant correlation between enzyme-inhibiting and enzyme-stabilizing effect. The Orthosiphon flavonoids show radical-scavenging activity towards the diphenylpicrylhydrazyl radical. This is correlated to their enzyme-stabilizing effect, but not to their inhibitory activity towards 15-lipoxygenase. When the enzyme is inactivated by air bubbling, a loss of sulfhydryl groups is observed. Sinensetin, a poor stabilizer of the enzyme, shows less efficiency in protecting sulfhydryl groups than tetramethylscutellarein, which stabilizes the enzyme more efficiently. Thus, oxidation of sulfhydryl groups may contribute to the observed air-induced inactivation of 15-lipoxygenase.

Technological advances and applications of hydrolytic enzymes for valorization of lignocellulosic biomass.

PubMed

Manisha; Yadav, Sudesh Kumar

2017-12-01

Hydrolytic enzymes are indispensable tools in the production of various foodstuffs, drugs, and consumables owing to their applications in almost every industrial process nowadays. One of the foremost areas of interest involving the use of hydrolytic enzymes is in the transformation of lignocellulosic biomass into value added products. However, limitations of the processes due to inadequate enzyme activity and stability with a narrow range of pH and temperature optima often limit their effective usage. The innovative technologies, involving manipulation of enzyme activity and stability through mutagenesis, genetic engineering and metagenomics lead to a major leap in all the fields using hydrolytic enzymes. This article provides recent advancement towards the isolation and use of microbes for lignocellulosic biomass utilisation, microbes producing the hydrolytic enzymes, the modern age technologies used to manipulate and enhance the hydrolytic enzyme activity and the applications of such enzymes in value added products development from lignocellulosic biomass. Copyright © 2017 Elsevier Ltd. All rights reserved.

An improved 96-well turbidity assay for T4 lysozyme activity

DTIC Science & Technology

2015-05-13

enzyme present in the reaction, resulting in a measure of activity in Unitsmg1. 10. To improve the reliability of the activity values, perform the...quantification of lysozyme activity with significantly lower enzyme concentrations, and the signal intensity can be enhanced by using greater amounts of... enzyme at the expense of a shorter linear reaction time. Several parameters of the assay are critical for obtaining reproducible activity

Antioxidative capacity and enzyme activity in Haematococcus pluvialis cells exposed to superoxide free radicals

NASA Astrophysics Data System (ADS)

Liu, Jianguo; Zhang, Xiaoli; Sun, Yanhong; Lin, Wei

2010-01-01

The antioxidative capacity of astaxanthin and enzyme activity of reactive oxygen eliminating enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were studied in three cell types of Haematococcus pluvialis exposed to high concentrations of a superoxide anion radical (O{2/-}). The results show that defensive enzymes and astaxanthin-related mechanisms were both active in H. pluvialis during exposure to reactive oxygen species (ROS) such as O{2/-}. Astaxanthin reacted with ROS much faster than did the protective enzymes, and had the strongest antioxidative capacity to protect against lipid peroxidation. The defensive mechanisms varied significantly between the three cell types and were related to the level of astaxanthin that had accumulated in those cells. Astaxanthin-enriched red cells had the strongest antioxidative capacity, followed by brown cells, and astaxanthin-deficient green cells. Although there was no significant increase in expression of protective enzymes, the malondialdehyde (MDA) content in red cells was sustained at a low level because of the antioxidative effect of astaxanthin, which quenched O{2/-} before the protective enzymes could act. In green cells, astaxanthin is very low or absent; therefore, scavenging of ROS is inevitably reliant on antioxidative enzymes. Accordingly, in green cells, these enzymes play the leading role in scavenging ROS, and the expression of these enzymes is rapidly increased to reduce excessive ROS. However, because ROS were constantly increased in this study, the enhance enzyme activity in the green cells was not able to repair the ROS damage, leading to elevated MDA content. Of the four defensive enzymes measured in astaxanthin-deficient green cells, SOD eliminates O{2/-}, POD eliminates H2O2, which is a by-product of SOD activity, and APX and CAT are then initiated to scavenge excessive ROS.

Homology to peptide pattern for annotation of carbohydrate-active enzymes and prediction of function.

PubMed

Busk, P K; Pilgaard, B; Lezyk, M J; Meyer, A S; Lange, L

2017-04-12

Carbohydrate-active enzymes are found in all organisms and participate in key biological processes. These enzymes are classified in 274 families in the CAZy database but the sequence diversity within each family makes it a major task to identify new family members and to provide basis for prediction of enzyme function. A fast and reliable method for de novo annotation of genes encoding carbohydrate-active enzymes is to identify conserved peptides in the curated enzyme families followed by matching of the conserved peptides to the sequence of interest as demonstrated for the glycosyl hydrolase and the lytic polysaccharide monooxygenase families. This approach not only assigns the enzymes to families but also provides functional prediction of the enzymes with high accuracy. We identified conserved peptides for all enzyme families in the CAZy database with Peptide Pattern Recognition. The conserved peptides were matched to protein sequence for de novo annotation and functional prediction of carbohydrate-active enzymes with the Hotpep method. Annotation of protein sequences from 12 bacterial and 16 fungal genomes to families with Hotpep had an accuracy of 0.84 (measured as F1-score) compared to semiautomatic annotation by the CAZy database whereas the dbCAN HMM-based method had an accuracy of 0.77 with optimized parameters. Furthermore, Hotpep provided a functional prediction with 86% accuracy for the annotated genes. Hotpep is available as a stand-alone application for MS Windows. Hotpep is a state-of-the-art method for automatic annotation and functional prediction of carbohydrate-active enzymes.

32 CFR 701.120 - Processing requests that cite or imply PA, Freedom of Information (FOIA), or PA/FOIA.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Privacy Program § 701.120 Processing requests that cite or imply PA, Freedom of Information (FOIA), or PA... maximum release of information allowed under the Acts. (d) Processing time limits. DON activities shall... 32 National Defense 5 2010-07-01 2010-07-01 false Processing requests that cite or imply PA...

Cellulolytic enzymes, nucleic acids encoding them and methods for making and using them

DOEpatents

Gray, Kevin A [San Diego, CA; Zhao, Lishan [Emeryville, CA; Cayouette, Michelle H [San Diego, CA

2012-01-24

The invention provides polypeptides having any cellulolytic activity, e.g., a cellulase activity, a endoglucanase, a cellobiohydrolase, a beta-glucosidase, a xylanase, a mannanse, a .beta.-xylosidase, an arabinofuranosidase, and/or an oligomerase activity, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. In one aspect, the invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. In one aspect, the invention provides polypeptides having an oligomerase activity, e.g., enzymes that convert recalcitrant soluble oligomers to fermentable sugars in the saccharification of biomass. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

Effects of culture conditions on monosaccharide composition of Ganoderma lucidum exopolysaccharide and on activities of related enzymes.

PubMed

Peng, Lin; Qiao, Shuangkui; Xu, Zhenghong; Guan, Feng; Ding, Zhongyang; Gu, Zhenghua; Zhang, Liang; Shi, Guiyang

2015-11-20

We investigated the relationship between monosaccharide composition of Ganoderma lucidum exopolysaccharide (EPS) and activities of EPS synthesis enzymes under various culture temperatures and initial pH values. The mole percentages of three major EPS monosaccharides, glucose, galactose and mannose, varied depending on culture conditions and the resulting EPS displayed differing anti-tumor activities. In nine tested enzymes, higher enzyme activities were correlated with higher temperature and lower initial pH. Altered mole percentages of galactose and mannose under various culture conditions were associated with activities of α-phosphoglucomutase (PGM) and phosphoglucose isomerase (PGI), respectively, and that of mannose was also associated with phosphomannose isomerase (PMI) activity only under various pH. Our findings suggest that mole percentages of G. lucidum EPS monosaccharides can be manipulated by changes of culture conditions that affect enzyme activities, and that novel fermentation strategies based on this approach may enhance production and biological activity of EPS. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

The allosteric activator ATP induces a substrate-dependent alteration of the quaternary structure of a mutant aspartate transcarbamoylase impaired in active site closure.

PubMed Central

Baker, D. P.; Fetler, L.; Vachette, P.; Kantrowitz, E. R.

1996-01-01

Aspartate transcarbamoylase from Escherichia coli shows homotropic cooperativity for aspartate as well as heterotropic regulation by nucleotides. Structurally, it consists of two trimeric catalytic subunits and three dimeric regulatory subunits, each chain being comprised of two domains. Glu-50 and Ser-171 are involved in stabilizing the closed conformation of the catalytic chain. Replacement of Glu-50 or Ser-171 by Ala in the holoenzyme has been shown previously to result in marked decreases in the maximal observed specific activity, homotropic cooperativity, and affinity for aspartate (Dembowski NJ, Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:3716-3723; Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444-1451). We have constructed a double mutant enzyme combining both mutations. The resulting Glu-50/ser-171-->Ala enzyme is 9-fold less active than the Ser-171-->Ala enzyme, 69-fold less active than the Glu-50-->Ala enzyme, and shows 1.3-fold and 1.6-fold increases in the [S]0.5Asp as compared to the Ser-171-->Ala and Glu-50-->Ala enzymes, respectively. However, the double mutant enzyme exhibits some enhancement of homotropic cooperativity with respect to aspartate, relative to the single mutant enzymes. At subsaturating concentrations of aspartate, the Glu-50/Ser-171 -->Ala enzyme is activated less by ATP than either the Glu-50-->Ala or Ser-171-->Ala enzyme, whereas CTP inhibition is intermediate between that of the two single mutants. As opposed to the wild-type enzyme, the Glu-50/Ser-171 -->Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Ser-171-->Ala and Glu-50/Ser-171-->Ala enzymes by solution X-ray scattering indicates that both mutants exist in the same T quaternary structure as the wild-type enzyme in the absence of ligands, and in the same R quaternary structure in the presence of saturating N-(phosphonoacetyl)-L-aspartate. However, saturating concentrations of carbamoyl phosphate and succinate are unable to convert a significant fraction of either mutant enzyme population to the R quaternary structure, as has been observed previously for the Glu-50-->Ala enzyme. The curves for both the Ser-171-->Ala and Glu-50/Ser-171-->Ala enzymes obtained in the presence of substoichiometric amounts of PALA are linear combinations of the two extreme T and R states. The structural consequences of nucleotide binding to these two enzymes were also investigated. Most surprisingly, the direction and amplitude of the effect of ATP upon the double mutant enzyme were shown to vary depending upon the substrate analogue used. PMID:8931146

Static and dynamic half-life and lifetime molecular turnover of enzymes.

PubMed

Miyawaki, Osato; Kanazawa, Tsukasa; Maruyama, Chika; Dozen, Michiko

2017-01-01

The static half-life of an enzyme is the half-life of a free enzyme not working without substrate and the dynamic half-life is that of an active enzyme working with plenty amount of substrate. These two half-lives were measured and compared for glucoamylase (GA) and β-galactosidase (BG). The dynamic half-life was much longer than the static half-life by one to three orders of magnitude for both enzymes. For BG, the half-life of the enzyme physically entrapped in a membrane reactor was also measured. In this case also, the half-life of BG in the membrane reactor was much longer than the free enzyme without substrate. These results suggest the large difference in stabilities between the free enzyme and the enzyme-substrate complex. This may be related to the natural enzyme metabolism. According to the difference in half-life, the lifetime molecular turnover (LMT), which is the number of product molecules produced by a single molecule of enzyme until it loses its activity completely, was much higher by one to four orders of magnitude for the active enzyme than the free enzyme. The concept of LMT, proposed here, will be important in bioreactor operations with or without immobilization. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Immobilization of polygalacturonase from Aspergillus niger onto activated polyethylene and its application in apple juice clarification.

PubMed

Saxena, Shivalika; Shukla, Surendra; Thakur, Akhilesh; Gupta, Reena

2008-03-01

The present work is focused on efficient immobilization of polygalacturonase on polyethylene matrix, followed by its application in apple juice clarification. Immobilization of polygalacturonase on activated polyethylene and its use in apple juice clarification was not reported so far. Aspergillus niger Van Tieghem (MTCC 3323) produced polygalacturonase when grown in modified Riviere's medium containing pectin as single carbon source by fed-batch culture. The enzyme was precipitated with ethanol and purified by gel filtration chromatography (Sephacryl S-100) and immobilized onto glutaraldehyde-activated polyethylene. The method is very simple and time saving for enzyme immobilization. Various characteristics of immobilized enzyme such as optimum reaction temperature and pH, temperature and pH stability, binding kinetics, efficiency of binding, reusability and metal ion effect on immobilized enzymes were evaluated in comparison to the free enzyme. Both the free and immobilized enzyme showed maximum activity at a temperature of 45 degrees C and pH 4.8. Maximum binding efficiency was 38%. The immobilized enzyme was reusable for 3 cycles with 50% loss of activity after the third cycle. Twenty-four U of immobilized enzyme at 45 degrees C and 1 h incubation time increased the transmittance of the apple juice by about 55% at 650 nm. The immobilized enzyme can be of industrial advantage in terms of sturdiness, availability, inertness, low price, reusability and temperature stability.

Biochemical characterization of a thermostable endonuclease V from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5.

PubMed

Wang, Yuxiao; Zhang, Likui; Zhu, Xinyuan; Li, Yuting; Shi, Haoqiang; Oger, Philippe; Yang, Zhihui

2018-05-22

Endonuclease V (Endo V) is an important enzyme for repairing deoxyinosine in DNA. While bacterial and eukaryotic endo Vs have been well studied, knowledge of archaeal endo Vs is limited. Here, we first presented biochemical characterization of a thermostable endonuclease V from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba endo V). The recombinant enzyme possessed optimal endonuclease activity for cleaving deoxyinosine-containing DNA at 70-90 °C. Furthermore, Tba endo V can withstand 100 °C for 120 min without significant loss of its activity, suggesting the enzyme is thermostable. Tba endo V exhibited varying cleavage efficiencies at various pH levels from 6.0 to 11.0, among which an optimal pH for the enzyme was 8.0-9.0. In addition, a divalent metal ion was required for the enzyme to cleave DNA. Mn 2+ and Mg 2+ were optimal ions for the enzyme's activity whereas Ca 2+ , Zn 2+ and Co 2+ inhibited the enzyme activity. Moreover, the enzyme activity was suppressed by high NaCl concentration. Tba endo V bound to all DNA substrates; however, the enzyme exhibited a higher affinity for binding to deoxyinosine-containing DNA than normal DNA. Our work provides valuable information for revealing the role of Tba endo V in the base excision repair pathway for deoxyinosine repair in Thermococcus. Copyright © 2018. Published by Elsevier B.V.

Characterization of the receptor-destroying enzyme activity from infectious salmon anaemia virus.

PubMed

Kristiansen, Marianne; Frøystad, Marianne K; Rishovd, Anne Lise; Gjøen, Tor

2002-11-01

Infectious salmon anaemia virus (ISAV) infects cells via the endocytic pathway and, like many other enveloped viruses, ISAV contains a receptor-destroying enzyme. We have analysed this acetylesterase activity with respect to substrate specificity, enzyme kinetics, inhibitors, temperature and pH stability. The ISAV acetylesterase was inhibited by di-isopropyl fluorophosphate (DFP) in a dose-dependent fashion but not by other known hydrolase inhibitors, suggesting that a serine residue is part of the active site. The pH optimum of the enzyme was in the range 7.5-8.0 and the enzymatic activity was lessened at temperatures above 40 degrees C. The effect of DFP on agglutination/elution of erythrocytes by ISAV demonstrated that the acetylesterase activity is the bona fide receptor-destroying enzyme. A haemadsorption assay was used to analyse whether the esterase was active on the surface of infected cells or not.

Characterization of Purified Staphylococcal Lipase1

PubMed Central

Vadehra, D. V.; Harmon, L. G.

1967-01-01

Purified staphylococcal lipase had an optimal pH of 8.3 for activity at 37 C, and an optimal temperature of 45 C at pH 8.0. During storage, the enzyme lost less than 10% of the activity over a period of 21 days at 4 and -23 C. The enzyme retained 93% of the activity when heated for 30 min at 50 C and was 95% destroyed in 30 min at 70 C. The purified lipase was capable of hydrolyzing a variety of natural fats and oils. However, the enzyme was three times more active on nonhydrogenated soybean oil than on hydrogenated soybean oil with an iodine value of <3.0. The enzyme was also capable of hydrolyzing fatty acids on the α, β, and α′ positions of a synthetic mixed triglyceride. In general, the presence of oxidizing agents increased the activity and the presence of reducing agents decreased the activity of the lipase enzyme. PMID:6035042

Release of enzymes from cells: transport and distribution within the extracellular space.

PubMed

Mattenheimer, H; Friedel, R

1977-01-01

The distribution in the extracellular space of enzymes released from organ cells was investigated using three models: (1) comparison of enzyme activities in blood plasma and lymph of the ductus thoracicus (dog) and plasma and intestinal lymph (rat); (2) i.v. injection of heterologous, homologous and autologous enzymes in order to increase acutely the activities and to measure the rate constants for the distribution and elimination of the enzymes (rat); or (3) plasmapheresis in order to create an enzyme activity gradient from the interstitial space and to determine the rate constants for the reestablishment of the equilibrium between the extra and intravascular compartments (rat). The results suggest that the enzymes are mainly released into the interstitial fluid and transported via the lymph into the intravascular compartment. From there the enzymes diffuse back into the interstitial compartment and are eliminated by a yet unknown mechanism. Transport of enzymes across the capillary membranes in both directions depends on (1) the permeability of the capillary membranes, which varies from region to region and (2) the molecular seizes of the enzymes.

Elevation of Glucose 6-Phosphate Dehydrogenase Activity Induced by Amplified Insulin Response in Low Glutathione Levels in Rat Liver

PubMed Central

Taniguchi, Misako; Mori, Nobuko; Iramina, Chizuru

2016-01-01

Weanling male Wistar rats were fed on a 10% soybean protein isolate (SPI) diet for 3 weeks with or without supplementing 0.3% sulfur-containing amino acids (SAA; methionine or cystine) to examine relationship between glutathione (GSH) levels and activities of NADPH-producing enzymes, glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME), in the liver. Of rats on the 10% SPI diet, GSH levels were lower and the enzyme activities were higher than of those fed on an SAA-supplemented diet. Despite the lower GSH level, γ-glutamylcysteine synthetase (γ-GCS) activity was higher in the 10% SPI group than other groups. Examination of mRNAs of G6PD and ME suggested that the GSH-suppressing effect on enzyme induction occurred prior to and/or at transcriptional levels. Gel electrophoresis of G6PD indicated that low GSH status caused a decrease in reduced form and an increase in oxidized form of the enzyme, suggesting an accelerated turnover rate of the enzyme. In primary cultured hepatocytes, insulin response to induce G6PD activity was augmented in low GSH levels manipulated in the presence of buthionine sulfoximine. These findings indicated that elevation of the G6PD activity in low GSH levels was caused by amplified insulin response for expression of the enzyme and accelerated turnover rate of the enzyme molecule. PMID:27597985

Statistical Optimization of Fibrinolytic Enzyme Production Using Agroresidues by Bacillus cereus IND1 and Its Thrombolytic Activity In Vitro

PubMed Central

Prakash Vincent, Samuel Gnana

2014-01-01

A potent fibrinolytic enzyme-producing Bacillus cereus IND1 was isolated from the Indian food, rice. Solid-state fermentation was carried out using agroresidues for the production of fibrinolytic enzyme. Among the substrates, wheat bran supported more enzyme production and has been used for the optimized enzyme production by statistical approach. Two-level full-factorial design demonstrated that moisture, supplementation of beef extract, and sodium dihydrogen phosphate have significantly influenced enzyme production (P < 0.05). A central composite design resulted in the production of 3699 U/mL of enzyme in the presence of 0.3% (w/w) beef extract and 0.05% (w/w) sodium dihydrogen phosphate, at 100% (v/w) moisture after 72 h of fermentation. The enzyme production increased fourfold compared to the original medium. This enzyme was purified to homogeneity by ammonium sulfate precipitation, diethylaminoethyl-cellulose ion-exchange chromatography, Sephadex G-75 gel filtration chromatography, and casein-agarose affinity chromatography and had an apparent molecular mass of 29.5 kDa. The optimum pH and temperature for the activity of fibrinolytic enzyme were found to be 8.0 and 60°C, respectively. This enzyme was highly stable at wide pH range (7.0–9.0) and showed 27% ± 6% enzyme activity after initial denaturation at 60°C for 1 h. In vitro assays revealed that the enzyme could activate plasminogen and significantly degraded the fibrin net of blood clot, which suggests its potential as an effective thrombolytic agent. PMID:25003130

Analysis of the activation of acetylcholinesterase by carbon nanoparticles using a monolithic immobilized enzyme microreactor: role of the water molecules in the active site gorge.

PubMed

Ibrahim, Firas; Andre, Claire; Iutzeler, Anne; Guillaume, Yves Claude

2013-10-01

A biochromatographic system was used to study the direct effect of carbon nanoparticles (CNPs) on the acetylcholinesterase (AChE) activity. The AChE enzyme was covalently immobilized on a monolithic CIM-disk via its NH2 residues. Our results showed an increase in the AChE activity in presence of CNPs. The catalytic constant (k(cat)) was increased while the Michaelis constant (K(m)) was slightly decreased. This indicated an increase in the enzyme efficiency with increase of the substrate affinity to the active site. The thermodynamic data of the activation mechanism of the enzyme, i.e. ΔH* and ΔS*, showed no change in the substrate interaction mechanism with the anionic binding site. The increase of the enthalpy (ΔH*) and the entropy (ΔS*) with decrease in the free energy of activation (Ea) was related to structural conformation change in the active site gorge. This affected the stability of water molecules in the active site gorge and facilitated water displacement by substrate for entering to the active site of the enzyme.

Functional screening of pharmacological chaperones via restoration of enzyme activity upon denaturation.

PubMed

Shanmuganathan, Meera; Britz-McKibbin, Philip

2012-10-02

Pharmacological chaperones (PCs) are small molecules that stabilize and promote protein folding. Enzyme inhibition is widely used for PC selection; however, it does not accurately reflect chaperone activity. We introduce a functional assay for characterization of PCs based on their capacity to restore enzyme activity that is abolished upon chemical denaturation. Dose-dependent activity curves were performed as a function of urea to assess the chaperone potency of various ligands to β-glucocerebrosidase as a model system. Restoration of enzyme activity upon denaturation allows direct screening of PCs for treatment of genetic disorders associated with protein deficiency, such as Gaucher disease.

Tetrachloro-p-benzoquinone induces hepatic oxidative damage and inflammatory response, but not apoptosis in mouse: The prevention of curcumin

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

Xu, Demei; Hu, Lihua; Su, Chuanyang

2014-10-15

This study investigated the protective effects of curcumin on tetrachloro-p-benzoquinone (TCBQ)-induced hepatotoxicity in mice. TCBQ-treatment causes significant liver injury (the elevation of serum AST and ALT activities, histopathological changes in liver section including centrilobular necrosis and inflammatory cells), oxidative stress (the elevation of TBAR level and the inhibition of SOD and catalase activities) and inflammation (up-regulation of iNOS, COX-2, IL-1β, IL-6, TNF-α and NF-κB). However, these changes were alleviated upon pretreatment with curcumin. Interestingly, TCBQ has no effect on caspase family genes or B-cell lymphoma 2 (Bcl-2)/Bcl-2 associated X (Bax) protein expressions, which implied that TCBQ-induced hepatotoxicity is independent ofmore » apoptosis. Moreover, curcumin was shown to induce phase II detoxifying/antioxidant enzymes HO-1 and NQO1 through the activation of nuclear factor erythroid-derived 2-like 2 (Nrf2). In summary, the protective mechanisms of curcumin against TCBQ-induced hepatoxicity may be related to the attenuation of oxidative stress, along with the inhibition of inflammatory response via the activation of Nrf2 signaling. - Highlights: • TCBQ-intoxication significantly increased AST and ALT activities. • TCBQ-intoxication induced oxidative stress in mice liver. • TCBQ-intoxication induced inflammatory response in mice liver. • TCBQ-intoxication induced hepatotoxicity is independent of apoptosis. • Curcumin relieved TCBQ-induced liver damage remarkably.« less

PhAP protease from Pseudoalteromonas haloplanktis TAC125: Gene cloning, recombinant production in E. coli and enzyme characterization

NASA Astrophysics Data System (ADS)

de Pascale, D.; Giuliani, M.; De Santi, C.; Bergamasco, N.; Amoresano, A.; Carpentieri, A.; Parrilli, E.; Tutino, M. L.

2010-08-01

Cold-adapted proteases have been found to be the dominant activity throughout the cold marine environment, indicating their importance in bacterial acquisition of nitrogen-rich complex organic compounds. However, few extracellular proteases from marine organisms have been characterized so far, and the mechanisms that enable their activity in situ are still largely unknown. Aside from their ecological importance and use as model enzyme for structure/function investigations, cold-active proteolytic enzymes offer great potential for biotechnological applications. Our studies on cold adapted proteases were performed on exo-enzyme produced by the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125. By applying a proteomic approach, we identified several proteolytic activities from its culture supernatant. PhAP protease was selected for further investigations. The encoding gene was cloned and the protein was recombinantly produced in E. coli cells. The homogeneous product was biochemically characterised and it turned out that the enzyme is a Zn-dependent aminopeptidase, with an activity dependence from assay temperature typical of psychrophilic enzymes.

Antioxidant enzyme activities are affected by salt content and temperature and influence muscle lipid oxidation during dry-salted bacon processing.

PubMed

Jin, Guofeng; He, Lichao; Yu, Xiang; Zhang, Jianhao; Ma, Meihu

2013-12-01

Fresh pork bacon belly was used as material and manufactured into dry-salted bacon through salting and drying-ripening. During processing both oxidative stability and antioxidant enzyme stability were evaluated by assessing peroxide value (PV), thiobarbituric acid reactive substances (TBARS) and activities of catalase, glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), and their correlations were also analysed. The results showed that all antioxidant enzyme activities decreased (p<0.05) until the end of process; GSH-Px was the most unstable one followed by catalase. Antioxidant enzyme activities were negatively correlated with TBARS (p<0.05), but the correlations were decreased with increasing process temperature. Salt showed inhibitory effect on all antioxidant enzyme activities and was concentration dependent. These results indicated that when process temperature and salt content were low at the same time during dry-salted bacon processing, antioxidant enzymes could effectively control lipid oxidation. Copyright © 2013 Elsevier Ltd. All rights reserved.

'Enzyme Test Bench': A biochemical application of the multi-rate modeling

NASA Astrophysics Data System (ADS)

Rachinskiy, K.; Schultze, H.; Boy, M.; Büchs, J.

2008-11-01

In the expanding field of 'white biotechnology' enzymes are frequently applied to catalyze the biochemical reaction from a resource material to a valuable product. Evolutionary designed to catalyze the metabolism in any life form, they selectively accelerate complex reactions under physiological conditions. Modern techniques, such as directed evolution, have been developed to satisfy the increasing demand on enzymes. Applying these techniques together with rational protein design, we aim at improving of enzymes' activity, selectivity and stability. To tap the full potential of these techniques, it is essential to combine them with adequate screening methods. Nowadays a great number of high throughput colorimetric and fluorescent enzyme assays are applied to measure the initial enzyme activity with high throughput. However, the prediction of enzyme long term stability within short experiments is still a challenge. A new high throughput technique for enzyme characterization with specific attention to the long term stability, called 'Enzyme Test Bench', is presented. The concept of the Enzyme Test Bench consists of short term enzyme tests conducted under partly extreme conditions to predict the enzyme long term stability under moderate conditions. The technique is based on the mathematical modeling of temperature dependent enzyme activation and deactivation. Adapting the temperature profiles in sequential experiments by optimum non-linear experimental design, the long term deactivation effects can be purposefully accelerated and detected within hours. During the experiment the enzyme activity is measured online to estimate the model parameters from the obtained data. Thus, the enzyme activity and long term stability can be calculated as a function of temperature. The results of the characterization, based on micro liter format experiments of hours, are in good agreement with the results of long term experiments in 1L format. Thus, the new technique allows for both: the enzyme screening with regard to the long term stability and the choice of the optimal process temperature. The presented article gives a successful example for the application of multi-rate modeling, experimental design and parameter estimation within biochemical engineering. At the same time, it shows the limitations of the methods at the state of the art and addresses the current problems to the applied mathematics community.

A Simple and Accurate Method for Measuring Enzyme Activity.

ERIC Educational Resources Information Center

Yip, Din-Yan

1997-01-01

Presents methods commonly used for investigating enzyme activity using catalase and presents a new method for measuring catalase activity that is more reliable and accurate. Provides results that are readily reproduced and quantified. Can also be used for investigations of enzyme properties such as the effects of temperature, pH, inhibitors,…

Microbial Interactions Associated with Biofilms Attached to Trichodesmium spp. and Detrital Particles in the Ocean

DTIC Science & Technology

2010-06-01

AHLs enhanced certain organic-matter degrading hydrolytic enzyme activities. My results suggest that AHL-QS is a factor regulating...biogeochemically relevant enzyme activities on sinking POC and within the biofilms attached to Trichodesmium colonies and thereby may impact the timing and... Enzyme activity assays ......................................................................................................................232 RESULTS

Activities of Tricarboxylic Acid Cycle Enzymes, Glyoxylate Cycle Enzymes, and Fructose Diphosphatase in Bakers' Yeast During Adaptation to Acetate Oxidation

PubMed Central

Gosling, J. P.; Duggan, P. F.

1971-01-01

Bakers' yeast oxidizes acetate at a high rate only after an adaptation period during which the capacity of the glyoxylate cycle is found to increase. There was apparently no necessity for the activity of acetyl-coenzyme A synthetase, the capacity of the tricarboxylic acid cycle, or the concentrations of the cytochromes to increase for this adaptation to occur. Elevation of fructose 1,6 diphosphatase occurred only when acetate oxidation was nearly maximal. Cycloheximide almost completely inhibited adaptation as well as increases in the activities of isocitrate lyase and aconitate hydratase, the only enzymes assayed. p-Fluorophenylalanine was partially effective and chloramphenicol did not inhibit at all. The presence of ammonium, which considerably delayed adaptation of the yeast to acetate oxidation, inhibited the increases in the activities of the glyoxylate cycle enzymes to different degrees, demonstrating noncoordinate control of these enzymes. Under the various conditions, the only enzyme activity increase consistently related to the rising oxygen uptake rate was that of isocitrate lyase which apparently limited the activity of the cycle. PMID:5557595

Reduction of nuclear encoded enzymes of mitochondrial energy metabolism in cells devoid of mitochondrial DNA

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

Mueller, Edith E., E-mail: ed.mueller@salk.at; Mayr, Johannes A., E-mail: h.mayr@salk.at; Zimmermann, Franz A., E-mail: f.zimmermann@salk.at

2012-01-20

Highlights: Black-Right-Pointing-Pointer We examined OXPHOS and citrate synthase enzyme activities in HEK293 cells devoid of mtDNA. Black-Right-Pointing-Pointer Enzymes partially encoded by mtDNA show reduced activities. Black-Right-Pointing-Pointer Also the entirely nuclear encoded complex II and citrate synthase exhibit reduced activities. Black-Right-Pointing-Pointer Loss of mtDNA induces a feedback mechanism that downregulates complex II and citrate synthase. -- Abstract: Mitochondrial DNA (mtDNA) depletion syndromes are generally associated with reduced activities of oxidative phosphorylation (OXPHOS) enzymes that contain subunits encoded by mtDNA. Conversely, entirely nuclear encoded mitochondrial enzymes in these syndromes, such as the tricarboxylic acid cycle enzyme citrate synthase (CS) and OXPHOS complexmore » II, usually exhibit normal or compensatory enhanced activities. Here we report that a human cell line devoid of mtDNA (HEK293 {rho}{sup 0} cells) has diminished activities of both complex II and CS. This finding indicates the existence of a feedback mechanism in {rho}{sup 0} cells that downregulates the expression of entirely nuclear encoded components of mitochondrial energy metabolism.« less

Development of radiometric assays for quantification of enzyme activities of the key enzymes of thyroid hormones metabolism.

PubMed

Pavelka, S

2014-01-01

We newly elaborated and adapted several radiometric enzyme assays for the determination of activities of the key enzymes engaged in the biosynthesis (thyroid peroxidase, TPO) and metabolic transformations (conjugating enzymes and iodothyronine deiodinases, IDs) of thyroid hormones (THs) in the thyroid gland and in peripheral tissues, especially in white adipose tissue (WAT). We also elaborated novel, reliable radiometric methods for extremely sensitive determination of enzyme activities of IDs of types 1, 2 and 3 in microsomal fractions of different rat and human tissues, as well as in homogenates of cultured mammalian cells. The use of optimized TLC separation of radioactive products from the unconsumed substrates and film-less autoradiography of radiochromatograms, taking advantage of storage phosphor screens, enabled us to determine IDs enzyme activities as low as 10(-18) katals. In studies of the interaction of fluoxetine (Fluox) with the metabolism of THs, we applied adapted radiometric enzyme assays for iodothyronine sulfotransferases (ST) and uridine 5'-diphospho-glucuronyltransferase (UDP-GT). Fluox is the most frequently used representative of a new group of non-tricyclic antidepressant drugs--selective serotonin re-uptake inhibitors. We used the elaborated assays for quantification the effects of Fluox and for the assessment of the degree of potential induction of rat liver ST and/or UDP-GT enzyme activities by Fluox alone or in combination with T(3). Furthermore, we studied possible changes in IDs activities in murine adipose tissue under the conditions that promoted either tissue hypertrophy (obesogenic treatment) or involution (caloric restriction), and in response to leptin, using our newly developed radiometric enzyme assays for IDs. Our results suggest that deiodinase D1 has a functional role in WAT, with D1 possibly being involved in the control of adipose tissue metabolism and/or accumulation of the tissue. Significant positive correlation between specific enzyme activity of D1 in WAT and plasma leptin levels was found. The newly developed and adapted radiometric enzyme assays proved to be very useful tools for studies of factors modulating THs metabolism, not only in model animals but also in clinical studies of human obesity.

Screening and Characterization of Polygalacturonase as Potential Enzyme for Keprok Garut Orange (Citrus nobilis var. chrysocarpa) Juice Clarification

NASA Astrophysics Data System (ADS)

Widowati, E.; Utami, R.; Kalistyatika, K.

2017-11-01

Use of thermostable enzyme from bacilli for industrial application is significant. This research aimed to isolate thermophilic pectinolytic bacteria from orange peel and vegetable waste which produced thermostable polygalacturonase, to investigate the polygalacturonase ability in clarifying keprok Garut orange juice, and to characterize polygalacturonase based on pH optimum, temperature optimum, enzyme stability, enzyme kinetics KM, and Vmax. Obtained, 14 isolates that further selected to 4 best isolates based on highest polygalacturonase activity and keprok Garut orange juice clarification ability. Four selected enzyme isolates were AR 2, AR 4, KK 4, and KK 5 had ability to increase juice transmittance, decrease juice viscosity and also reduce total soluble solid. Furthermore 4 selected isolates were partially purified by ammonium sulphate precipitation and dialysis method. Four partially purified enzymes were known that enzyme character of AR 2 optimum at pH 6; AR 4 optimum at pH 5.5; KK 4 optimum at pH 6; and KK 5 optimum at pH 4.5. Four enzymes were optimum at temperature 60°C thus stable at temperature 50-60°C, this characteristic indicate that enzymes were thermostable. AR 2 showed active activity stable at pH 4-7; AR 4 showed active activity stable at pH 6-7; KK 4 showed active activity stable at pH 4-6; however KK 5 stable at pH 4-5. Enzyme AR 2 and KK 4 was getting inactive at pH 11, thus AR 4 and KK 5 inactive at pH 12. KM value of AR 2, AR 4, KK 4, and KK 5 was 0.0959; 0.0974; 0.0966; and 0.178 mg/ml respectively. Vmax of AR 2, AR 4, KK 4, and KK 5 was 0.0203; 0.0202; 0.0185; and 0.0229 U/ml respectively. Enzyme AR 2 was the most compatible enzyme to be applied in keprok Garut orange juice clarification for it had the lowest KM value.

The 46 kDa dimeric protein from Variovorax paradoxus shows faster methotrexate degrading activity in its nanoform compare to the native enzyme.

PubMed

Bayineni, Venkata Krishna; Venkatesh, Krishna; Sahu, Chandan Kumar; Kadeppagari, Ravi-Kumar

2016-04-01

Methotrexate degrading enzymes are required to overcome the toxicity of the methotrexate while treating the cancer. The enzyme from Variovorax paradoxus converts the methotrexate in to non toxic products. Methotrexate degrading enzyme from V. paradoxus is a dimeric protein with a molecular mass of 46 kDa and it acts on casein and gelatin. This enzyme is optimally active at pH 7.5 and 40°C and nanoparticles of this enzyme were prepared by desolvation-crosslinking method. Enzyme nanoparticles could degrade methotrexate faster than the native enzyme and they show lower Km compare to the native enzyme. Enzyme nanoparticles show better thermostability and they were stable for much longer time in the serum compare to the native enzyme. Enzyme nanoparticles show better functionality than the native enzyme while clearing the methotrexate added to the serum suggesting their advantage over the native enzyme for the therapeutic and biotechnological applications. Copyright © 2016 Elsevier Inc. All rights reserved.