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.

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

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…

Functional cooperation between exonucleases and endonucleases—basis for the evolution of restriction enzymes

PubMed Central

Raghavendra, Nidhanapathi K.; Rao, Desirazu N.

2003-01-01

Many types of restriction enzymes cleave DNA away from their recognition site. Using the type III restriction enzyme, EcoP15I, which cleaves DNA 25–27 bp away from its recognition site, we provide evidence to show that an intact recognition site on the cleaved DNA sequesters the restriction enzyme and decreases the effective concentration of the enzyme. EcoP15I restriction enzyme is shown here to perform only a single round of DNA cleavage. Significantly, we show that an exonuclease activity is essential for EcoP15I restriction enzyme to perform multiple rounds of DNA cleavage. This observation may hold true for all restriction enzymes cleaving DNA sufficiently far away from their recognition site. Our results highlight the importance of functional cooperation in the modulation of enzyme activity. Based on results presented here and other data on well-characterised restriction enzymes, a functional evolutionary hierarchy of restriction enzymes is discussed. PMID:12655005

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

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

Expression, function and regulation of mouse cytochrome P450 enzymes: comparison with human P450 enzymes.

PubMed

Hrycay, E G; Bandiera, S M

2009-12-01

The present review focuses on the expression, function and regulation of mouse cytochrome P450 (Cyp) enzymes. Information compiled for mouse Cyp enzymes is compared with data collected for human CYP enzymes. To date, approximately 40 pairs of orthologous mouse-human CYP genes have been identified that encode enzymes performing similar metabolic functions. Recent knowledge concerning the tissue expression of mouse Cyp enzymes from families 1 to 51 is summarized. The catalytic activities of microsomal, mitochondrial and recombinant mouse Cyp enzymes are discussed and their involvement in the metabolism of exogenous and endogenous compounds is highlighted. The role of nuclear receptors, such as the aryl hydrocarbon receptor, constitutive androstane receptor and pregnane X receptor, in regulating the expression of mouse Cyp enzymes is examined. Targeted disruption of selected Cyp genes has generated numerous Cyp null mouse lines used to decipher the role of Cyp enzymes in metabolic, toxicological and biological processes. In conclusion, the laboratory mouse is an indispensable model for exploring human CYP-mediated activities.

DNA in a Tunnel: A Comfy Spot for Recognition - or -The Structure of BsoBI Complexed with DNA. What can we Learn about Function via Structure Determination and how can this be Applied to Bone or Muscle Biology?

NASA Technical Reports Server (NTRS)

vanderWoerd, Mark

2004-01-01

The structure and function of a biologically active molecule are related. To understand its function, it is necessary (but not always sufficient) to know the structure of the molecule. There are many ways of relating the molecular function with the structure. Mutation analysis can identify pertinent amino acids of an enzyme, or alternatively structure comparison of the of two similar molecules with different function may lead to understanding which parts are responsible for a functional aspect, or a series of "structural cartoons" - enzyme structure, enzyme plus substrate, enzyme with transition state analog, and enzyme with product - may give insight in the function of a molecule. As an example we will discuss the structure and function of the restriction enzyme BsoBI from Bacillus stearothemzophilus in complex with its cognate DNA. The enzyme forms a unique complex with DNA in that it completely encircles the DNA. The structure reveals the enzyme-DNA contacts, how the DNA is distorted compared with the canonical forms, and elegantly shows how two distinct DNA sequences can be recognized with the same efficiency. Based on the structure we may also propose a hypothesis how the enzymatic mechanism works. The knowledge gained thru studies such as this one can be used to alter the function by changing the molecular structure. Usually this is done by design of inhibitors specifically active against and fitting into an active site of the enzyme of choice. In the case of BsoBI one of the objectives of the study was to alter the enzyme specificity. In bone biology there are many candidates available for molecular study in order to explain, alter, or (temporarily) suspend activity. For example, the understanding of a pathway that negatively regulates bone formation may be a good target for drug design to stimulate bone formation and have good potential as the basis for new countermeasures against bone loss. In principle the same approach may aid muscle atrophy, radiation damage, immune response changes and other risks identified for long-duration Space travel.

Disconnect of microbial structure and function: enzyme activities and bacterial communities in nascent stream corridors.

PubMed

Frossard, Aline; Gerull, Linda; Mutz, Michael; Gessner, Mark O

2012-03-01

A fundamental issue in microbial and general ecology is the question to what extent environmental conditions dictate the structure of communities and the linkages with functional properties of ecosystems (that is, ecosystem function). We approached this question by taking advantage of environmental gradients established in soil and sediments of small stream corridors in a recently created, early successional catchment. Specifically, we determined spatial and temporal patterns of bacterial community structure and their linkages with potential microbial enzyme activities along the hydrological flow paths of the catchment. Soil and sediments were sampled in a total of 15 sites on four occasions spread throughout a year. Denaturing gradient gel electrophoresis (DGGE) was used to characterize bacterial communities, and substrate analogs linked to fluorescent molecules served to track 10 different enzymes as specific measures of ecosystem function. Potential enzyme activities varied little among sites, despite contrasting environmental conditions, especially in terms of water availability. Temporal changes, in contrast, were pronounced and remarkably variable among the enzymes tested. This suggests much greater importance of temporal dynamics than spatial heterogeneity in affecting specific ecosystem functions. Most strikingly, bacterial community structure revealed neither temporal nor spatial patterns. The resulting disconnect between bacterial community structure and potential enzyme activities indicates high functional redundancy within microbial communities even in the physically and biologically simplified stream corridors of early successional landscapes.

Horseradish peroxidase-nanoclay hybrid particles of high functional and colloidal stability.

PubMed

Pavlovic, Marko; Rouster, Paul; Somosi, Zoltan; Szilagyi, Istvan

2018-08-15

Highly stable dispersions of enzyme-clay nanohybrids of excellent horseradish peroxidase activity were developed. Layered double hydroxide nanoclay was synthesized and functionalized with heparin polyelectrolyte to immobilize the horseradish peroxidase enzyme. The formation of a saturated heparin layer on the platelets led to charge inversion of the positively charged bare nanoclay and to highly stable aqueous dispersions. Great affinity of the enzyme to the surface modified platelets resulted in strong horseradish peroxidase adsorption through electrostatic and hydrophobic interactions as well as hydrogen bonding network and prevented enzyme leakage from the obtained material. The enzyme kept its functional integrity upon immobilization and showed excellent activity in decomposition of hydrogen peroxide and oxidation of an aromatic compound in the test reactions. In addition, remarkable long term functional stability of the enzyme-nanoclay hybrid was observed making the developed colloidal system a promising antioxidant candidate in biomedical treatments and industrial processes. Copyright © 2018 Elsevier Inc. All rights reserved.

Evolutionarily conserved linkage between enzyme fold, flexibility, and catalysis.

PubMed

Ramanathan, Arvind; Agarwal, Pratul K

2011-11-01

Proteins are intrinsically flexible molecules. The role of internal motions in a protein's designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function. Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease) that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 Å away) are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity) that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme-substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme-substrate interactions, thereby impacting the mechanism of catalyzed chemistry. These results have implications for understanding the mechanism of allostery, and for protein engineering and drug design.

Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains.

PubMed

Ishikawa, Fumihiro; Miyamoto, Kengo; Konno, Sho; Kasai, Shota; Kakeya, Hideaki

2015-12-18

A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique and adenylating enzymes together using a combination of active site-directed probes for the A domains in NRPSs should accelerate both the functional characterization and manipulation of the A domains in NRPSs.

Genetics Home Reference: ALG1-congenital disorder of glycosylation

MedlinePlus

... and lipids so they can fully perform their functions. The enzyme produced from the ALG1 gene transfers a simple ... abnormal enzyme with reduced activity. The poorly functioning enzyme cannot add ... many organs and tissues. Learn more about ...

Scaffolding Function of PI3Kgamma Emerges from Enzyme's Shadow.

PubMed

Mohan, Maradumane L; Naga Prasad, Sathyamangla V

2017-03-24

Traditionally, an enzyme is a protein that mediates biochemical action by binding to the substrate and by catalyzing the reaction that translates external cues into biological responses. Sequential dissemination of information from one enzyme to another facilitates signal transduction in biological systems providing for feed-forward and feed-back mechanisms. Given this viewpoint, an enzyme without its catalytic activity is generally considered to be an inert organizational protein without catalytic function and has classically been termed as pseudo-enzymes. However, pseudo-enzymes still have biological function albeit non-enzymatic like serving as a chaperone protein or an interactive platform between proteins. In this regard, majority of the studies have focused solely on the catalytic role of enzymes in biological function, overlooking the potentially critical non-enzymatic roles. Increasing evidence from recent studies implicate that the scaffolding function of enzymes could be as important in signal transduction as its catalytic activity, which is an antithesis to the definition of enzymes. Recognition of non-enzymatic functions could be critical, as these unappreciated roles may hold clues to the ineffectiveness of kinase inhibitors in pathology, which is characteristically associated with increased enzyme expression. Using an established enzyme phosphoinositide 3-kinase γ, we discuss the insights obtained from the scaffolding function and how this non-canonical role could contribute to/alter the outcomes in pathology like cancer and heart failure. Also, we hope that with this review, we provide a forum and a starting point to discuss the idea that catalytic function alone may not account for all the actions observed with increased expression of the enzyme. Copyright © 2017 Elsevier Ltd. All rights reserved.

Can enzyme engineering benefit from the modulation of protein motions? Lessons learned from NMR relaxation dispersion experiments.

PubMed

Doucet, Nicolas

2011-04-01

Despite impressive progress in protein engineering and design, our ability to create new and efficient enzyme activities remains a laborious and time-consuming endeavor. In the past few years, intricate combinations of rational mutagenesis, directed evolution and computational methods have paved the way to exciting engineering examples and are now offering a new perspective on the structural requirements of enzyme activity. However, these structure-function analyses are usually guided by the time-averaged static models offered by enzyme crystal structures, which often fail to describe the functionally relevant 'invisible states' adopted by proteins in space and time. To alleviate such limitations, NMR relaxation dispersion experiments coupled to mutagenesis studies have recently been applied to the study of enzyme catalysis, effectively complementing 'structure-function' analyses with 'flexibility-function' investigations. In addition to offering quantitative, site-specific information to help characterize residue motion, these NMR methods are now being applied to enzyme engineering purposes, providing a powerful tool to help characterize the effects of controlling long-range networks of flexible residues affecting enzyme function. Recent advancements in this emerging field are presented here, with particular attention to mutagenesis reports highlighting the relevance of NMR relaxation dispersion tools in enzyme engineering.

Functional Metagenomics: Construction and High-Throughput Screening of Fosmid Libraries for Discovery of Novel Carbohydrate-Active Enzymes.

PubMed

Ufarté, Lisa; Bozonnet, Sophie; Laville, Elisabeth; Cecchini, Davide A; Pizzut-Serin, Sandra; Jacquiod, Samuel; Demanèche, Sandrine; Simonet, Pascal; Franqueville, Laure; Veronese, Gabrielle Potocki

2016-01-01

Activity-based metagenomics is one of the most efficient approaches to boost the discovery of novel biocatalysts from the huge reservoir of uncultivated bacteria. In this chapter, we describe a highly generic procedure of metagenomic library construction and high-throughput screening for carbohydrate-active enzymes. Applicable to any bacterial ecosystem, it enables the swift identification of functional enzymes that are highly efficient, alone or acting in synergy, to break down polysaccharides and oligosaccharides.

Metatranscriptomics Reveals the Functions and Enzyme Profiles of the Microbial Community in Chinese Nong-Flavor Liquor Starter

PubMed Central

Huang, Yuhong; Yi, Zhuolin; Jin, Yanling; Huang, Mengjun; He, Kaize; Liu, Dayu; Luo, Huibo; Zhao, Dong; He, Hui; Fang, Yang; Zhao, Hai

2017-01-01

Chinese liquor is one of the world's best-known distilled spirits and is the largest spirit category by sales. The unique and traditional solid-state fermentation technology used to produce Chinese liquor has been in continuous use for several thousand years. The diverse and dynamic microbial community in a liquor starter is the main contributor to liquor brewing. However, little is known about the ecological distribution and functional importance of these community members. In this study, metatranscriptomics was used to comprehensively explore the active microbial community members and key transcripts with significant functions in the liquor starter production process. Fungi were found to be the most abundant and active community members. A total of 932 carbohydrate-active enzymes, including highly expressed auxiliary activity family 9 and 10 proteins, were identified at 62°C under aerobic conditions. Some potential thermostable enzymes were identified at 50, 62, and 25°C (mature stage). Increased content and overexpressed key enzymes involved in glycolysis and starch, pyruvate and ethanol metabolism were detected at 50 and 62°C. The key enzymes of the citrate cycle were up-regulated at 62°C, and their abundant derivatives are crucial for flavor generation. Here, the metabolism and functional enzymes of the active microbial communities in NF liquor starter were studied, which could pave the way to initiate improvements in liquor quality and to discover microbes that produce novel enzymes or high-value added products. PMID:28955318

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

Ramanathan, Arvind; Agarwal, Pratul K

Proteins are intrinsically flexible molecules. The role of internal motions in a protein's designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function.more » Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease) that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 away) are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity) that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme substrate interactions, thereby impacting the mechanism of catalyzed chemistry. These results have implications for understanding the mechanism of allostery, and for protein engineering and drug design.« less

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

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

Identification of Functionally Related Enzymes by Learning-to-Rank Methods.

PubMed

Stock, Michiel; Fober, Thomas; Hüllermeier, Eyke; Glinca, Serghei; Klebe, Gerhard; Pahikkala, Tapio; Airola, Antti; De Baets, Bernard; Waegeman, Willem

2014-01-01

Enzyme sequences and structures are routinely used in the biological sciences as queries to search for functionally related enzymes in online databases. To this end, one usually departs from some notion of similarity, comparing two enzymes by looking for correspondences in their sequences, structures or surfaces. For a given query, the search operation results in a ranking of the enzymes in the database, from very similar to dissimilar enzymes, while information about the biological function of annotated database enzymes is ignored. In this work, we show that rankings of that kind can be substantially improved by applying kernel-based learning algorithms. This approach enables the detection of statistical dependencies between similarities of the active cleft and the biological function of annotated enzymes. This is in contrast to search-based approaches, which do not take annotated training data into account. Similarity measures based on the active cleft are known to outperform sequence-based or structure-based measures under certain conditions. We consider the Enzyme Commission (EC) classification hierarchy for obtaining annotated enzymes during the training phase. The results of a set of sizeable experiments indicate a consistent and significant improvement for a set of similarity measures that exploit information about small cavities in the surface of enzymes.

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

DOE PAGES

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

2017-02-28

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

Stepwise Loop Insertion Strategy for Active Site Remodeling to Generate Novel Enzyme Functions.

PubMed

Hoque, Md Anarul; Zhang, Yong; Chen, Liuqing; Yang, Guangyu; Khatun, Mst Afroza; Chen, Haifeng; Hao, Liu; Feng, Yan

2017-05-19

The remodeling of active sites to generate novel biocatalysts is an attractive and challenging task. We developed a stepwise loop insertion strategy (StLois), in which randomized residue pairs are inserted into active site loops. The phosphotriesterase-like lactonase from Geobacillus kaustophilus (GkaP-PLL) was used to investigate StLois's potential for changing enzyme function. By inserting six residues into active site loop 7, the best variant ML7-B6 demonstrated a 16-fold further increase in catalytic efficiency toward ethyl-paraoxon compared with its initial template, that is a 609-fold higher, >10 7 fold substrate specificity shift relative to that of wild-type lactonase. The remodeled variants displayed 760-fold greater organophosphate hydrolysis activity toward the organophosphates parathion, diazinon, and chlorpyrifos. Structure and docking computations support the source of notably inverted enzyme specificity. Considering the fundamental importance of active site loops, the strategy has potential for the rapid generation of novel enzyme functions by loop remodeling.

Functional Trade-Offs in Promiscuous Enzymes Cannot Be Explained by Intrinsic Mutational Robustness of the Native Activity.

PubMed

Kaltenbach, Miriam; Emond, Stephane; Hollfelder, Florian; Tokuriki, Nobuhiko

2016-10-01

The extent to which an emerging new function trades off with the original function is a key characteristic of the dynamics of enzyme evolution. Various cases of laboratory evolution have unveiled a characteristic trend; a large increase in a new, promiscuous activity is often accompanied by only a mild reduction of the native, original activity. A model that associates weak trade-offs with "evolvability" was put forward, which proposed that enzymes possess mutational robustness in the native activity and plasticity in promiscuous activities. This would enable the acquisition of a new function without compromising the original one, reducing the benefit of early gene duplication and therefore the selection pressure thereon. Yet, to date, no experimental study has examined this hypothesis directly. Here, we investigate the causes of weak trade-offs by systematically characterizing adaptive mutations that occurred in two cases of evolutionary transitions in enzyme function: (1) from phosphotriesterase to arylesterase, and (2) from atrazine chlorohydrolase to melamine deaminase. Mutational analyses in various genetic backgrounds revealed that, in contrast to the prevailing model, the native activity is less robust to mutations than the promiscuous activity. For example, in phosphotriesterase, the deleterious effect of individual mutations on the native phosphotriesterase activity is much larger than their positive effect on the promiscuous arylesterase activity. Our observations suggest a revision of the established model: weak trade-offs are not caused by an intrinsic robustness of the native activity and plasticity of the promiscuous activity. We propose that upon strong adaptive pressure for the new activity without selection against the original one, selected mutations will lead to the largest possible increases in the new function, but whether and to what extent they decrease the old function is irrelevant, creating a bias towards initially weak trade-offs and the emergence of generalist enzymes.

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.

The biodiscovery potential of marine bacteria: an investigation of phylogeny and function

PubMed Central

Mühling, Martin; Joint, Ian; Willetts, Andrew J

2013-01-01

Summary A collection of marine bacteria isolated from a temperate coastal zone has been screened in a programme of biodiscovery. A total of 34 enzymes with biotechnological potential were screened in 374 isolates of marine bacteria. Only two enzymes were found in all isolates while the majority of enzyme activities were present in a smaller proportion of the isolates. A cluster analysis demonstrated no significant correlation between taxonomy and enzyme function. However, there was evidence of co-occurrence of some enzyme activity in the same isolate. In this study marine Proteobacteria had a higher complement of enzymes with biodiscovery potential than Actinobacteria; this contrasts with the terrestrial environment where the Actinobacteria phylum is a proven source of enzymes with important industrial applications. In addition, a number of novel enzyme functions were more abundant in this marine culture collection than would be expected on the basis of knowledge from terrestrial bacteria. There is a strong case for future investigation of marine bacteria as a source for biodiscovery. PMID:23557256

Structure-Function Perturbation and Dissociation of Tetrameric Urate Oxidase by High Hydrostatic Pressure

PubMed Central

Girard, Eric; Marchal, Stéphane; Perez, Javier; Finet, Stéphanie; Kahn, Richard; Fourme, Roger; Marassio, Guillaume; Dhaussy, Anne-Claire; Prangé, Thierry; Giffard, Marion; Dulin, Fabienne; Bonneté, Françoise; Lange, Reinhard; Abraini, Jacques H.; Mezouar, Mohamed; Colloc'h, Nathalie

2010-01-01

Abstract Structure-function relationships in the tetrameric enzyme urate oxidase were investigated using pressure perturbation. As the active sites are located at the interfaces between monomers, enzyme activity is directly related to the integrity of the tetramer. The effect of hydrostatic pressure on the enzyme was investigated by x-ray crystallography, small-angle x-ray scattering, and fluorescence spectroscopy. Enzymatic activity was also measured under pressure and after decompression. A global model, consistent with all measurements, discloses structural and functional details of the pressure-induced dissociation of the tetramer. Before dissociating, the pressurized protein adopts a conformational substate characterized by an expansion of its substrate binding pocket at the expense of a large neighboring hydrophobic cavity. This substate should be adopted by the enzyme during its catalytic mechanism, where the active site has to accommodate larger intermediates and product. The approach, combining several high-pressure techniques, offers a new (to our knowledge) means of exploring structural and functional properties of transient states relevant to protein mechanisms. PMID:20483346

Substrate-permeable encapsulation of enzymes maintains effective activity, stabilizes against denaturation, and protects against proteolytic degradation.

PubMed

Nasseau, M; Boublik, Y; Meier, W; Winterhalter, M; Fournier, D

2001-12-05

How can enzymes be protected against denaturation and proteolysis while keeping them in a fully functional state? One solution is to encapsulate the enzymes into liposomes, which enhances their stability against denaturation and proteases. However, the permeability barrier of the lipid membrane drastically reduces the activity of enzyme entrapped in the liposome by reducing the internal concentration of the substrate. To overcome this problem, we permeabilized the wall of the liposome by reconstitution of a porin from Escherichia coli. In this way, we recovered the full functionality of the enzyme while retaining the protection against denaturation and proteolytic enzymes. Copyright 2001 John Wiley & Sons, Inc.

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.

Evolutionarily Conserved Linkage between Enzyme Fold, Flexibility, and Catalysis

PubMed Central

Ramanathan, Arvind; Agarwal, Pratul K.

2011-01-01

Proteins are intrinsically flexible molecules. The role of internal motions in a protein's designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function. Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease) that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 Å away) are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity) that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme–substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme–substrate interactions, thereby impacting the mechanism of catalyzed chemistry. These results have implications for understanding the mechanism of allostery, and for protein engineering and drug design. PMID:22087074

Mycorrhizas and soil ecosystem function of co-existing woody vegetation islands at the alpine tree line.

PubMed

Wang, Lixia; Otgonsuren, Burenjargal; Godbold, Douglas L

2017-01-01

Picea abies , Pinus mugo and Rhododendron ferrugineum co-exist at the alpine tree line, and can have different mycorrhizal communities. The activity and diversity of mycorrhizal fungi are considered to be important factors in regulation of soil function. At a tree line site and a lower elevation site in the Austrian Alps, the community structure of ectomycorrhiza on Picea abies and Pinus mugo was determined. The activity of surface enzymes was determined on ectomycorrhizal and ericoid mycorrhizal roots. In soils, the activity of a range of enzymes, nitrogen (N) mineralization and biomass decomposition were determined. The community structure of the ectomycorrhizal community of Picea abies and Pinus mugo differed strongly, but the average activity of surface enzymes of the ectomycorrhizal communities was similar. A lower root surface enzyme activity was determined on Rhododendron ferrugineum . Soil N-mineralization under Rhododendron ferrugineum was significantly lower than under Picea abies and Pinus mugo . In soil, the activity of a range of enzymes did not differ at the tree line but differed between the tree line and the lower elevation sites. The different ectomycorrhizal communities on Picea abies and Pinus mugo and ericoid mycorrhizas on Rhododendron ferrugineum support similar ecosystem functions in soil.

Discovery of novel enzymes with industrial potential from a cold and alkaline environment by a combination of functional metagenomics and culturing

PubMed Central

2014-01-01

Background The use of cold-active enzymes has many advantages, including reduced energy consumption and easy inactivation. The ikaite columns of SW Greenland are permanently cold (4-6°C) and alkaline (above pH 10), and the microorganisms living there and their enzymes are adapted to these conditions. Since only a small fraction of the total microbial diversity can be cultured in the laboratory, a combined approach involving functional screening of a strain collection and a metagenomic library was undertaken for discovery of novel enzymes from the ikaite columns. Results A strain collection with 322 cultured isolates was screened for enzymatic activities identifying a large number of enzyme producers, with a high re-discovery rate to previously characterized strains. A functional expression library established in Escherichia coli identified a number of novel cold-active enzymes. Both α-amylases and β-galactosidases were characterized in more detail with respect to temperature and pH profiles and one of the β-galactosidases, BGalI17E2, was able to hydrolyze lactose at 5°C. A metagenome sequence of the expression library indicated that the majority of enzymatic activities were not detected by functional expression. Phylogenetic analysis showed that different bacterial communities were targeted with the culture dependent and independent approaches and revealed the bias of multiple displacement amplification (MDA) of DNA isolated from complex microbial communities. Conclusions Many cold- and/or alkaline-active enzymes of industrial relevance were identified in the culture based approach and the majority of the enzyme-producing isolates were closely related to previously characterized strains. The function-based metagenomic approach, on the other hand, identified several enzymes (β-galactosidases, α-amylases and a phosphatase) with low homology to known sequences that were easily expressed in the production host E. coli. The β-galactosidase BGalI17E2 was able to hydrolyze lactose at low temperature, suggesting a possibly use in the dairy industry for this enzyme. The two different approaches complemented each other by targeting different microbial communities, highlighting the usefulness of combining methods for bioprospecting. Finally, we document here that ikaite columns constitute an important source of cold- and/or alkaline-active enzymes with industrial application potential. PMID:24886068

Discovery of novel enzymes with industrial potential from a cold and alkaline environment by a combination of functional metagenomics and culturing.

PubMed

Vester, Jan Kjølhede; Glaring, Mikkel Andreas; Stougaard, Peter

2014-05-20

The use of cold-active enzymes has many advantages, including reduced energy consumption and easy inactivation. The ikaite columns of SW Greenland are permanently cold (4-6°C) and alkaline (above pH 10), and the microorganisms living there and their enzymes are adapted to these conditions. Since only a small fraction of the total microbial diversity can be cultured in the laboratory, a combined approach involving functional screening of a strain collection and a metagenomic library was undertaken for discovery of novel enzymes from the ikaite columns. A strain collection with 322 cultured isolates was screened for enzymatic activities identifying a large number of enzyme producers, with a high re-discovery rate to previously characterized strains. A functional expression library established in Escherichia coli identified a number of novel cold-active enzymes. Both α-amylases and β-galactosidases were characterized in more detail with respect to temperature and pH profiles and one of the β-galactosidases, BGalI17E2, was able to hydrolyze lactose at 5°C. A metagenome sequence of the expression library indicated that the majority of enzymatic activities were not detected by functional expression. Phylogenetic analysis showed that different bacterial communities were targeted with the culture dependent and independent approaches and revealed the bias of multiple displacement amplification (MDA) of DNA isolated from complex microbial communities. Many cold- and/or alkaline-active enzymes of industrial relevance were identified in the culture based approach and the majority of the enzyme-producing isolates were closely related to previously characterized strains. The function-based metagenomic approach, on the other hand, identified several enzymes (β-galactosidases, α-amylases and a phosphatase) with low homology to known sequences that were easily expressed in the production host E. coli. The β-galactosidase BGalI17E2 was able to hydrolyze lactose at low temperature, suggesting a possibly use in the dairy industry for this enzyme. The two different approaches complemented each other by targeting different microbial communities, highlighting the usefulness of combining methods for bioprospecting. Finally, we document here that ikaite columns constitute an important source of cold- and/or alkaline-active enzymes with industrial application potential.

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.

An activity transition from NADH dehydrogenase to NADH oxidase during protein denaturation.

PubMed

Huston, Scott; Collins, John; Sun, Fangfang; Zhang, Ting; Vaden, Timothy D; Zhang, Y-H Percival; Fu, Jinglin

2018-05-01

A decrease in the specific activity of an enzyme is commonly observed when the enzyme is inappropriately handled or is stored over an extended period. Here, we reported a functional transition of an FMN-bound diaphorase (FMN-DI) that happened during the long-term storage process. It was found that FMN-DI did not simply lose its β-nicotinamide adenine diphosphate (NADH) dehydrogenase activity after a long-time storage, but obtained a new enzyme activity of NADH oxidase. Further mechanistic studies suggested that the alteration of the binding strength of an FMN cofactor with a DI protein could be responsible for this functional switch of the enzyme. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

(/sup 11/C)clorgyline and (/sup 11/C)-L-deprenyl and their use in measuring functional monoamine oxidase activity in the brain using positron emission tomography

DOEpatents

Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.

1986-04-17

This invention involves a new strategy for imaging the activity of the enzyme monoamine oxidase in the living body by using /sup 11/C-labeled enzyme inhibitors which bind irreversibly to an enzyme as a result of catalysis. 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.

Substrate-binding specificity of chitinase and chitosanase as revealed by active-site architecture analysis.

PubMed

Liu, Shijia; Shao, Shangjin; Li, Linlin; Cheng, Zhi; Tian, Li; Gao, Peiji; Wang, Lushan

2015-12-11

Chitinases and chitosanases, referred to as chitinolytic enzymes, are two important categories of glycoside hydrolases (GH) that play a key role in degrading chitin and chitosan, two naturally abundant polysaccharides. Here, we investigate the active site architecture of the major chitosanase (GH8, GH46) and chitinase families (GH18, GH19). Both charged (Glu, His, Arg, Asp) and aromatic amino acids (Tyr, Trp, Phe) are observed with higher frequency within chitinolytic active sites as compared to elsewhere in the enzyme structure, indicating significant roles related to enzyme function. Hydrogen bonds between chitinolytic enzymes and the substrate C2 functional groups, i.e. amino groups and N-acetyl groups, drive substrate recognition, while non-specific CH-π interactions between aromatic residues and substrate mainly contribute to tighter binding and enhanced processivity evident in GH8 and GH18 enzymes. For different families of chitinolytic enzymes, the number, type, and position of substrate atoms bound in the active site vary, resulting in different substrate-binding specificities. The data presented here explain the synergistic action of multiple enzyme families at a molecular level and provide a more reasonable method for functional annotation, which can be further applied toward the practical engineering of chitinases and chitosanases. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structural and Functional Survey of Environmental Aminoglycoside Acetyltransferases Reveals Functionality of Resistance Enzymes.

PubMed

Xu, Zhiyu; Stogios, Peter J; Quaile, Andrew T; Forsberg, Kevin J; Patel, Sanket; Skarina, Tatiana; Houliston, Scott; Arrowsmith, Cheryl; Dantas, Gautam; Savchenko, Alexei

2017-09-08

Aminoglycoside N-acetyltransferases (AACs) confer resistance against the clinical use of aminoglycoside antibiotics. The origin of AACs can be traced to environmental microbial species representing a vast reservoir for new and emerging resistance enzymes, which are currently undercharacterized. Here, we performed detailed structural characterization and functional analyses of four metagenomic AAC (meta-AACs) enzymes recently identified in a survey of agricultural and grassland soil microbiomes ( Forsberg et al. Nature 2014 , 509 , 612 ). These enzymes are new members of the Gcn5-Related-N-Acetyltransferase superfamily and confer resistance to the aminoglycosides gentamicin C, sisomicin, and tobramycin. Moreover, the meta-AAC0020 enzyme demonstrated activity comparable with an AAC(3)-I enzyme that serves as a model AAC enzyme identified in a clinical bacterial isolate. The crystal structure of meta-AAC0020 in complex with sisomicin confirmed an unexpected AAC(6') regiospecificity of this enzyme and revealed a drug binding mechanism distinct from previously characterized AAC(6') enzymes. Together, our data highlights the presence of highly active antibiotic-modifying enzymes in the environmental microbiome and reveals unexpected diversity in substrate specificity. These observations of additional AAC enzymes must be considered in the search for novel aminoglycosides less prone to resistance.

Histoplasma capsulatum encodes a dipeptidyl peptidase active against the mammalian immunoregulatory peptide, substance P.

PubMed

Cooper, Kendal G; Zarnowski, Robert; Woods, Jon P

2009-01-01

The pathogenic fungus Histoplasma capsulatum secretes dipeptidyl peptidase (Dpp) IV enzyme activity and has two putative DPPIV homologs (HcDPPIVA and HcDPPIVB). We previously showed that HcDPPIVB is the gene responsible for the majority of secreted DppIV activity in H. capsulatum culture supernatant, while we could not detect any functional contribution from HcDPPIVA. In order to determine whether HcDPPIVA encodes a functional DppIV enzyme, we expressed HcDPPIVA in Pichia pastoris and purified the recombinant protein. The recombinant enzyme cleaved synthetic DppIV substrates and had similar biochemical properties to other described DppIV enzymes, with temperature and pH optima of 42 degrees C and 8, respectively. Recombinant HcDppIVA cleaved the host immunoregulatory peptide substance P, indicating the enzyme has the potential to affect the immune response during infection. Expression of HcDPPIVA under heterologous regulatory sequences in H. capsulatum resulted in increased secreted DppIV activity, indicating that the encoded protein can be expressed and secreted by its native organism. However, HcDPPIVA was not required for virulence in a murine model of histoplasmosis. This work reports a fungal enzyme that can function to cleave the immunomodulatory host peptide substance P.

RUTBC1 Functions as a GTPase-activating Protein for Rab32/38 and Regulates Melanogenic Enzyme Trafficking in Melanocytes*

PubMed Central

Marubashi, Soujiro; Shimada, Hikaru; Fukuda, Mitsunori; Ohbayashi, Norihiko

2016-01-01

Two cell type-specific Rab proteins, Rab32 and Rab38 (Rab32/38), have been proposed as regulating the trafficking of melanogenic enzymes, including tyrosinase and tyrosinase-related protein 1 (Tyrp1), to melanosomes in melanocytes. Like other GTPases, Rab32/38 function as switch molecules that cycle between a GDP-bound inactive form and a GTP-bound active form; the cycle is thought to be regulated by an activating enzyme, guanine nucleotide exchange factor (GEF), and an inactivating enzyme, GTPase-activating protein (GAP), which stimulates the GTPase activity of Rab32/38. Although BLOC-3 has already been identified as a Rab32/38-specific GEF that regulates the trafficking of tyrosinase and Tyrp1, no physiological GAP for Rab32/38 in melanocytes has ever been identified, and it has remained unclear whether Rab32/38 is involved in the trafficking of dopachrome tautomerase, another melanogenic enzyme, in mouse melanocytes. In this study we investigated RUTBC1, which was originally characterized as a Rab9-binding protein and GAP for Rab32 and Rab33B in vitro, and the results demonstrated that RUTBC1 functions as a physiological GAP for Rab32/38 in the trafficking of all three melanogenic enzymes in mouse melanocytes. The results of this study also demonstrated the involvement of Rab9A in the regulation of the RUTBC1 localization and in the trafficking of all three melanogenic enzymes. We discovered that either excess activation or inactivation of Rab32/38 achieved by manipulating RUTBC1 inhibits the trafficking of all three melanogenic enzymes. These results collectively indicate that proper spatiotemporal regulation of Rab32/38 is essential for the trafficking of all three melanogenic enzymes in mouse melanocytes. PMID:26620560

Role of conformational dynamics in the evolution of novel enzyme function.

PubMed

Maria-Solano, Miguel A; Serrano-Hervás, Eila; Romero-Rivera, Adrian; Iglesias-Fernández, Javier; Osuna, Sílvia

2018-05-21

The free energy landscape concept that describes enzymes as an ensemble of differently populated conformational sub-states in dynamic equilibrium is key for evaluating enzyme activity, enantioselectivity, and specificity. Mutations introduced in the enzyme sequence can alter the populations of the pre-existing conformational states, thus strongly modifying the enzyme ability to accommodate alternative substrates, revert its enantiopreferences, and even increase the activity for some residual promiscuous reactions. In this feature article, we present an overview of the current experimental and computational strategies to explore the conformational free energy landscape of enzymes. We provide a series of recent publications that highlight the key role of conformational dynamics for the enzyme evolution towards new functions and substrates, and provide some perspectives on how conformational dynamism should be considered in future computational enzyme design protocols.

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.

X-Ray Crystallography as a Tool to Determine Three-Dimensional Structures of Commercial Enzymes Subjected to Treatment in Pressurized Fluids.

PubMed

Feiten, Mirian Cristina; Di Luccio, Marco; Santos, Karine F; de Oliveira, Débora; Oliveira, J Vladimir

2017-06-01

The study of enzyme function often involves a multi-disciplinary approach. Several techniques are documented in the literature towards determining secondary and tertiary structures of enzymes, and X-ray crystallography is the most explored technique for obtaining three-dimensional structures of proteins. Knowledge of three-dimensional structures is essential to understand reaction mechanisms at the atomic level. Additionally, structures can be used to modulate or improve functional activity of enzymes by the production of small molecules that act as substrates/cofactors or by engineering selected mutants with enhanced biological activity. This paper presentes a short overview on how to streamline sample preparation for crystallographic studies of treated enzymes. We additionally revise recent developments on the effects of pressurized fluid treatment on activity and stability of commercial enzymes. Future directions and perspectives on the the role of crystallography as a tool to access the molecular mechanisms underlying enzymatic activity modulation upon treatment in pressurized fluids are also addressed.

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.

Mesoporous silica-encapsulated gold nanoparticles as artificial enzymes for self-activated cascade catalysis.

PubMed

Lin, Youhui; Li, Zhenhua; Chen, Zhaowei; Ren, Jinsong; Qu, Xiaogang

2013-04-01

A significant challenge in chemistry is to create synthetic structures that mimic the complexity and function of natural systems. Here, a self-activated, enzyme-mimetic catalytic cascade has been realized by utilizing expanded mesoporous silica-encapsulated gold nanoparticles (EMSN-AuNPs) as both glucose oxidase- and peroxidase-like artificial enzymes. Specifically, EMSN helps the formation of a high degree of very small and well-dispersed AuNPs, which exhibit an extraordinarily stability and dual enzyme-like activities. Inspired by these unique and attractive properties, we further piece them together into a self-organized artificial cascade reaction, which is usually completed by the oxidase-peroxidase coupled enzyme system. Our finding may pave the way to use matrix as the structural component for the design and development of biomimetic catalysts and to apply enzyme mimics for realizing higher functions. Copyright © 2013 Elsevier Ltd. All rights reserved.

[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.

Comparative Characterization of CTX-M-64 and CTX-M-14 Provides Insights into the Structure and Catalytic Activity of the CTX-M Class of Enzymes.

PubMed

He, Dandan; Chiou, Jiachi; Zeng, Zhenling; Chan, Edward Wai-Chi; Liu, Jian-Hua; Chen, Sheng

2016-10-01

Clinical isolates producing hybrid CTX-M β-lactamases, presumably due to recombination between the blaCTX-M-15 and blaCTX-M-14 elements, have emerged in recent years. Among the hybrid enzymes, CTX-M-64 and CTX-M-14 display the most significant difference in catalytic activity. This study aims to investigate the mechanisms underlying such differential enzymatic activities in order to provide insight into the structure/function relationship of this class of enzymes. Sequence alignment analysis showed that the major differences between the amino acid composition of CTX-M-64 and CTX-M-14 lie at both the N and C termini of the enzymes. Single or multiple amino acid substitutions introduced into CTX-M-64 and CTX-M-14 were found to produce only minor effects on hydrolytic functions; such a finding is consistent with the notion that the discrepancy between the functional activities of the two enzymes is not the result of only a few amino acid changes but is attributable to interactions between a unique set of amino acid residues in each enzyme. This theory is supported by the results of the thermal stability assay, which confirmed that CTX-M-64 is significantly more stable than CTX-M-14. Our data confirmed that, in addition to the important residues located in the active site, residues distal to the active site also contribute to the catalytic activity of the enzyme through stabilizing its structural integrity. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Comparative Characterization of CTX-M-64 and CTX-M-14 Provides Insights into the Structure and Catalytic Activity of the CTX-M Class of Enzymes

PubMed Central

He, Dandan; Chiou, Jiachi; Zeng, Zhenling; Chan, Edward Wai-Chi

2016-01-01

Clinical isolates producing hybrid CTX-M β-lactamases, presumably due to recombination between the blaCTX-M-15 and blaCTX-M-14 elements, have emerged in recent years. Among the hybrid enzymes, CTX-M-64 and CTX-M-14 display the most significant difference in catalytic activity. This study aims to investigate the mechanisms underlying such differential enzymatic activities in order to provide insight into the structure/function relationship of this class of enzymes. Sequence alignment analysis showed that the major differences between the amino acid composition of CTX-M-64 and CTX-M-14 lie at both the N and C termini of the enzymes. Single or multiple amino acid substitutions introduced into CTX-M-64 and CTX-M-14 were found to produce only minor effects on hydrolytic functions; such a finding is consistent with the notion that the discrepancy between the functional activities of the two enzymes is not the result of only a few amino acid changes but is attributable to interactions between a unique set of amino acid residues in each enzyme. This theory is supported by the results of the thermal stability assay, which confirmed that CTX-M-64 is significantly more stable than CTX-M-14. Our data confirmed that, in addition to the important residues located in the active site, residues distal to the active site also contribute to the catalytic activity of the enzyme through stabilizing its structural integrity. PMID:27480856

The GH51 α-l-arabinofuranosidase from Paenibacillus sp. THS1 is multifunctional, hydrolyzing main-chain and side-chain glycosidic bonds in heteroxylans.

PubMed

Bouraoui, Hanen; Desrousseaux, Marie-Laure; Ioannou, Eleni; Alvira, Pablo; Manaï, Mohamed; Rémond, Caroline; Dumon, Claire; Fernandez-Fuentes, Narcis; O'Donohue, Michael J

2016-01-01

Conceptually, multi-functional enzymes are attractive because in the case of complex polymer hydrolysis having two or more activities defined by a single enzyme offers the possibility of synergy and reduced enzyme cocktail complexity. Nevertheless, multi-functional enzymes are quite rare and are generally multi-domain assemblies with each activity being defined by a separate protein module. However, a recent report described a GH51 arabinofuranosidase from Alicyclobacillus sp. A4 that displays both α-l-arabinofuranosidase and β-d-xylanase activities, which are defined by a single active site. Following on from this, we describe in detail another multi-functional GH51 arabinofuranosidase and discuss the molecular basis of multifunctionality. THSAbf is a GH51 α-l-arabinofuranosidase. Characterization revealed that THSAbf is active up to 75 °C, stable at 60 °C and active over a broad pH range (4-7). THSAbf preferentially releases para-nitrophenyl from the l-arabinofuranoside (k cat/K M = 1050 s(-1) mM(-1)) and to some extent from d-galactofuranoside and d-xyloside. THSAbf is active on 4-O-methylglucuronoxylans from birch and beechwood (10.8 and 14.4 U mg(-1), respectively) and on sugar beet branched and linear arabinans (1.1 ± 0.24 and 1.8 ± 0.1 U mg(-1)). Further investigation revealed that like the Alicyclobacillus sp. A4 α-l-arabinofuranosidase, THSAbf also displays endo-xylanase activity, cleaving β-1,4 bonds in heteroxylans. The optimum pH for THASAbf activity is substrate dependent, but ablation of the catalytic nucleophile caused a general loss of activity, indicating the involvement of a single active center. Combining the α-l-arabinofuranosidase with a GH11 endoxylanase did not procure synergy. The molecular modeling of THSAbf revealed a wide active site cleft and clues to explain multi-functionality. The discovery of single active site, multifunctional enzymes such as THSAbf opens up exciting avenues for enzyme engineering and the development of new biomass-degrading cocktails that could considerably reduce enzyme production costs.

O 2 Activation by Non-Heme Iron Enzymes

DOE PAGES

Solomon, Edward I.; Goudarzi, Serra; Sutherlin, Kyle D.

2016-10-28

The non-heme Fe enzymes are ubiquitous in nature and perform a wide range of functions involving O 2 activation. These had been difficult to study relative to heme enzymes; however, spectroscopic methods have now been developed that provide significant insight into the correlation of structure with function. This Current Topics article summarizes both the molecular mechanism these enzymes use to control O 2 activation in the presence of cosubstrates and the oxygen intermediates these reactions generate. Three types of O 2 activation are observed. First, non-heme reactivity is shown to be different from heme chemistry where a low-spin Fe III-OOHmore » non-heme intermediate directly reacts with substrate. Also, two subclasses of non-heme Fe enzymes generate high-spin Fe IV=O intermediates that provide both σ and π frontier molecular orbitals that can control selectivity. Lastly, for several subclasses of non-heme Fe enzymes, substrate binding to the Fe II site leads to the one electron reductive activation of O 2 to an Fe III-superoxide capable of H-atom abstraction and electrophilic attack.« less

Influence of Tribulus terrestris on testicular enzyme in fresh water ornamental fish Poecilia latipinna.

PubMed

Kavitha, P; Subramanian, P

2011-12-01

The influence of Tribulus terrestris on the activities of testicular enzyme in Poecilia latipinna was assessed in lieu of reproductive manipulation. Different concentrations of (100, 150, 200, 250, and 300 mg) Tribulus terrestris extract and of a control were tested for testicular activity of enzymes in Poecilia latipinna for 2 months. The testis and liver were homogenized separately in 0.1 mol/l potassium phosphate buffer (0.1 mol/l, pH 7.2). The crude homogenate was centrifuged, and supernatant obtained was used as an enzyme extract for determination of activities. The activities of testicular functional enzyme ALP, ACP, SDH, LDH, and G6PDH levels were changed to different extent in treated groups compared with that of the control. The total body weight and testis weight were increased with the Tribulus terrestris-treated fish (Poecilia latipinna). These results suggest that Tribulus terrestris induced the testicular enzyme activity that may aid in the male reproductive functions. It is discernible from the present study that Tribulus terrestris has the inducing effect on reproductive system of Poecilia latipinna.

O 2 Activation by Non-Heme Iron Enzymes

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

Solomon, Edward I.; Goudarzi, Serra; Sutherlin, Kyle D.

The non-heme Fe enzymes are ubiquitous in nature and perform a wide range of functions involving O 2 activation. These had been difficult to study relative to heme enzymes; however, spectroscopic methods have now been developed that provide significant insight into the correlation of structure with function. This Current Topics article summarizes both the molecular mechanism these enzymes use to control O 2 activation in the presence of cosubstrates and the oxygen intermediates these reactions generate. Three types of O 2 activation are observed. First, non-heme reactivity is shown to be different from heme chemistry where a low-spin Fe III-OOHmore » non-heme intermediate directly reacts with substrate. Also, two subclasses of non-heme Fe enzymes generate high-spin Fe IV=O intermediates that provide both σ and π frontier molecular orbitals that can control selectivity. Lastly, for several subclasses of non-heme Fe enzymes, substrate binding to the Fe II site leads to the one electron reductive activation of O 2 to an Fe III-superoxide capable of H-atom abstraction and electrophilic attack.« less

Co-immobilization of cellulase and lysozyme on amino-functionalized magnetic nanoparticles: An activity-tunable biocatalyst for extraction of lipids from microalgae.

PubMed

Chen, Qingtai; Liu, Dong; Wu, Chongchong; Yao, Kaisheng; Li, Zhiheng; Shi, Nan; Wen, Fushan; Gates, Ian D

2018-05-03

An activity-tunable biocatalyst for Nannochloropsis sp. cell-walls degradation was prepared by co-immobilization of cellulase and lysozyme on the surface of amino-functionalized magnetic nanoparticles (MNPs) employing glutaraldehyde. The competition between cellulase and lysozyme during immobilization was caused by the limited active sites of the MNPs. The maximum recovery of activities (cellulase: 78.9% and lysozyme: 69.6%) were achieved due to synergistic effects during dual-enzyme co-immobilization. The thermal stability in terms of half-life of the co-immobilized enzymes was three times higher than that in free form and had higher catalytic efficiency for hydrolysis of cell walls. Moreover, the co-immobilized enzymes showed greater thermal stability and wider pH tolerance than free enzymes under harsh conditions. Furthermore, the co-immobilized enzymes retained up to 60% of the residual activity after being recycled 6 times. This study provides a feasible approach for the industrialization of enzyme during cell-walls disruption and lipids extraction from Nannochloropsis sp. Copyright © 2018. Published by Elsevier Ltd.

Removing the Active-Site Flap in Lipase A from Candida antarctica Produces a Functional Enzyme without Interfacial Activation.

PubMed

Wikmark, Ylva; Engelmark Cassimjee, Karim; Lihammar, Richard; Bäckvall, Jan-E

2016-01-01

A mobile region is proposed to be a flap that covers the active site of Candida antarctica lipase A. Removal of the mobile region retains the functional properties of the enzyme. Interestingly interfacial activation, required for the wild-type enzyme, was not observed for the truncated variant, although stability, activity, and stereoselectivity were very similar for the wild-type and variant enzymes. The variant followed classical Michaelis-Menten kinetics, unlike the wild type. Both gave the same relative specificity in the transacylation of a primary and a secondary alcohol in organic solvent. Furthermore, both showed the same enantioselectivity in transacylation of alcohols and the hydrolysis of alcohol esters, as well as in the hydrolysis of esters chiral at the acid part. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Dissecting enzyme function with microfluidic-based deep mutational scanning.

PubMed

Romero, Philip A; Tran, Tuan M; Abate, Adam R

2015-06-09

Natural enzymes are incredibly proficient catalysts, but engineering them to have new or improved functions is challenging due to the complexity of how an enzyme's sequence relates to its biochemical properties. Here, we present an ultrahigh-throughput method for mapping enzyme sequence-function relationships that combines droplet microfluidic screening with next-generation DNA sequencing. We apply our method to map the activity of millions of glycosidase sequence variants. Microfluidic-based deep mutational scanning provides a comprehensive and unbiased view of the enzyme function landscape. The mapping displays expected patterns of mutational tolerance and a strong correspondence to sequence variation within the enzyme family, but also reveals previously unreported sites that are crucial for glycosidase function. We modified the screening protocol to include a high-temperature incubation step, and the resulting thermotolerance landscape allowed the discovery of mutations that enhance enzyme thermostability. Droplet microfluidics provides a general platform for enzyme screening that, when combined with DNA-sequencing technologies, enables high-throughput mapping of enzyme sequence space.

A novel, extremely alkaliphilic and cold-active esterase from Antarctic desert soil.

PubMed

Hu, Xiao Ping; Heath, Caroline; Taylor, Mark Paul; Tuffin, Marla; Cowan, Don

2012-01-01

A novel, cold-active and highly alkaliphilic esterase was isolated from an Antarctic desert soil metagenomic library by functional screening. The 1,044 bp gene sequence contained several conserved regions common to lipases/esterases, but lacked clear classification based on sequence analysis alone. Moderate (<40%) amino acid sequence similarity to known esterases was apparent (the closest neighbour being a hypothetical protein from Chitinophaga pinensis), despite phylogenetic distance to many of the lipolytic "families". The enzyme functionally demonstrated activity towards shorter chain p-nitrophenyl esters with the optimal activity recorded towards p-nitrophenyl propionate (C3). The enzyme possessed an apparent T(opt) at 20°C and a pH optimum at pH 11. Esterases possessing such extreme alkaliphily are rare and so this enzyme represents an intriguing novel locus in protein sequence space. A metagenomic approach has been shown, in this case, to yield an enzyme with quite different sequential/structural properties to known lipases. It serves as an excellent candidate for analysis of the molecular mechanisms responsible for both cold and alkaline activity and novel structure-function relationships of esterase activity.

Cell endogenous activities of fukutin and FKRP coexist with the ribitol xylosyltransferase, TMEM5.

PubMed

Nishihara, Ryuta; Kobayashi, Kazuhiro; Imae, Rieko; Tsumoto, Hiroki; Manya, Hiroshi; Mizuno, Mamoru; Kanagawa, Motoi; Endo, Tamao; Toda, Tatsushi

2018-03-18

Dystroglycanopathies are a group of muscular dystrophies that are caused by abnormal glycosylation of dystroglycan; currently 18 causative genes are known. Functions of the dystroglycanopathy genes fukutin, fukutin-related protein (FKRP), and transmembrane protein 5 (TMEM5) were most recently identified; fukutin and FKRP are ribitol-phosphate transferases and TMEM5 is a ribitol xylosyltransferase. In this study, we show that fukutin, FKRP, and TMEM5 form a complex while maintaining each of their enzyme activities. Immunoprecipitation and immunofluorescence experiments demonstrated protein interactions between these 3 proteins. A protein complex consisting of endogenous fukutin and FKRP, and exogenously expressed TMEM5 exerts activities of each enzyme. Our data showed for the first time that endogenous fukutin and FKRP enzyme activities coexist with TMEM5 enzyme activity, and suggest the possibility that formation of this enzyme complex may contribute to specific and prompt biosynthesis of glycans that are required for dystroglycan function. Copyright © 2018 Elsevier Inc. All rights reserved.

Identification and modification of dynamical regions in proteins for alteration of enzyme catalytic effect

DOEpatents

Agarwal, Pratul K.

2015-11-24

A method for analysis, control, and manipulation for improvement of the chemical reaction rate of a protein-mediated reaction is provided. Enzymes, which typically comprise protein molecules, are very efficient catalysts that enhance chemical reaction rates by many orders of magnitude. Enzymes are widely used for a number of functions in chemical, biochemical, pharmaceutical, and other purposes. The method identifies key protein vibration modes that control the chemical reaction rate of the protein-mediated reaction, providing identification of the factors that enable the enzymes to achieve the high rate of reaction enhancement. By controlling these factors, the function of enzymes may be modulated, i.e., the activity can either be increased for faster enzyme reaction or it can be decreased when a slower enzyme is desired. This method provides an inexpensive and efficient solution by utilizing computer simulations, in combination with available experimental data, to build suitable models and investigate the enzyme activity.

Identification and modification of dynamical regions in proteins for alteration of enzyme catalytic effect

DOEpatents

Agarwal, Pratul K.

2013-04-09

A method for analysis, control, and manipulation for improvement of the chemical reaction rate of a protein-mediated reaction is provided. Enzymes, which typically comprise protein molecules, are very efficient catalysts that enhance chemical reaction rates by many orders of magnitude. Enzymes are widely used for a number of functions in chemical, biochemical, pharmaceutical, and other purposes. The method identifies key protein vibration modes that control the chemical reaction rate of the protein-mediated reaction, providing identification of the factors that enable the enzymes to achieve the high rate of reaction enhancement. By controlling these factors, the function of enzymes may be modulated, i.e., the activity can either be increased for faster enzyme reaction or it can be decreased when a slower enzyme is desired. This method provides an inexpensive and efficient solution by utilizing computer simulations, in combination with available experimental data, to build suitable models and investigate the enzyme activity.

[Specific features of digestive function development in larvae of some salmonid fish].

PubMed

Ershova, T S; Volkova, I V; Zaĭtseva, V F

2004-01-01

We studied the activities of digestive enzymes responsible for the digestion of food carbohydrate and protein components in plant-eating fish at various stages of larval development. The activities of all digestive enzymes tend to rise during larval development. Species specific features of the alimentary canal functioning have been described.

Enzymatically Regulated Peptide Pairing and Catalysis for the Bioanalysis of Extracellular Prometastatic Activities of Functionally Linked Enzymes

NASA Astrophysics Data System (ADS)

Li, Hao; Huang, Yue; Yu, Yue; Li, Tianqi; Li, Genxi; Anzai, Jun-Ichi

2016-05-01

Diseases such as cancer arise from systematical reconfiguration of interactions of exceedingly large numbers of proteins in cell signaling. The study of such complicated molecular mechanisms requires multiplexed detection of the inter-connected activities of several proteins in a disease-associated context. However, the existing methods are generally not well-equipped for this kind of application. Here a method for analyzing functionally linked protein activities is developed based on enzyme controlled pairing between complementary peptide helix strands, which simultaneously enables elaborate regulation of catalytic activity of the paired peptides. This method has been used to detect three different types of protein modification enzymes that participate in the modification of extracellular matrix and the formation of invasion front in tumour. In detecting breast cancer tissue samples using this method, up-regulated activity can be observed for two of the assessed enzymes, while the third enzyme is found to have a subtle fluctuation of activity. These results may point to the application of this method in evaluating prometastatic activities of proteins in tumour.

The evolution of new enzyme function: lessons from xenobiotic metabolizing bacteria versus insecticide-resistant insects

PubMed Central

Russell, Robyn J; Scott, Colin; Jackson, Colin J; Pandey, Rinku; Pandey, Gunjan; Taylor, Matthew C; Coppin, Christopher W; Liu, Jian-Wei; Oakeshott, John G

2011-01-01

Here, we compare the evolutionary routes by which bacteria and insects have evolved enzymatic processes for the degradation of four classes of synthetic chemical insecticide. For insects, the selective advantage of such degradative activities is survival on exposure to the insecticide, whereas for the bacteria the advantage is simply a matter of access to additional sources of nutrients. Nevertheless, bacteria have evolved highly efficient enzymes from a wide variety of enzyme families, whereas insects have relied upon generalist esterase-, cytochrome P450- and glutathione-S-transferase-dependent detoxification systems. Moreover, the mutant insect enzymes are less efficient kinetically and less diverged in sequence from their putative ancestors than their bacterial counterparts. This presumably reflects several advantages that bacteria have over insects in the acquisition of new enzymatic functions, such as a broad biochemical repertoire from which new functions can be evolved, large population sizes, high effective mutation rates, very short generation times and access to genetic diversity through horizontal gene transfer. Both the insect and bacterial systems support recent theory proposing that new biochemical functions often evolve from ‘promiscuous’ activities in existing enzymes, with subsequent mutations then enhancing those activities. Study of the insect enzymes will help in resistance management, while the bacterial enzymes are potential bioremediants of insecticide residues in a range of contaminated environments. PMID:25567970

Effects of argan oil on the mitochondrial function, antioxidant system and the activity of NADPH- generating enzymes in acrylamide treated rat brain.

PubMed

Aydın, Birsen

2017-03-01

Argan oil (AO) is rich in minor compounds such as polyphenols and tocopherols which are powerful antioxidants. Acrylamide (ACR) has been classified as a neurotoxic agent in animals and humans. Mitochondrial oxidative stress and dysfunction is one of the most probable molecular mechanisms of neurodegenerative diseases. Female Sprague Dawley rats were exposed to ACR (50mg/kg i.p. three times a week), AO (6ml/kg,o.p, per day) or together for 30days. The activities of cytosolic enzymes such as xanthine oxidase (XO), glucose 6-phosphate dehydrogenase (G6PDH), glutathione-S-transferase (GST), mitochondrial oxidative stress, oxidative phosphorylation (OXPHOS) and tricarboxylic acid cycle (TCA) enzymes, mitochondrial metabolic function, adenosine triphosphate (ATP) level and acetylcholinesterase (AChE) activity were assessed in rat brain. Cytosolic and mitochondrial antioxidant enzymes were significantly diminished in the brains of rats treated with ACR compared to those in control. Besides, ACR treatment resulted in a significant reduction in brain ATP level, mitochondrial metabolic function, OXPHOS and TCA enzymes. Administration of AO restored both the cytosolic and mitochondrial oxidative stress by normalizing nicotinamide adenine dinucleotide phosphate (NADPH) generating enzymes. In addition, improved mitochondrial function primarily enhancing nicotinamide adenine dinucleotide (NADH) generated enzymes activities and ATP level in the mitochondria. The reason for AO's obvious beneficial effects in this study may be due to synergistic effects of its different bioactive compounds which is especially effective on mitochondria. Modulation of the brain mitochondrial functions and antioxidant systems by AO may lead to the development of new mitochondria-targeted antioxidants in the future. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Relationship between global structural parameters and Enzyme Commission hierarchy: implications for function prediction.

PubMed

Boareto, Marcelo; Yamagishi, Michel E B; Caticha, Nestor; Leite, Vitor B P

2012-10-01

In protein databases there is a substantial number of proteins structurally determined but without function annotation. Understanding the relationship between function and structure can be useful to predict function on a large scale. We have analyzed the similarities in global physicochemical parameters for a set of enzymes which were classified according to the four Enzyme Commission (EC) hierarchical levels. Using relevance theory we introduced a distance between proteins in the space of physicochemical characteristics. This was done by minimizing a cost function of the metric tensor built to reflect the EC classification system. Using an unsupervised clustering method on a set of 1025 enzymes, we obtained no relevant clustering formation compatible with EC classification. The distance distributions between enzymes from the same EC group and from different EC groups were compared by histograms. Such analysis was also performed using sequence alignment similarity as a distance. Our results suggest that global structure parameters are not sufficient to segregate enzymes according to EC hierarchy. This indicates that features essential for function are rather local than global. Consequently, methods for predicting function based on global attributes should not obtain high accuracy in main EC classes prediction without relying on similarities between enzymes from training and validation datasets. Furthermore, these results are consistent with a substantial number of studies suggesting that function evolves fundamentally by recruitment, i.e., a same protein motif or fold can be used to perform different enzymatic functions and a few specific amino acids (AAs) are actually responsible for enzyme activity. These essential amino acids should belong to active sites and an effective method for predicting function should be able to recognize them. Copyright © 2012 Elsevier Ltd. All rights reserved.

Insights into the evolution of enzyme substrate promiscuity after the discovery of (βα)₈ isomerase evolutionary intermediates from a diverse metagenome.

PubMed

Noda-García, Lianet; Juárez-Vázquez, Ana L; Ávila-Arcos, María C; Verduzco-Castro, Ernesto A; Montero-Morán, Gabriela; Gaytán, Paul; Carrillo-Tripp, Mauricio; Barona-Gómez, Francisco

2015-06-10

Current sequence-based approaches to identify enzyme functional shifts, such as enzyme promiscuity, have proven to be highly dependent on a priori functional knowledge, hampering our ability to reconstruct evolutionary history behind these mechanisms. Hidden Markov Model (HMM) profiles, broadly used to classify enzyme families, can be useful to distinguish between closely related enzyme families with different specificities. The (βα)8-isomerase HisA/PriA enzyme family, involved in L-histidine (HisA, mono-substrate) biosynthesis in most bacteria and plants, but also in L-tryptophan (HisA/TrpF or PriA, dual-substrate) biosynthesis in most Actinobacteria, has been used as model system to explore evolutionary hypotheses and therefore has a considerable amount of evolutionary, functional and structural knowledge available. We searched for functional evolutionary intermediates between the HisA and PriA enzyme families in order to understand the functional divergence between these families. We constructed a HMM profile that correctly classifies sequences of unknown function into the HisA and PriA enzyme sub-families. Using this HMM profile, we mined a large metagenome to identify plausible evolutionary intermediate sequences between HisA and PriA. These sequences were used to perform phylogenetic reconstructions and to identify functionally conserved amino acids. Biochemical characterization of one selected enzyme (CAM1) with a mutation within the functionally essential N-terminus phosphate-binding site, namely, an alanine instead of a glycine in HisA or a serine in PriA, showed that this evolutionary intermediate has dual-substrate specificity. Moreover, site-directed mutagenesis of this alanine residue, either backwards into a glycine or forward into a serine, revealed the robustness of this enzyme. None of these mutations, presumably upon functionally essential amino acids, significantly abolished its enzyme activities. A truncated version of this enzyme (CAM2) predicted to adopt a (βα)6-fold, and thus entirely lacking a C-terminus phosphate-binding site, was identified and shown to have HisA activity. As expected, reconstruction of the evolution of PriA from HisA with HMM profiles suggest that functional shifts involve mutations in evolutionarily intermediate enzymes of otherwise functionally essential residues or motifs. These results are in agreement with a link between promiscuous enzymes and intragenic epistasis. HMM provides a convenient approach for gaining insights into these evolutionary processes.

Functional analysis of variant lysosomal acid glycosidases of Anderson-Fabry and Pompe disease in a human embryonic kidney epithelial cell line (HEK 293 T).

PubMed

Ebrahim, Hatim Y; Baker, Robert J; Mehta, Atul B; Hughes, Derralynn A

2012-03-01

The functional significance of missense mutations in genes encoding acid glycosidases of lysosomal storage disorders (LSDs) is not always clear. Here we describe a method of investigating functional properties of variant enzymes in vitro using a human embryonic kidney epithelial cell line. Site-directed mutagenesis was performed on the parental plasmids containing cDNA encoding for alpha-galactosidase A (α-Gal A) and acid maltase (α-Glu) to prepare plasmids encoding relevant point mutations. Mutant plasmids were transfected into HEK 293 T cells, and transient over-expression of variant enzymes was measured after 3 days. We have illustrated the method by examining enzymatic activities of four unknown α-Gal A and one α-Glu variants identified in our patients with Anderson-Fabry disease and Pompe diseases respectively. Comparison with control variants known to be either pathogenic or non-pathogenic together with over-expression of wild-type enzyme allowed determination of the pathogenicity of the mutation. One leader sequence novel variant of α-Gal A (p.A15T) was shown not to significantly reduce enzyme activity, whereas three other novel α-Gal A variants (p.D93Y, p.L372P and p.T410I) were shown to be pathogenic as they resulted in significant reduction of enzyme activity. A novel α-Glu variant (p.L72R) was shown to be pathogenic as this significantly reduced enzyme activity. Certain acid glycosidase variants that have been described in association with late-onset LSDs and which are known to have variable residual plasma and leukocyte enzyme activity in patients appear to show intermediate to low enzyme activity (p.N215S and p.Q279E α-Gal A respectively) in the over-expression system.

Reciprocal regulation of p53 and malic enzymes modulates metabolism and senescence.

PubMed

Jiang, Peng; Du, Wenjing; Mancuso, Anthony; Wellen, Kathryn E; Yang, Xiaolu

2013-01-31

Cellular senescence both protects multicellular organisms from cancer and contributes to their ageing. The pre-eminent tumour suppressor p53 has an important role in the induction and maintenance of senescence, but how it carries out this function remains poorly understood. In addition, although increasing evidence supports the idea that metabolic changes underlie many cell-fate decisions and p53-mediated tumour suppression, few connections between metabolic enzymes and senescence have been established. Here we describe a new mechanism by which p53 links these functions. We show that p53 represses the expression of the tricarboxylic-acid-cycle-associated malic enzymes ME1 and ME2 in human and mouse cells. Both malic enzymes are important for NADPH production, lipogenesis and glutamine metabolism, but ME2 has a more profound effect. Through the inhibition of malic enzymes, p53 regulates cell metabolism and proliferation. Downregulation of ME1 and ME2 reciprocally activates p53 through distinct MDM2- and AMP-activated protein kinase-mediated mechanisms in a feed-forward manner, bolstering this pathway and enhancing p53 activation. Downregulation of ME1 and ME2 also modulates the outcome of p53 activation, leading to strong induction of senescence, but not apoptosis, whereas enforced expression of either malic enzyme suppresses senescence. Our findings define physiological functions of malic enzymes, demonstrate a positive-feedback mechanism that sustains p53 activation, and reveal a connection between metabolism and senescence mediated by p53.

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

Directed evolution of new and improved enzyme functions using an evolutionary intermediate and multidirectional search.

PubMed

Porter, Joanne L; Boon, Priscilla L S; Murray, Tracy P; Huber, Thomas; Collyer, Charles A; Ollis, David L

2015-02-20

The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse α/β hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.

Transformation of cyclodextrin glucanotransferase (CGTase) from aqueous suspension to fine solid particles via electrospraying.

PubMed

Saallah, Suryani; Naim, M Nazli; Mokhtar, Mohd Noriznan; Abu Bakar, Noor Fitrah; Gen, Masao; Lenggoro, I Wuled

2014-10-01

In this study, the potential of electrohydrodynamic atomization or electrospraying to produce nanometer-order CGTase particles from aqueous suspension was demonstrated. CGTase enzyme was prepared in acetate buffer solution (1% v/v), followed by electrospraying in stable Taylor cone-jet mode. The deposits were collected on aluminium foil (collector) at variable distances from the tip of spraying needle, ranging from 10 to 25 cm. The Coulomb fission that occurs during electrospraying process successfully transformed the enzyme to the solid state without any functional group deterioration. The functional group verification was conducted by FTIR analysis. Comparison between the deposit and the as-received enzyme in dry state indicates almost identical spectra. By increasing the distance of the collector from the needle tip, the average particle size of the solidified enzyme was reduced from 200±117 nm to 75±34 nm. The average particle sizes produced from the droplet fission were in agreement with the scaling law models. Enzyme activity analysis showed that the enzyme retained its initial activity after the electrospraying process. The enzyme particles collected at the longest distance (25 cm) demonstrated the highest enzyme activity, which indicates that the activity was controlled by the enzyme particle size. Copyright © 2014 Elsevier Inc. All rights reserved.

Evolution of enzymes in a series is driven by dissimilar functional demands.

PubMed

Salvador, Armindo; Savageau, Michael A

2006-02-14

That distinct enzyme activities in an unbranched metabolic pathway are evolutionarily tuned to a single functional requirement is a pervasive assumption. Here we test this assumption by examining the activities of two consecutively acting enzymes in human erythrocytes with an approach to quantitative evolutionary design that avoids the above-mentioned assumption. We previously found that avoidance of NADPH depletion during the pulses of oxidative load to which erythrocytes are normally exposed is the main functional requirement mediating selection for high glucose-6-phosphate dehydrogenase activity. In the present study, we find that, in contrast, the maintenance of oxidized glutathione at low concentrations is the main functional requirement mediating selection for high glutathione reductase activity. The results in this case show that, contrary to the assumption of a single functional requirement, natural selection for the normal activities of the distinct enzymes in the pathway is mediated by different requirements. On the other hand, the results agree with the more general principles that underlie our approach. Namely, that (i) the values of biochemical parameters evolve so as to fulfill the various performance requirements that are relevant to achieve high fitness, and (ii) these performance requirements can be inferred from quantitative systems theory considerations, informed by knowledge of specific aspects of the biochemistry, physiology, genetics, and ecology of the organism.

Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes.

PubMed

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

2018-01-29

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

Structural and Biochemical Characterization of Acinetobacter spp. Aminoglycoside Acetyltransferases Highlights Functional and Evolutionary Variation among Antibiotic Resistance Enzymes.

PubMed

Stogios, Peter J; Kuhn, Misty L; Evdokimova, Elena; Law, Melissa; Courvalin, Patrice; Savchenko, Alexei

2017-02-10

Modification of aminoglycosides by N-acetyltransferases (AACs) is one of the major mechanisms of resistance to these antibiotics in human bacterial pathogens. More than 50 enzymes belonging to the AAC(6') subfamily have been identified in Gram-negative and Gram-positive clinical isolates. Our understanding of the molecular function and evolutionary origin of these resistance enzymes remains incomplete. Here we report the structural and enzymatic characterization of AAC(6')-Ig and AAC(6')-Ih from Acinetobacter spp. The crystal structure of AAC(6')-Ig in complex with tobramycin revealed a large substrate-binding cleft remaining partially unoccupied by the substrate, which is in stark contrast with the previously characterized AAC(6')-Ib enzyme. Enzymatic analysis indicated that AAC(6')-Ig and -Ih possess a broad specificity against aminoglycosides but with significantly lower turnover rates as compared to other AAC(6') enzymes. Structure- and function-informed phylogenetic analysis of AAC(6') enzymes led to identification of at least three distinct subfamilies varying in oligomeric state, active site composition, and drug recognition mode. Our data support the concept of AAC(6') functionality originating through convergent evolution from diverse Gcn5-related-N-acetyltransferase (GNAT) ancestral enzymes, with AAC(6')-Ig and -Ih representing enzymes that may still retain ancestral nonresistance functions in the cell as provided by their particular active site properties.

Functional Analogy in Human Metabolism: Enzymes with Different Biological Roles or Functional Redundancy?

PubMed Central

Piergiorge, Rafael Mina; de Miranda, Antonio Basílio; Catanho, Marcos

2017-01-01

Abstract Since enzymes catalyze almost all chemical reactions that occur in living organisms, it is crucial that genes encoding such activities are correctly identified and functionally characterized. Several studies suggest that the fraction of enzymatic activities in which multiple events of independent origin have taken place during evolution is substantial. However, this topic is still poorly explored, and a comprehensive investigation of the occurrence, distribution, and implications of these events has not been done so far. Fundamental questions, such as how analogous enzymes originate, why so many events of independent origin have apparently occurred during evolution, and what are the reasons for the coexistence in the same organism of distinct enzymatic forms catalyzing the same reaction, remain unanswered. Also, several isofunctional enzymes are still not recognized as nonhomologous, even with substantial evidence indicating different evolutionary histories. In this work, we begin to investigate the biological significance of the cooccurrence of nonhomologous isofunctional enzymes in human metabolism, characterizing functional analogous enzymes identified in metabolic pathways annotated in the human genome. Our hypothesis is that the coexistence of multiple enzymatic forms might not be interpreted as functional redundancy. Instead, these enzymatic forms may be implicated in distinct (and probably relevant) biological roles. PMID:28854631

Nanobio interfaces: charge control of enzyme/inorganic interfaces for advanced biocatalysis.

PubMed

Deshapriya, Inoka K; Kumar, Challa V

2013-11-19

Specific approaches to the rational design of nanobio interfaces for enzyme and protein binding to nanomaterials are vital for engineering advanced, functional nanobiomaterials for biocatalysis, sensing, and biomedical applications. This feature article presents an overview of our recent discoveries on structural, functional, and mechanistic details of how enzymes interact with inorganic nanomaterials and how they can be controlled in a systematic manner using α-Zr(IV)phosphate (α-ZrP) as a model system. The interactions of a number of enzymes having a wide array of surface charges, sizes, and functional groups are investigated. Interactions are carefully controlled to screen unfavorable repulsions and enhance favorable interactions for high affinity, structure retention, and activity preservation. In specific cases, catalytic activities and substrate selectivities are improved over those of the pristine enzymes, and two examples of high activity near the boiling point of water have been demonstrated. Isothermal titration calorimetric studies indicated that enzyme binding is coupled to ion sequestration or release to or from the nanobio interface, and binding is controlled in a rational manner. We learned that (1) bound enzyme stabilities are improved by lowering the entropy of the denatured state; (2) maximal loadings are obtained by matching charge footprints of the enzyme and the nanomaterial surface; (3) binding affinities are improved by ion sequestration at the nanobio interface; and (4) maximal enzyme structure retention is obtained by biophilizing the nanobio interface with protein glues. The chemical and physical manipulations of the nanobio interface are significant not only for understanding the complex behaviors of enzymes at biological interfaces but also for desiging better functional nanobiomaterials for a wide variety of practical applications.

Enzyme dehydration using Microglassification™ preserves the protein's structure and function.

PubMed

Aniket; Gaul, David A; Bitterfield, Deborah L; Su, Jonathan T; Li, Victoria M; Singh, Ishita; Morton, Jackson; Needham, David

2015-02-01

Controlled enzyme dehydration using a new processing technique of Microglassification™ has been investigated. Aqueous solution microdroplets of lysozyme, α-chymotrypsin, catalase, and horseradish peroxidase were dehydrated in n-pentanol, n-octanol, n-decanol, triacetin, or butyl lactate, and changes in their structure and function were analyzed upon rehydration. Water solubility and microdroplet dissolution rate in each solvent decreased in the order: butyl lactate > n-pentanol > triacetin > n-octanol > n-decanol. Enzymes Microglassified™ in n-pentanol retained higher activity (93%-98%) than n-octanol (78%-85%) or n-decanol (75%-89%), whereas those Microglassified™ in triacetin (36%-75%) and butyl lactate (48%-79%) retained markedly lower activity. FTIR spectroscopy analyses showed α-helix to β-sheet transformation for all enzymes upon Microglassification™, reflecting a loss of bound water in the dried state; however, the enzymes reverted to native-like conformation upon rehydration. Accelerated stressed-storage tests using Microglassified™ lysozyme showed a significant (p < 0.01) decrease in enzymatic activity from 46,560 ± 2736 to 31,060 ± 4327 units/mg after 3 months of incubation; however, it was comparable to the activity of the lyophilized formulation throughout the test period. These results establish Microglassification™ as a viable technique for enzyme preservation without affecting its structure or function. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Histoplasma capsulatum Encodes a Dipeptidyl Peptidase Active against the Mammalian Immunoregulatory Peptide, Substance P

PubMed Central

Cooper, Kendal G.; Zarnowski, Robert; Woods, Jon P.

2009-01-01

The pathogenic fungus Histoplasma capsulatum secretes dipeptidyl peptidase (Dpp) IV enzyme activity and has two putative DPPIV homologs (HcDPPIVA and HcDPPIVB). We previously showed that HcDPPIVB is the gene responsible for the majority of secreted DppIV activity in H. capsulatum culture supernatant, while we could not detect any functional contribution from HcDPPIVA. In order to determine whether HcDPPIVA encodes a functional DppIV enzyme, we expressed HcDPPIVA in Pichia pastoris and purified the recombinant protein. The recombinant enzyme cleaved synthetic DppIV substrates and had similar biochemical properties to other described DppIV enzymes, with temperature and pH optima of 42°C and 8, respectively. Recombinant HcDppIVA cleaved the host immunoregulatory peptide substance P, indicating the enzyme has the potential to affect the immune response during infection. Expression of HcDPPIVA under heterologous regulatory sequences in H. capsulatum resulted in increased secreted DppIV activity, indicating that the encoded protein can be expressed and secreted by its native organism. However, HcDPPIVA was not required for virulence in a murine model of histoplasmosis. This work reports a fungal enzyme that can function to cleave the immunomodulatory host peptide substance P. PMID:19384411

Angiotensin-converting enzyme 2 activation improves endothelial function.

PubMed

Fraga-Silva, Rodrigo A; Costa-Fraga, Fabiana P; Murça, Tatiane M; Moraes, Patrícia L; Martins Lima, Augusto; Lautner, Roberto Q; Castro, Carlos H; Soares, Célia Maria A; Borges, Clayton L; Nadu, Ana Paula; Oliveira, Marilene L; Shenoy, Vinayak; Katovich, Michael J; Santos, Robson A S; Raizada, Mohan K; Ferreira, Anderson J

2013-06-01

Diminished release and function of endothelium-derived nitric oxide coupled with increases in reactive oxygen species production is critical in endothelial dysfunction. Recent evidences have shown that activation of the protective axis of the renin-angiotensin system composed by angiotensin-converting enzyme 2, angiotensin-(1-7), and Mas receptor promotes many beneficial vascular effects. This has led us to postulate that activation of intrinsic angiotensin-converting enzyme 2 would improve endothelial function by decreasing the reactive oxygen species production. In the present study, we tested 1-[[2-(dimetilamino)etil]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT), a small molecule angiotensin-converting enzyme 2 activator, on endothelial function to validate this hypothesis. In vivo treatment with XNT (1 mg/kg per day for 4 weeks) improved the endothelial function of spontaneously hypertensive rats and of streptozotocin-induced diabetic rats when evaluated through the vasorelaxant responses to acetylcholine/sodium nitroprusside. Acute in vitro incubation with XNT caused endothelial-dependent vasorelaxation in aortic rings of rats. This vasorelaxation effect was attenuated by the Mas antagonist D-pro7-Ang-(1-7), and it was reduced in Mas knockout mice. These effects were associated with reduction in reactive oxygen species production. In addition, Ang II-induced reactive oxygen species production in human aortic endothelial cells was attenuated by preincubation with XNT. These results showed that chronic XNT administration improves the endothelial function of hypertensive and diabetic rat vessels by attenuation of the oxidative stress. Moreover, XNT elicits an endothelial-dependent vasorelaxation response, which was mediated by Mas. Thus, this study indicated that angiotensin-converting enzyme 2 activation promotes beneficial effects on the endothelial function and it is a potential target for treating cardiovascular disease.

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.

The Ω-loop lid domain of phosphoenolpyruvate carboxykinase is essential for catalytic function

PubMed Central

Johnson, Troy A.; Holyoak, Todd

2012-01-01

Phosphoenolpyruvate carboxykinase (PEPCK) is an essential metabolic enzyme operating in the gluconeogenesis and glyceroneogenesis pathways. Recent studies have demonstrated that the enzyme contains a mobile active site lid domain that transitions between an open/disorded conformation to a closed/ordered conformation as the enzyme progresses through the catalytic cycle. The understanding of how this mobile domain functions in catalysis is incomplete. Previous studies show that the closure of the lid domain stabilizes the reaction intermediate and protects the reactive intermediate from spurious protonation and thus contributes to the fidelity of the enzyme. In order to more fully investigate the roles of the lid domain in PEPCK function we created three mutations that replaced the 11-residue lid domain with one, two or three glycine residues. Kinetic analysis of the mutant enzymes demonstrates that none of the enzyme constructs exhibit any measurable kinetic activity resulting in a decrease in the catalytic parameters by at least 106. Structural characterization of the mutants in complexes representing the catalytic cycle suggest that the inactivity is due to a role for the lid domain in the formation of the fully closed state of the enzyme that is required for catalytic function. In the absence of the lid domain, the enzyme is unable to achieve the fully closed state and is rendered inactive despite possessing all of the residues and substrates required for catalytic function. This work demonstrates how enzyme catalytic function can be abolished through the alteration of conformational equilibria despite all elements required for chemical conversion of substrates to products remaining intact. PMID:23127136

Long-term fertilization, but not warming, shifts rates of ectomycorrhizal nutrient cycling in Arctic tussock tundra.

NASA Astrophysics Data System (ADS)

Dunleavy, H.; Mack, M. C.

2017-12-01

The role of ectomycorrhizae (ECM) in Arctic nutrient cycling may be changing as temperature, nutrient availability, and ECM shrub abundance and size increase. A shift in ECM function has been proposed as a possible mechanism for shrub expansion. While several studies demonstrate a higher abundance of ECM as well as community compositional shifts in response to long-term experimental warming and fertilization, direct measurements of functional responses are missing. To understand the potential role of ECM in soil biogeochemical processes of the changing Arctic, we investigated the functional response of ECM to 30 years of summer warming and increased nutrient availability by measuring potential activities of extracellular enzymes associated with nitrogen (N) and phosphorous (P) acquisition on ECM root tips. We hypothesize ECM enzyme activities will be higher with warmer temperatures. Conversely, fertilization will lower ECM enzyme activities as N and P become less limiting to host plants. Preliminary results strongly support our latter hypothesis, but not the first. Warming decreased hydrolytic P-associated and labile N-associated enzyme activities on individual root tips (pmol/min/mm2 root tip) by 30% and 83%, respectively. However, warming increased ECM abundance and did not alter community-level activities (pmol/min/cm3 soil). Fertilization decreased hydrolytic and oxidative enzymatic activities on individual root tips by 34 to 80% as well as on a community level by 67 to 93%, even though ECM shrubs were almost monodominant. The combined effect of warming and fertilization decreased labile N-associated enzyme activity by 82%, but had little effect on oxidative and other hydrolytic enzyme activities. Although both warming and fertilization decreased root tip activities, reflecting a potential reduction in plant allocation to mycorrhizal nutrient acquisition, only fertilization lowered rates of ECM nutrient cycling. The indirect relationship between ECM abundance and individual root tip activity highlights the importance of measuring ECM function to assess the role of this symbiosis in nutrient cycling.

STRUCTURAL AND FUNCTIONAL CONSEQUENCES OF CIRCULAR PERMUTATION ON THE ACTIVE SITE OF OLD YELLOW ENZYME.

PubMed

Daugherty, Ashley B; Horton, John R; Cheng, Xiaodong; Lutz, Stefan

2015-02-06

Circular permutation of the NADPH-dependent oxidoreductase Old Yellow Enzyme from Saccharomyces pastorianus (OYE1) can significantly enhance the enzyme's catalytic performance. Termini relocation into four regions of the protein (sectors I-IV) near the active site has proven effective in altering enzyme function. To better understand the structural consequences and rationalize the observed functional gains in these OYE1 variants, we selected representatives from sectors I-III for further characterization by biophysical methods and X-ray crystallography. These investigations not only show trends in enzyme stability and quaternary structure as a function of termini location, but also provide a possible explanation for the catalytic gains in our top-performing OYE variant (new N-terminus at residue 303; sector III). Crystallographic analysis indicates that termini relocation into sector III affects the loop β6 region (amino acid positions: 290-310) of OYE1 which forms a lid over the active site. Peptide backbone cleavage greatly enhances local flexibility, effectively converting the loop into a tether and consequently increasing the environmental exposure of the active site. Interestingly, such active site remodeling does not negatively impact the enzyme's activity and stereoselectivity, nor does it perturb the conformation of other key active site residues with the exception of Y375. These observations were confirmed in truncation experiments, deleting all residues of the loop β6 region in our OYE variant. Intrigued by the finding that circular permutation leaves most of the key catalytic residues unchanged, we also tested OYE permutants for possible additive or synergistic effects of amino acid substitutions. Distinct functional changes in these OYE variants were detected upon mutations at W116, known in native OYE1 to cause inversion of diastereo-selectivity for ( S )-carvone reduction. Our findings demonstrate the contribution of loop β6 toward determining the stereoselectivity of OYE1, an important insight for future OYE engineering efforts.

Cajanus cajan Linn. (Leguminosae) prevents alcohol-induced rat liver damage and augments cytoprotective function.

PubMed

Kundu, Rakesh; Dasgupta, Suman; Biswas, Anindita; Bhattacharya, Anirban; Pal, Bikas C; Bandyopadhyay, Debashis; Bhattacharya, Shelley; Bhattacharya, Samir

2008-08-13

Cajanus cajan Linn. (Leguminosae) is a nontoxic edible herb, widely used in Indian folk medicine for the prevention of various liver disorders. In the present study we have demonstrated that methanol-aqueous fraction (MAF2) of Cajanus cajan leaf extract could prevent the chronically treated alcohol induced rat liver damage. Chronic doses of alcohol (3.7 g/ kg) orally administered to rats for 28 days and liver function marker enzymes such as GPT, GOT, ALP and anti-oxidant enzyme activities were determined. Effect of MAF2 at a dose of 50mg/kg body weight on alcohol treated rats was noted. Alcohol effected significant increase in liver marker enzyme activities and reduced the activities of anti-oxidant enzymes. Co-administration of MAF2 reversed the liver damage due to alcohol; it decreased the activities of liver marker enzymes and augmented antioxidant enzyme activities. We also demonstrate significant decrease of the phase II detoxifying enzyme, UDP-glucuronosyl transferase (UGT) activity along with a three- and two-fold decrease of UGT2B gene and protein expression respectively. MAF2 co-administration normalized UGT activity and revived the expression of UGT2B with a concomitant expression and nuclear translocation of Nrf2, a transcription factor that regulates the expression of many cytoprotective genes. Cajanus cajan extract therefore shows a promise in therapeutic use in alcohol induced liver dysfunction.

Structure-Function Studies with the Unique Hexameric Form II Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) from Rhodopseudomonas palustris*

PubMed Central

Satagopan, Sriram; Chan, Sum; Perry, L. Jeanne; Tabita, F. Robert

2014-01-01

The first x-ray crystal structure has been solved for an activated transition-state analog-bound form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This enzyme, from Rhodopseudomonas palustris, assembles as a unique hexamer with three pairs of catalytic large subunit homodimers around a central 3-fold symmetry axis. This oligomer arrangement is unique among all known Rubisco structures, including the form II homolog from Rhodospirillum rubrum. The presence of a transition-state analog in the active site locked the activated enzyme in a “closed” conformation and revealed the positions of critical active site residues during catalysis. Functional roles of two form II-specific residues (Ile165 and Met331) near the active site were examined via site-directed mutagenesis. Substitutions at these residues affect function but not the ability of the enzyme to assemble. Random mutagenesis and suppressor selection in a Rubisco deletion strain of Rhodobacter capsulatus identified a residue in the amino terminus of one subunit (Ala47) that compensated for a negative change near the active site of a neighboring subunit. In addition, substitution of the native carboxyl-terminal sequence with the last few dissimilar residues from the related R. rubrum homolog increased the enzyme's kcat for carboxylation. However, replacement of a longer carboxyl-terminal sequence with termini from either a form III or a form I enzyme, which varied both in length and sequence, resulted in complete loss of function. From these studies, it is evident that a number of subtle interactions near the active site and the carboxyl terminus account for functional differences between the different forms of Rubiscos found in nature. PMID:24942737

Structure-function studies with the unique hexameric form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodopseudomonas palustris.

PubMed

Satagopan, Sriram; Chan, Sum; Perry, L Jeanne; Tabita, F Robert

2014-08-01

The first x-ray crystal structure has been solved for an activated transition-state analog-bound form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This enzyme, from Rhodopseudomonas palustris, assembles as a unique hexamer with three pairs of catalytic large subunit homodimers around a central 3-fold symmetry axis. This oligomer arrangement is unique among all known Rubisco structures, including the form II homolog from Rhodospirillum rubrum. The presence of a transition-state analog in the active site locked the activated enzyme in a "closed" conformation and revealed the positions of critical active site residues during catalysis. Functional roles of two form II-specific residues (Ile(165) and Met(331)) near the active site were examined via site-directed mutagenesis. Substitutions at these residues affect function but not the ability of the enzyme to assemble. Random mutagenesis and suppressor selection in a Rubisco deletion strain of Rhodobacter capsulatus identified a residue in the amino terminus of one subunit (Ala(47)) that compensated for a negative change near the active site of a neighboring subunit. In addition, substitution of the native carboxyl-terminal sequence with the last few dissimilar residues from the related R. rubrum homolog increased the enzyme's kcat for carboxylation. However, replacement of a longer carboxyl-terminal sequence with termini from either a form III or a form I enzyme, which varied both in length and sequence, resulted in complete loss of function. From these studies, it is evident that a number of subtle interactions near the active site and the carboxyl terminus account for functional differences between the different forms of Rubiscos found in nature. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteomic analyses for profiling regulated proteins/enzymes by Fucus vesiculosus fucoidan in B16 melanoma cells: A combination of enzyme kinetics functional study.

PubMed

Wang, Zhi-Jiang; Zheng, Li; Yang, Jun-Mo; Kang, Yani; Park, Yong-Doo

2018-06-01

Fucoidans are complex sulfated polysaccharides that have a wide range of biological activities. Previously, we reported the various effects of Fucus vesiculosus fucoidan on tyrosinase and B16 melanoma cells. In this study, to identify fucoidan-targeted proteins in B16 melanoma cells, we performed a proteomics study and integrated enzyme kinetics. We detected 19 candidate proteins dysregulated by fucoidan treatment. Among the probed proteins, the enzyme kinetics of two candidate enzymes, namely lactate dehydrogenase (LDH) as an upregulated protein and superoxide dismutase (SOD) as a downregulated enzyme, were determined. The enzyme kinetics results showed that Fucus vesiculosus fucoidan significantly inhibited LDH catalytic function while it did not affect SOD activity even at a high dose, while only slightly decreased activity (up to 10%) at a low dose. Based on our previous and present observations, fucoidan could inhibit B16 melanoma cells growth via regulating proteins/enzymes expression levels such as LDH and SOD known as cell survival biomarkers. Interestingly, both expression level and enzyme catalytic activity of LDH were regulated by fucoidan, which could directly induce the apoptotic effect on B16 melanoma cells along with SOD downregulation. This study highlights how combining proteomics with enzyme kinetics can yield valuable insights into fucoidan targets. Copyright © 2018 Elsevier B.V. All rights reserved.

A single molecule perspective on the functional diversity of in vitro evolved β-glucuronidase.

PubMed

Liebherr, Raphaela B; Renner, Max; Gorris, Hans H

2014-04-23

The mechanisms that drive the evolution of new enzyme activity have been investigated by comparing the kinetics of wild-type and in vitro evolved β-glucuronidase (GUS) at the single molecule level. Several hundred single GUS molecules were separated in large arrays of 62,500 ultrasmall reaction chambers etched into the surface of a fused silica slide to observe their individual substrate turnover rates in parallel by fluorescence microscopy. Individual GUS molecules feature long-lived but divergent activity states, and their mean activity is consistent with classic Michaelis-Menten kinetics. The large number of single molecule substrate turnover rates is representative of the activity distribution within an entire enzyme population. Partially evolved GUS displays a much broader activity distribution among individual enzyme molecules than wild-type GUS. The broader activity distribution indicates a functional division of work between individual molecules in a population of partially evolved enzymes that-as so-called generalists-are characterized by their promiscuous activity with many different substrates.

Structural similarities and functional differences clarify evolutionary relationships between tRNA healing enzymes and the myelin enzyme CNPase.

PubMed

Muruganandam, Gopinath; Raasakka, Arne; Myllykoski, Matti; Kursula, Inari; Kursula, Petri

2017-05-16

Eukaryotic tRNA splicing is an essential process in the transformation of a primary tRNA transcript into a mature functional tRNA molecule. 5'-phosphate ligation involves two steps: a healing reaction catalyzed by polynucleotide kinase (PNK) in association with cyclic phosphodiesterase (CPDase), and a sealing reaction catalyzed by an RNA ligase. The enzymes that catalyze tRNA healing in yeast and higher eukaryotes are homologous to the members of the 2H phosphoesterase superfamily, in particular to the vertebrate myelin enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). We employed different biophysical and biochemical methods to elucidate the overall structural and functional features of the tRNA healing enzymes yeast Trl1 PNK/CPDase and lancelet PNK/CPDase and compared them with vertebrate CNPase. The yeast and the lancelet enzymes have cyclic phosphodiesterase and polynucleotide kinase activity, while vertebrate CNPase lacks PNK activity. In addition, we also show that the healing enzymes are structurally similar to the vertebrate CNPase by applying synchrotron radiation circular dichroism spectroscopy and small-angle X-ray scattering. We provide a structural analysis of the tRNA healing enzyme PNK and CPDase domains together. Our results support evolution of vertebrate CNPase from tRNA healing enzymes with a loss of function at its N-terminal PNK-like domain.

Functional Role of Tyr12 in the Catalytic Activity of Novel Zeta-like Glutathione S-transferase from Acidovorax sp. KKS102.

PubMed

Shehu, Dayyabu; Alias, Zazali

2018-05-19

Glutathione S-transferases (GSTs) are a family of enzymes that function in the detoxification of variety of electrophilic substrates. In the present work, we report a novel zeta-like GST (designated as KKSG9) from the biphenyl/polychlorobiphenyl degrading organism Acidovorax sp. KKS102. KKSG9 possessed low sequence similarity but similar biochemical properties to zeta class GSTs. Functional analysis showed that the enzyme exhibits wider substrate specificity compared to most zeta class GSTs by reacting with 1-chloro-2,4-dinitrobenzene (CDNB), p-nitrobenzyl chloride (NBC), ethacrynic acid (EA), hydrogen peroxide, and cumene hydroperoxide. The enzyme also displayed dehalogenation function against dichloroacetate, permethrin, and dieldrin. The functional role of Tyr12 was also investigated by site-directed mutagenesis. The mutant (Y12C) displayed low catalytic activity and dehalogenation function against all the substrates when compared with the wild type. Kinetic analysis using NBC and GSH as substrates showed that the mutant (Y12C) displayed a higher affinity for NBC when compared with the wild type, however, no significant change in GSH affinity was observed. These findings suggest that the presence of tyrosine residue in the motif might represent an evolutionary trend toward improving the catalytic activity of the enzyme. The enzyme as well could be useful in the bioremediation of various types of organochlorine pollutants.

Evolutionary dynamics of enzymes.

PubMed

Demetrius, L

1995-08-01

This paper codifies and rationalizes the large diversity in reaction rates and substrate specificity of enzymes in terms of a model which postulates that the kinetic properties of present-day enzymes are the consequence of the evolutionary force of mutation and selection acting on a class of primordial enzymes with poor catalytic activity and broad substrate specificity. Enzymes are classified in terms of their thermodynamic parameters, activation enthalpy delta H* and activation entropy delta S*, in their kinetically significant transition states as follows: type 1, delta H* > 0, delta S* < 0; type 2, delta H* < or = 0, delta S* < or = 0; type 3, delta H* > 0, delta S* > 0. We study the evolutionary dynamics of these three classes of enzymes subject to mutation, which acts at the level of the gene which codes for the enzyme and selection, which acts on the organism that contains the enzyme. Our model predicts the following evolutionary trends in the reaction rate and binding specificity for the three classes of molecules. In type 1 enzymes, evolution results in random, non-directional changes in the reaction rate and binding specificity. In type 2 and 3 enzymes, evolution results in a unidirectional increase in both the reaction rate and binding specificity. We exploit these results in order to codify the diversity in functional properties of present-day enzymes. Type 1 molecules will be described by intermediate reaction rates and broad substrate specificity. Type 2 enzymes will be characterized by diffusion-controlled rates and absolute substrate specificity. The type 3 catalysts can be further subdivided in terms of their activation enthalpy into two classes: type 3a (delta H* small) and type 3b (delta H* large). We show that type 3a will be represented by the same functional properties that identify type 2, namely, diffusion-controlled rates and absolute substrate specificity, whereas type 3b will be characterized by non-diffusion-controlled rates and absolute substrate specificity. We infer from this depiction of the three classes of enzymes, a general relation between the two functional properties, reaction rate and substrate specificity, namely, enzymes with diffusion-controlled rates have absolute substrate specificity. By appealing to energetic considerations, we furthermore show that enzymes with diffusion-controlled rates (types 2 and 3a) form a small subset of the class of all enzymes. This codification of present-day enzymes derived from an evolutionary model, essentially relates the structural properties of enzymes, as described by their thermodynamic parameters, to their functional properties, as represented by the reaction rate and substrate specificity.

RUTBC1 Functions as a GTPase-activating Protein for Rab32/38 and Regulates Melanogenic Enzyme Trafficking in Melanocytes.

PubMed

Marubashi, Soujiro; Shimada, Hikaru; Fukuda, Mitsunori; Ohbayashi, Norihiko

2016-01-15

Two cell type-specific Rab proteins, Rab32 and Rab38 (Rab32/38), have been proposed as regulating the trafficking of melanogenic enzymes, including tyrosinase and tyrosinase-related protein 1 (Tyrp1), to melanosomes in melanocytes. Like other GTPases, Rab32/38 function as switch molecules that cycle between a GDP-bound inactive form and a GTP-bound active form; the cycle is thought to be regulated by an activating enzyme, guanine nucleotide exchange factor (GEF), and an inactivating enzyme, GTPase-activating protein (GAP), which stimulates the GTPase activity of Rab32/38. Although BLOC-3 has already been identified as a Rab32/38-specific GEF that regulates the trafficking of tyrosinase and Tyrp1, no physiological GAP for Rab32/38 in melanocytes has ever been identified, and it has remained unclear whether Rab32/38 is involved in the trafficking of dopachrome tautomerase, another melanogenic enzyme, in mouse melanocytes. In this study we investigated RUTBC1, which was originally characterized as a Rab9-binding protein and GAP for Rab32 and Rab33B in vitro, and the results demonstrated that RUTBC1 functions as a physiological GAP for Rab32/38 in the trafficking of all three melanogenic enzymes in mouse melanocytes. The results of this study also demonstrated the involvement of Rab9A in the regulation of the RUTBC1 localization and in the trafficking of all three melanogenic enzymes. We discovered that either excess activation or inactivation of Rab32/38 achieved by manipulating RUTBC1 inhibits the trafficking of all three melanogenic enzymes. These results collectively indicate that proper spatiotemporal regulation of Rab32/38 is essential for the trafficking of all three melanogenic enzymes in mouse melanocytes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Letterman Army Institute of Research Annual Research Progress Report, FY 1981.

DTIC Science & Technology

1981-10-01

with physiology and mech- anisms of skin damage and repair. The mechanisms by which nerve agents and vesicants produce physiologic aberration and...enzyme vital for nerve function. Organophos- phates react rapidly and covalently with the enzyme to produce an in- active enzyme. Reactivation of the...warfare agents . These organic molecules may also alter the natural defense mechanism by activating or deactivating enzymes in the skin that destroy

Gold core/ceria shell-based redox active nanozyme mimicking the biological multienzyme complex phenomenon

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

Bhagat, Stuti; Srikanth Vallabani, N. V.; Shutthanandan, Vaithiyalingam

Catalytically active individual gold (Au) and cerium oxide (CeO2) nanoparticles are well known to exhibit specific enzyme-like activities, such as natural catalase, oxidase, superoxide dismutase, and peroxidase enzymes. These activities have been maneuvered to design several biological applications such as immunoassays, glucose detection, radiation and free radical protection and tissue engineering. A functional nanozyme depicting multienzyme like properties that functions as a synthetic super enzyme has eluded the researchers in the nanoscience community for past decade. In current report, we have designed a functional multienzyme in the form of Gold (core)-CeO2 (shell) nanoparticles (Au@CeO2 CSNPs) exhibiting excellent peroxidase, catalase andmore » superoxide dismutase enzyme-like activities that are controlled simply by tuning the pH. The reaction kinetic parameters reveal that the peroxidase-like activity of this core shell nanozyme is comparable to natural HRP enzyme. Unlike peroxidase-like activity exhibited by other nanomaterials, Au@CeO2 CSNPs showed decrease in hydroxyl radical formation, suggesting that the bio catalytic reactions are performed by efficient electron transfers. A significant enzyme-like activity of this core shell nanoparticle was conserved at extreme pH (2 – 11) and temperatures (up to 90 °C), clearly suggesting the superiority over natural enzymes. Further, the utility of peroxidase-like activity of this core shell nanoparticles was extended for the detection of glucose, which showed a linear range of detection between (100 µM – 1 mM). It is hypothesized that the proximity of the redox potentials of Au+/Au and Ce (III)/Ce (IV) may result in a redox couple promoting the multienzyme activity of core shell nanoparticles. Au@CeO2 CSNPs may open new directions for development of single platform sensors in multiple biosensing applications.« less

RNA as an Enzyme.

ERIC Educational Resources Information Center

Cech, Thomas R.

1986-01-01

Reviews current findings that explain RNA's function as an enzyme in addition to being an informational molecule. Highlights recent research efforts and notes changes in the information base on RNA activity. Includes models and diagrams of RNA activity. (ML)

Coupling between Catalytic Loop Motions and Enzyme Global Dynamics

PubMed Central

Kurkcuoglu, Zeynep; Bakan, Ahmet; Kocaman, Duygu; Bahar, Ivet; Doruker, Pemra

2012-01-01

Catalytic loop motions facilitate substrate recognition and binding in many enzymes. While these motions appear to be highly flexible, their functional significance suggests that structure-encoded preferences may play a role in selecting particular mechanisms of motions. We performed an extensive study on a set of enzymes to assess whether the collective/global dynamics, as predicted by elastic network models (ENMs), facilitates or even defines the local motions undergone by functional loops. Our dataset includes a total of 117 crystal structures for ten enzymes of different sizes and oligomerization states. Each enzyme contains a specific functional/catalytic loop (10–21 residues long) that closes over the active site during catalysis. Principal component analysis (PCA) of the available crystal structures (including apo and ligand-bound forms) for each enzyme revealed the dominant conformational changes taking place in these loops upon substrate binding. These experimentally observed loop reconfigurations are shown to be predominantly driven by energetically favored modes of motion intrinsically accessible to the enzyme in the absence of its substrate. The analysis suggests that robust global modes cooperatively defined by the overall enzyme architecture also entail local components that assist in suitable opening/closure of the catalytic loop over the active site. PMID:23028297

Biocatalysts based on nanozeolite-enzyme complexes: Effects of alkoxysilane surface functionalization and biofuel production using microalgae lipids feedstock.

PubMed

de Vasconcellos, Adriano; Miller, Alex Henrique; Aranda, Donato A G; Nery, José Geraldo

2018-05-01

Nanozeolites with different crystallographic structures (Nano/TS1, Nano/GIS, Nano/LTA, Nano/BEA, Nano/X, and Nano-X/Ni), functionalized with (3-aminopropyl)trimethoxysilane (APTMS) and crosslinked with glutaraldehyde (GA), were studied as solid supports for Thermomyces lanuginosus lipase (TLL) immobilization. Physicochemical characterizations of the surface-functionalized nanozeolites and nanozeolite-enzyme complexes were performed using XRD, SEM, AFM, ATR-FTIR, and zeta potential measurements. The experimental enzymatic activity results indicated that the nanozeolitic supports functionalized with APTMS and GA immobilized larger amounts of enzymes and provided higher enzymatic activities, compared to unfunctionalized supports. Correlations were observed among the nanozeolite surface charges, the enzyme immobilization efficiencies, and the biocatalyst activities. The catalytic performance and reusability of these enzyme-nanozeolite complexes were evaluated in the ethanolysis transesterification of microalgae oil to fatty acid ethyl esters (FAEEs). TLL immobilized on the nanozeolite supports functionalized with APTMS and GA provided the most efficient biocatalysis, with FAEEs yields above 93% and stability during five reaction cycles. Lower FAEEs yields and poorer catalytic stability were found for nanozeolite-enzyme complexes prepared only by physical adsorption. The findings indicated the viability of designing highly efficient biocatalysts for biofuel production by means of chemical modulation of nanozeolite surfaces. The high biocatalyst catalytic efficiency observed in ethanolysis reactions using a lipid feedstock that does not compete with food production is an advantage that should encourage the industrial application of these biocatalysts. Copyright © 2018 Elsevier B.V. All rights reserved.

Radiofrequency treatment enhances the catalytic function of an immobilized nanobiohybrid catalyst

NASA Astrophysics Data System (ADS)

San, Boi Hoa; Ha, Eun-Ju; Paik, Hyun-Jong; Kim, Kyeong Kyu

2014-05-01

Biocatalysis, the use of enzymes in chemical transformation, has undergone intensive development for a wide range of applications. As such, maximizing the functionality of enzymes for biocatalysis is a major priority to enable industrial use. To date, many innovative technologies have been developed to address the future demand of enzymes for these purposes, but maximizing the catalytic activity of enzymes remains a challenge. In this study, we demonstrated that the functionality of a nanobiocatalyst could be enhanced by combining immobilization and radiofrequency (RF) treatment. Aminopeptidase PepA-encapsulating 2 nm platinum nanoparticles (PepA-PtNPs) with the catalytic activities of hydrolysis and hydrogenation were employed as multifunctional nanobiocatalysts. Immobilizing the nanobiocatalysts in a hydrogel using metal chelation significantly enhanced their functionalities, including catalytic power, thermal-stability, pH tolerance, organic solvent tolerance, and reusability. Most importantly, RF treatment of the hydrogel-immobilized PepA-PtNPs increased their catalytic power by 2.5 fold greater than the immobilized PepA. Our findings indicate that the catalytic activities and functionalities of PepA-PtNPs are greatly enhanced by the combination of hydrogel-immobilization and RF treatment. Based on our findings, we propose that RF treatment of nanobiohybrid catalysts immobilized on the bulk hydrogel represents a new strategy for achieving efficient biocatalysis.Biocatalysis, the use of enzymes in chemical transformation, has undergone intensive development for a wide range of applications. As such, maximizing the functionality of enzymes for biocatalysis is a major priority to enable industrial use. To date, many innovative technologies have been developed to address the future demand of enzymes for these purposes, but maximizing the catalytic activity of enzymes remains a challenge. In this study, we demonstrated that the functionality of a nanobiocatalyst could be enhanced by combining immobilization and radiofrequency (RF) treatment. Aminopeptidase PepA-encapsulating 2 nm platinum nanoparticles (PepA-PtNPs) with the catalytic activities of hydrolysis and hydrogenation were employed as multifunctional nanobiocatalysts. Immobilizing the nanobiocatalysts in a hydrogel using metal chelation significantly enhanced their functionalities, including catalytic power, thermal-stability, pH tolerance, organic solvent tolerance, and reusability. Most importantly, RF treatment of the hydrogel-immobilized PepA-PtNPs increased their catalytic power by 2.5 fold greater than the immobilized PepA. Our findings indicate that the catalytic activities and functionalities of PepA-PtNPs are greatly enhanced by the combination of hydrogel-immobilization and RF treatment. Based on our findings, we propose that RF treatment of nanobiohybrid catalysts immobilized on the bulk hydrogel represents a new strategy for achieving efficient biocatalysis. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr00407h

Extending enzyme molecular recognition with an expanded amino acid alphabet

PubMed Central

Windle, Claire L.; Simmons, Katie J.; Ault, James R.; Trinh, Chi H.; Nelson, Adam

2017-01-01

Natural enzymes are constructed from the 20 proteogenic amino acids, which may then require posttranslational modification or the recruitment of coenzymes or metal ions to achieve catalytic function. Here, we demonstrate that expansion of the alphabet of amino acids can also enable the properties of enzymes to be extended. A chemical mutagenesis strategy allowed a wide range of noncanonical amino acids to be systematically incorporated throughout an active site to alter enzymic substrate specificity. Specifically, 13 different noncanonical side chains were incorporated at 12 different positions within the active site of N-acetylneuraminic acid lyase (NAL), and the resulting chemically modified enzymes were screened for activity with a range of aldehyde substrates. A modified enzyme containing a 2,3-dihydroxypropyl cysteine at position 190 was identified that had significantly increased activity for the aldol reaction of erythrose with pyruvate compared with the wild-type enzyme. Kinetic investigation of a saturation library of the canonical amino acids at the same position showed that this increased activity was not achievable with any of the 20 proteogenic amino acids. Structural and modeling studies revealed that the unique shape and functionality of the noncanonical side chain enabled the active site to be remodeled to enable more efficient stabilization of the transition state of the reaction. The ability to exploit an expanded amino acid alphabet can thus heighten the ambitions of protein engineers wishing to develop enzymes with new catalytic properties. PMID:28196894

Characterising Complex Enzyme Reaction Data

PubMed Central

Rahman, Syed Asad; Thornton, Janet M.

2016-01-01

The relationship between enzyme-catalysed reactions and the Enzyme Commission (EC) number, the widely accepted classification scheme used to characterise enzyme activity, is complex and with the rapid increase in our knowledge of the reactions catalysed by enzymes needs revisiting. We present a manual and computational analysis to investigate this complexity and found that almost one-third of all known EC numbers are linked to more than one reaction in the secondary reaction databases (e.g., KEGG). Although this complexity is often resolved by defining generic, alternative and partial reactions, we have also found individual EC numbers with more than one reaction catalysing different types of bond changes. This analysis adds a new dimension to our understanding of enzyme function and might be useful for the accurate annotation of the function of enzymes and to study the changes in enzyme function during evolution. PMID:26840640

Activity-based protein profiling: from enzyme chemistry to proteomic chemistry.

PubMed

Cravatt, Benjamin F; Wright, Aaron T; Kozarich, John W

2008-01-01

Genome sequencing projects have provided researchers with a complete inventory of the predicted proteins produced by eukaryotic and prokaryotic organisms. Assignment of functions to these proteins represents one of the principal challenges for the field of proteomics. Activity-based protein profiling (ABPP) has emerged as a powerful chemical proteomic strategy to characterize enzyme function directly in native biological systems on a global scale. Here, we review the basic technology of ABPP, the enzyme classes addressable by this method, and the biological discoveries attributable to its application.

Agrobotanical attributes, nitrogen-fixation, enzyme activities and nutraceuticals and tyrosinase enzyme of hyacinth bean (Lablab purpureus L.) - a bio-functional medicinal legume.

USDA-ARS?s Scientific Manuscript database

Hyacinth bean (Lablab purpureus L.) accessions of different origins received from USDA, ARS, Plant Genetic Resources Conservation Unit, Griffin, GA, U.S.A. were evaluated for agrobotanical attributes, enzyme activities, nutraceuticals and quality in pot culture at AMU, Aligarh, Uttar Pradesh. Fresh ...

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues.

PubMed

Molenaar, Remco J; Khurshed, Mohammed; Hira, Vashendriya V V; Van Noorden, Cornelis J F

2018-05-26

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation. We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P) + and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase's activity. The formazan's absorbance is therefore a direct measure of the dehydrogenase's activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays. Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an indispensable technique to study metabolism at the cellular or tissue level. The technique is easy to adopt, provides in-depth, comprehensive and integrated metabolic information and enables rapid quantitative analysis.

Caenorhabditis elegans glutamylating enzymes function redundantly in male mating.

PubMed

Chawla, Daniel G; Shah, Ruchi V; Barth, Zachary K; Lee, Jessica D; Badecker, Katherine E; Naik, Anar; Brewster, Megan M; Salmon, Timothy P; Peel, Nina

2016-09-15

Microtubule glutamylation is an important modulator of microtubule function and has been implicated in the regulation of centriole stability, neuronal outgrowth and cilia motility. Glutamylation of the microtubules is catalyzed by a family of tubulin tyrosine ligase-like (TTLL) enzymes. Analysis of individual TTLL enzymes has led to an understanding of their specific functions, but how activities of the TTLL enzymes are coordinated to spatially and temporally regulate glutamylation remains relatively unexplored. We have undertaken an analysis of the glutamylating TTLL enzymes in C. elegans We find that although all five TTLL enzymes are expressed in the embryo and adult worm, loss of individual enzymes does not perturb microtubule function in embryonic cell divisions. Moreover, normal dye-filling, osmotic avoidance and male mating behavior indicate the presence of functional amphid cilia and male-specific neurons. A ttll-4(tm3310); ttll-11(tm4059); ttll-5(tm3360) triple mutant, however, shows reduced male mating efficiency due to a defect in the response step, suggesting that these three enzymes function redundantly, and that glutamylation is required for proper function of the male-specific neurons. © 2016. Published by The Company of Biologists Ltd.

The structure and function of Alzheimer's gamma secretase enzyme complex.

PubMed

Krishnaswamy, Sudarsan; Verdile, Giuseppe; Groth, David; Kanyenda, Limbikani; Martins, Ralph N

2009-01-01

The production and accumulation of the beta amyloid protein (Abeta) is a key event in the cascade of oxidative and inflammatory processes that characterizes Alzheimer's disease (AD). A multi-subunit enzyme complex, referred to as gamma (gamma) secretase, plays a pivotal role in the generation of Abeta from its parent molecule, the amyloid precursor protein (APP). Four core components (presenilin, nicastrin, aph-1, and pen-2) interact in a high-molecular-weight complex to perform intramembrane proteolysis on a number of membrane-bound proteins, including APP and Notch. Inhibitors and modulators of this enzyme have been assessed for their therapeutic benefit in AD. However, although these agents reduce Abeta levels, the majority have been shown to have severe side effects in pre-clinical animal studies, most likely due to the enzymes role in processing other proteins involved in normal cellular function. Current research is directed at understanding this enzyme and, in particular, at elucidating the roles that each of the core proteins plays in its function. In addition, a number of interacting proteins that are not components of gamma-secretase also appear to play important roles in modulating enzyme activity. This review will discuss the structural and functional complexity of the gamma-secretase enzyme and the effects of inhibiting its activity.

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.

A function-based screen for seeking RubisCO active clones from metagenomes: novel enzymes influencing RubisCO activity.

PubMed

Böhnke, Stefanie; Perner, Mirjam

2015-03-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a key enzyme of the Calvin cycle, which is responsible for most of Earth's primary production. Although research on RubisCO genes and enzymes in plants, cyanobacteria and bacteria has been ongoing for years, still little is understood about its regulation and activation in bacteria. Even more so, hardly any information exists about the function of metagenomic RubisCOs and the role of the enzymes encoded on the flanking DNA owing to the lack of available function-based screens for seeking active RubisCOs from the environment. Here we present the first solely activity-based approach for identifying RubisCO active fosmid clones from a metagenomic library. We constructed a metagenomic library from hydrothermal vent fluids and screened 1056 fosmid clones. Twelve clones exhibited RubisCO activity and the metagenomic fragments resembled genes from Thiomicrospira crunogena. One of these clones was further analyzed. It contained a 35.2 kb metagenomic insert carrying the RubisCO gene cluster and flanking DNA regions. Knockouts of twelve genes and two intergenic regions on this metagenomic fragment demonstrated that the RubisCO activity was significantly impaired and was attributed to deletions in genes encoding putative transcriptional regulators and those believed to be vital for RubisCO activation. Our new technique revealed a novel link between a poorly characterized gene and RubisCO activity. This screen opens the door to directly investigating RubisCO genes and respective enzymes from environmental samples.

Extremophilic Enzymatic Response for Protection against UV-Radiation Damage

DTIC Science & Technology

2012-09-17

superoxide dismutase from the thermophile E1 is a very active enzyme and extremely efficient in its function as antioxidant by capturing superoxide radicals...Ollivet-Besson, Papić, L., Blamey J.M. “Optimization of the antioxidant activity of the enzyme superoxide dismutase from the thermophile E1 induced by...antioxidant enzymes , superoxide dismutase and catalase, from selected microorganisms and the contribution of these enzymes to the resistance to extreme and

Changes of Serum Angiotensin-Converting Enzyme Activity During Treatment of Patients with Graves’ Disease*

PubMed Central

Lee, Dong Soo; Chung, June-Key; Cho, Bo Youn; Koh, Chang-Soon; Lee, Munho

1986-01-01

Serum angiotensin-converting enzyme activity was measured spectrophotometrically, and serum thyrotropin-binding-inhibitory immunoglobulin (TBII) activity was measured by radioreceptor assay in normal subjects and in patients with Graves’ disease serially before and during treatment, and these activities were compared with each other and with thyroid hormone levels in various thyroid functional status. Correlation between serum angiotensin-converting enzyme activity and serum thyroid hormone level was pursued with relation to the changes of thyroid functional status in patients with Graves’ disease during treatment. Serum angiotensin-converting enzyme activity was significantly elevated in patients with hyperthyroid Graves’ disease before the start of treatment (35 ± 13 nmol/min/ml, n=50), and not in patients with Graves’ disease, euthyroid state during treatment with antithyroid drugs or radioactive iodine (23 ± 9 nmol/min/ml, n=12), but decreased significantly in patients with Graves’ disease, hypothyroid state transiently during treatment (15 ± 4 nmol/min/ml, n=12), respectively in comparison with normal control subjects. Serum angiotensin-converting enzyme activity was positively correlated with the log value of serum T3 concentration (r=0.62, p<0.001, n=95), and with the log value of free thyroxine index (r=0.66, p<0.001, n=91) but not statistically significantly with serum TBII activity. Serum angiotensin-converting enzyme activity was followed in 11 patients with initially increased activity and the activity decreased in proportion to serum thyroid hormone level during treatment, irrespective of treatment modality. It is suggested that thyroid hormones play a role in the increase and decrease of serum angiotensin-converting enzyme activity directly or indirectly influencing the peripheral tissues (probably reticuloendothelial cells or peripheral endothelial cells) in patients with Graves’ disease. PMID:15759385

Relating Enzyme Function to Concepts of Dominance and Recessiveness.

ERIC Educational Resources Information Center

Lanza, Janet; Cress, Camille

2001-01-01

Points out the difficulties students have in making connections from the DNA molecule to a phenotype. Presents an activity in which students imitate the actions of functional and nonfunctional enzymes and examine the phenotype of organisms of different genotypes. (YDS)

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.

Exploiting fine-scale genetic and physiological variation of closely related microbes to reveal unknown enzyme functions.

PubMed

Badur, Ahmet H; Plutz, Matthew J; Yalamanchili, Geethika; Jagtap, Sujit Sadashiv; Schweder, Thomas; Unfried, Frank; Markert, Stephanie; Polz, Martin F; Hehemann, Jan-Hendrik; Rao, Christopher V

2017-08-04

Polysaccharide degradation by marine microbes represents one of the largest and most rapid heterotrophic transformations of organic matter in the environment. Microbes employ systems of complementary carbohydrate-specific enzymes to deconstruct algal or plant polysaccharides (glycans) into monosaccharides. Because of the high diversity of glycan substrates, the functions of these enzymes are often difficult to establish. One solution to this problem may lie within naturally occurring microdiversity; varying numbers of enzymes, due to gene loss, duplication, or transfer, among closely related environmental microbes create metabolic differences akin to those generated by knock-out strains engineered in the laboratory used to establish the functions of unknown genes. Inspired by this natural fine-scale microbial diversity, we show here that it can be used to develop hypotheses guiding biochemical experiments for establishing the role of these enzymes in nature. In this work, we investigated alginate degradation among closely related strains of the marine bacterium Vibrio splendidus One strain, V. splendidus 13B01, exhibited high extracellular alginate lyase activity compared with other V. splendidus strains. To identify the enzymes responsible for this high extracellular activity, we compared V. splendidus 13B01 with the previously characterized V. splendidus 12B01, which has low extracellular activity and lacks two alginate lyase genes present in V. splendidus 13B01. Using a combination of genomics, proteomics, biochemical, and functional screening, we identified a polysaccharide lyase family 7 enzyme that is unique to V. splendidus 13B01, secreted, and responsible for the rapid digestion of extracellular alginate. These results demonstrate the value of querying the enzymatic repertoires of closely related microbes to rapidly pinpoint key proteins with beneficial functions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Role of loop L5-6 connecting transmembrane segments M5 and M6 in biogenesis and functioning of yeast Pma1 H+-ATPase.

PubMed

Petrov, V V

2015-01-01

The L5-6 loop is a short extracytoplasmic stretch (714-DNSLDID) connecting transmembrane segments M5 and M6 and forming along with segments M4 and M8 the core through which cations are transported by H+-, Ca2+-, K+,Na+-, H+,K+-, and other P2-ATPases. To study structure-function relationships within this loop of the yeast plasma membrane Pma1 H+-ATPase, alanine- and cysteine-scanning mutagenesis has been employed. Ala and Cys substitutions for the most conserved residue (Leu717) led to complete block in biogenesis preventing the enzyme from reaching secretory vesicles. The Ala replacement at Asp714 led to five-fold decrease in the mutant expression and loss of its activity, while the Cys substitution blocked biogenesis completely. Replacements of other residues did not lead to loss of enzymatic activity. Additional replacements were made for Asp714 and Asp720 (Asp®Asn/Glu). Of the substitutions made at Asp714, only D714N partially restored the mutant enzyme biogenesis and functioning. However, all mutant enzymes with substituted Asp720 were active. The expressed mutants (34-95% of the wild-type level) showed activity high enough (35-108%) to be analyzed in detail. One of the mutants (I719A) had three-fold reduced coupling ratio between ATP hydrolysis and H+ transport; however, the I719C mutation was rather indistinguishable from the wild-type enzyme. Thus, substitutions at two of the seven positions seriously affected biogenesis and/or functioning of the enzyme. Taken together, these results suggest that the M5-M6 loop residues play an important role in protein stability and function, and they are probably responsible for proper arrangement of transmembrane segments M5 and M6 and other domains of the enzyme. This might also be important for the regulation of the enzyme.

A thermodynamic and theoretical view for enzyme regulation.

PubMed

Zhao, Qinyi

2015-01-01

Precise regulation is fundamental to the proper functioning of enzymes in a cell. Current opinions about this, such as allosteric regulation and dynamic contribution to enzyme regulation, are experimental models and substantially empirical. Here we proposed a theoretical and thermodynamic model of enzyme regulation. The main idea is that enzyme regulation is processed via the regulation of abundance of active conformation in the reaction buffer. The theoretical foundation, experimental evidence, and experimental criteria to test our model are discussed and reviewed. We conclude that basic principles of enzyme regulation are laws of protein thermodynamics and it can be analyzed using the concept of distribution curve of active conformations of enzymes.

Immobilization of Lipase from Penicillium sp. Section Gracilenta (CBMAI 1583) on Different Hydrophobic Supports: Modulation of Functional Properties.

PubMed

Turati, Daniela F M; Morais Júnior, Wilson G; Terrasan, César R F; Moreno-Perez, Sonia; Pessela, Benevides C; Fernandez-Lorente, Gloria; Guisan, Jose M; Carmona, Eleonora C

2017-02-22

Lipases are promising enzymes that catalyze the hydrolysis of triacylglycerol ester bonds at the oil/water interface. Apart from allowing biocatalyst reuse, immobilization can also affect enzyme structure consequently influencing its activity, selectivity, and stability. The lipase from Penicillium sp. section Gracilenta (CBMAI 1583) was successfully immobilized on supports bearing butyl, phenyl, octyl, octadecyl, and divinylbenzyl hydrophobic moieties wherein lipases were adsorbed through the highly hydrophobic opened active site. The highest activity in aqueous medium was observed for the enzyme adsorbed on octyl support, with a 150% hyperactivation regarding the soluble enzyme activity, and the highest adsorption strength was verified with the most hydrophobic support (octadecyl Sepabeads), requiring 5% Triton X-100 to desorb the enzyme from the support. Most of the derivatives presented improved properties such as higher stability to pH, temperature, and organic solvents than the covalently immobilized CNBr derivative (prepared under very mild experimental conditions and thus a reference mimicking free-enzyme behavior). A 30.8- and 46.3-fold thermostabilization was achieved in aqueous medium, respectively, by the octyl Sepharose and Toyopearl butyl derivatives at 60 °C, in relation to the CNBr derivative. The octyl- and phenyl-agarose derivatives retained 50% activity after four and seven cycles of p -nitrophenyl palmitate hydrolysis, respectively. Different derivatives exhibited different properties regarding their properties for fish oil hydrolysis in aqueous medium and ethanolysis in anhydrous medium. The most active derivative in ethanolysis of fish oil was the enzyme adsorbed on a surface covered by divinylbenzyl moieties and it was 50-fold more active than the enzyme adsorbed on octadecyl support. Despite having identical mechanisms of immobilization, different hydrophobic supports seem to promote different shapes of the adsorbed open active site of the lipase and hence different functional properties.

Self-Assembling Protein Nanostructures - Towards Active Functionality

DTIC Science & Technology

2011-04-22

the (alpha-beta)8 barrel. A model thermophilic enzyme , natively a tetramer, was engineered as a monomer, a dimer (submitted to Biochimica et...problem. Objective one successfully built high surface area nanoscaffolds from amyloid fibrils, and demonstrated that enzyme activity could be attached...quaternary structure, amyloid fibril, organophosphorous hydrolase, enzyme Juliet Gerrard University of Canterbury 20 Kirkwood Ave Ilam 8041 - REPORT

Liver function in cats with hyperthyroidism before and after 131I therapy.

PubMed

Berent, Allyson C; Drobatz, Kenneth J; Ziemer, Lisa; Johnson, Victoria S; Ward, Cynthia R

2007-01-01

The clinical significance of high serum concentration or activity of markers of liver damage in cats with hyperthyroidism is unknown. To evaluate serum markers of liver function and damage, and ultrasonographic changes in cats with hyperthyroidism and with high liver enzymes, and to determine if abnormalities resolve after treatment with 131I. Nineteen cats with hyperthyroidism (15 with high serum activities of liver enzymes) and 4 age-matched healthy control cats. Serum bile acids, albumin, ammonia, cholesterol, and blood urea nitrogen concentrations, and activities of liver-derived enzymes, and blood glucose concentrations were measured before and after 131I therapy. These values were compared with those of cats that were euthyroid. In addition, gross liver parenchymal changes detected by abdominal ultrasonographic examination, before and after 131I therapy were evaluated. High serum liver enzyme activities were not associated with abnormalities in hepatic parenchyma and liver functional variables, regardless of the degree of increase. Serum liver enzyme activities return to normal after control of hyperthyroidism with 131I therapy. Cats with hyperthyroidism have a significantly higher serum fasting ammonia concentration than cats who were euthyroid (P = .019). Cats with hyperthyroidism also have significantly lower serum cholesterol (P = .005) and glucose (P = .002) concentrations before compared with after 131I therapy. Nine of 19 cats with hyperthyroidism had trace ketonuria. These results demonstrate that extensive examination for hepatobiliary disease in most cats with hyperthyroidism is unnecessary.

Structure and Function in Homodimeric Enzymes: Simulations of Cooperative and Independent Functional Motions.

PubMed

Wells, Stephen A; van der Kamp, Marc W; McGeagh, John D; Mulholland, Adrian J

2015-01-01

Large-scale conformational change is a common feature in the catalytic cycles of enzymes. Many enzymes function as homodimers with active sites that contain elements from both chains. Symmetric and anti-symmetric cooperative motions in homodimers can potentially lead to correlated active site opening and/or closure, likely to be important for ligand binding and release. Here, we examine such motions in two different domain-swapped homodimeric enzymes: the DcpS scavenger decapping enzyme and citrate synthase. We use and compare two types of all-atom simulations: conventional molecular dynamics simulations to identify physically meaningful conformational ensembles, and rapid geometric simulations of flexible motion, biased along normal mode directions, to identify relevant motions encoded in the protein structure. The results indicate that the opening/closure motions are intrinsic features of both unliganded enzymes. In DcpS, conformational change is dominated by an anti-symmetric cooperative motion, causing one active site to close as the other opens; however a symmetric motion is also significant. In CS, we identify that both symmetric (suggested by crystallography) and asymmetric motions are features of the protein structure, and as a result the behaviour in solution is largely non-cooperative. The agreement between two modelling approaches using very different levels of theory indicates that the behaviours are indeed intrinsic to the protein structures. Geometric simulations correctly identify and explore large amplitudes of motion, while molecular dynamics simulations indicate the ranges of motion that are energetically feasible. Together, the simulation approaches are able to reveal unexpected functionally relevant motions, and highlight differences between enzymes.

Structure and Function in Homodimeric Enzymes: Simulations of Cooperative and Independent Functional Motions

PubMed Central

McGeagh, John D.; Mulholland, Adrian J.

2015-01-01

Large-scale conformational change is a common feature in the catalytic cycles of enzymes. Many enzymes function as homodimers with active sites that contain elements from both chains. Symmetric and anti-symmetric cooperative motions in homodimers can potentially lead to correlated active site opening and/or closure, likely to be important for ligand binding and release. Here, we examine such motions in two different domain-swapped homodimeric enzymes: the DcpS scavenger decapping enzyme and citrate synthase. We use and compare two types of all-atom simulations: conventional molecular dynamics simulations to identify physically meaningful conformational ensembles, and rapid geometric simulations of flexible motion, biased along normal mode directions, to identify relevant motions encoded in the protein structure. The results indicate that the opening/closure motions are intrinsic features of both unliganded enzymes. In DcpS, conformational change is dominated by an anti-symmetric cooperative motion, causing one active site to close as the other opens; however a symmetric motion is also significant. In CS, we identify that both symmetric (suggested by crystallography) and asymmetric motions are features of the protein structure, and as a result the behaviour in solution is largely non-cooperative. The agreement between two modelling approaches using very different levels of theory indicates that the behaviours are indeed intrinsic to the protein structures. Geometric simulations correctly identify and explore large amplitudes of motion, while molecular dynamics simulations indicate the ranges of motion that are energetically feasible. Together, the simulation approaches are able to reveal unexpected functionally relevant motions, and highlight differences between enzymes. PMID:26241964

New advances in cell physiology and pathophysiology of the exocrine pancreas.

PubMed

Mössner, Joachim

2010-01-01

This review provides some aspects on the physiology of stimulation and inhibition of pancreatic digestive enzyme secretion and the pathophysiology of pancreatic acinar cell function leading to pancreatitis. Cholecystokinin (CCK) stimulates both directly via CCK-A receptors on acinar cells and indirectly via CCK-B receptors on nerves, followed by acetylcholine release, pancreatic enzyme secretion. It is still not known whether CCK-A receptors exist in human acinar cells, in contrast to acinar cells of rodents where CCK-A receptors have been well described. CCK has numerous actions both in the periphery and in the central nervous systems. CCK inhibits gastric motility and regulates satiety. Another major function of CCK is stimulation of gallbladder contraction. This function enables that bile acids act simultaneously with pancreatic lipolytic enzymes. Secretin is a major stimulator of bicarbonate secretion. Trypsinogen is activated by the gut mucosal enzyme enterokinase. The other pancreatic proenzymes are activated by trypsin. Termination of enzyme secretion may be regulated by negative feedback mechanisms via destruction of CCK-releasing peptides by trypsin. Furthermore, the ileum may act as a brake by release of inhibitory hormones such as PYY and somatostatin. In the pathophysiology of acute pancreatitis, fusion of zymogen granules with lysosomes leading to intracellular activation of trypsinogen is regarded as an initiation step. This activation of trypsinogen may be caused by the lysosomal enzyme cathepsin B. However, autoactivation of trypsinogen itself may be a possibility in pathogenesis. Autoactivation is enhanced in certain mutations of trypsinogen. Furthermore, an imbalance of protease inhibitors and active proteases may be involved. The role of pancreatic lipolytic enzymes, the role of bicarbonate secretion, and toxic Ca(2+) signals by excessive liberation from the endoplasmic reticulum have to be discussed in the pathogenesis of acute pancreatitis. Copyright © 2011 S. Karger AG, Basel.

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

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

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

Effect of posttranslational modifications on enzyme function and assembly.

PubMed

Ryšlavá, Helena; Doubnerová, Veronika; Kavan, Daniel; Vaněk, Ondřej

2013-10-30

The detailed examination of enzyme molecules by mass spectrometry and other techniques continues to identify hundreds of distinct PTMs. Recently, global analyses of enzymes using methods of contemporary proteomics revealed widespread distribution of PTMs on many key enzymes distributed in all cellular compartments. Critically, patterns of multiple enzymatic and nonenzymatic PTMs within a single enzyme are now functionally evaluated providing a holistic picture of a macromolecule interacting with low molecular mass compounds, some of them being substrates, enzyme regulators, or activated precursors for enzymatic and nonenzymatic PTMs. Multiple PTMs within a single enzyme molecule and their mutual interplays are critical for the regulation of catalytic activity. Full understanding of this regulation will require detailed structural investigation of enzymes, their structural analogs, and their complexes. Further, proteomics is now integrated with molecular genetics, transcriptomics, and other areas leading to systems biology strategies. These allow the functional interrogation of complex enzymatic networks in their natural environment. In the future, one might envisage the use of robust high throughput analytical techniques that will be able to detect multiple PTMs on a global scale of individual proteomes from a number of carefully selected cells and cellular compartments. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Copyright © 2013 Elsevier B.V. All rights reserved.

Prediction and characterization of enzymatic activities guided by sequence similarity and genome neighborhood networks

DOE PAGES

Zhao, Suwen; Sakai, Ayano; Zhang, Xinshuai; ...

2014-06-30

Metabolic pathways in eubacteria and archaea often are encoded by operons and/or gene clusters (genome neighborhoods) that provide important clues for assignment of both enzyme functions and metabolic pathways. We describe a bioinformatic approach (genome neighborhood network; GNN) that enables large scale prediction of the in vitro enzymatic activities and in vivo physiological functions (metabolic pathways) of uncharacterized enzymes in protein families. We demonstrate the utility of the GNN approach by predicting in vitro activities and in vivo functions in the proline racemase superfamily (PRS; InterPro IPR008794). The predictions were verified by measuring in vitro activities for 51 proteins inmore » 12 families in the PRS that represent ~85% of the sequences; in vitro activities of pathway enzymes, carbon/nitrogen source phenotypes, and/or transcriptomic studies confirmed the predicted pathways. The synergistic use of sequence similarity networks3 and GNNs will facilitate the discovery of the components of novel, uncharacterized metabolic pathways in sequenced genomes.« less

Deletion of loop fragment adjacent to active site diminishes the stability and activity of exo-inulinase.

PubMed

Arjomand, Maryam Rezaei; Habibi-Rezaei, Mehran; Ahmadian, Gholamreza; Hassanzadeh, Malihe; Karkhane, Ali Asghar; Asadifar, Mandana; Amanlou, Massoud

2016-11-01

Inulinases are classified as hydrolases and widely used in the food and medical industries. Here, we report the deletion of a six-membered adjacent active site loop fragment ( 74 YGSDVT 79 sequence) from third Ω-loop of the exo-inulinase containing aspartate residue from Aspergillus niger to study its structural and functional importance. Site-directed mutagenesis was used to create the mutant of the exo-inulinase (Δ6SL). To investigate the stability of the region spanning this loop, MD simulations were performed 80ns for 20-85 residues. Molecular docking was performed to compare the interactions in the active sites of enzymes with fructose as a ligand. Accordingly, the functional thermostability of the exo-inulinase was significantly decreased upon loop fragment deletion. Evaluation of the kinetics parameters (V max , K m , k cat and, k cat /K m ) and activation energy (E a ) of the catalysis of enzymes indicated the importance of the deleted sequence on the catalytic performance of the enzyme. In conclusion, six-membered adjacent active site loop fragment not only plays a crucial role in the stability of the enzyme, but also it involves in the enzyme catalysis through lowering the activation energy of the catalysis and effective improving the catalytic performance. Copyright © 2016. Published by Elsevier B.V.

The prokaryotic FAD synthetase family: a potential drug target.

PubMed

Serrano, Ana; Ferreira, Patricia; Martínez-Júlvez, Marta; Medina, Milagros

2013-01-01

Disruption of cellular production of the flavin cofactors, flavin adenine mononucleotide (FMN) and flavin adenine dinucleotide(FAD) will prevent the assembly of a large number of flavoproteins and flavoenzymes involved in key metabolic processes in all types of organisms. The enzymes responsible for FMN and FAD production in prokaryotes and eukaryotes exhibit various structural characteristics to catalyze the same chemistry, a fact that converts the prokaryotic FAD synthetase (FADS) in a potential drug target for the development of inhibitors endowed with anti-pathogenic activity. The first step before searching for selective inhibitors of FADS is to understand the structural and functional mechanisms for the riboflavin kinase and FMN adenylyltransferase activities of the prokaryotic enzyme, and particularly to identify their differential functional characteristics with regard to the enzymes performing similar functions in other organisms, particularly humans. In this paper, an overview of the current knowledge of the structure-function relationships in prokaryotic FADS has been presented, as well as of the state of the art in the use of these enzymes as drug targets.

Functional Sites Induce Long-Range Evolutionary Constraints in Enzymes

PubMed Central

Jack, Benjamin R.; Meyer, Austin G.; Echave, Julian; Wilke, Claus O.

2016-01-01

Functional residues in proteins tend to be highly conserved over evolutionary time. However, to what extent functional sites impose evolutionary constraints on nearby or even more distant residues is not known. Here, we report pervasive conservation gradients toward catalytic residues in a dataset of 524 distinct enzymes: evolutionary conservation decreases approximately linearly with increasing distance to the nearest catalytic residue in the protein structure. This trend encompasses, on average, 80% of the residues in any enzyme, and it is independent of known structural constraints on protein evolution such as residue packing or solvent accessibility. Further, the trend exists in both monomeric and multimeric enzymes and irrespective of enzyme size and/or location of the active site in the enzyme structure. By contrast, sites in protein–protein interfaces, unlike catalytic residues, are only weakly conserved and induce only minor rate gradients. In aggregate, these observations show that functional sites, and in particular catalytic residues, induce long-range evolutionary constraints in enzymes. PMID:27138088

Mutations that Allow SIR2 Orthologs to Function in a NAD+-Depleted Environment.

PubMed

Ondracek, Caitlin R; Frappier, Vincent; Ringel, Alison E; Wolberger, Cynthia; Guarente, Leonard

2017-03-07

Sirtuin enzymes depend on NAD + to catalyze protein deacetylation. Therefore, the lowering of NAD + during aging leads to decreased sirtuin activity and may speed up aging processes in laboratory animals and humans. In this study, we used a genetic screen to identify two mutations in the catalytic domain of yeast Sir2 that allow the enzyme to function in an NAD + -depleted environment. These mutant enzymes give rise to a significant increase of yeast replicative lifespan and increase deacetylation by the Sir2 ortholog, SIRT1, in mammalian cells. Our data suggest that these mutations increase the stability of the conserved catalytic sirtuin domain, thereby increasing the catalytic efficiency of the mutant enzymes. Our approach to identifying sirtuin mutants that permit function in NAD + -limited environments may inform the design of small molecules that can maintain sirtuin activity in aging organisms. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Extracellular Enzyme Composition and Functional Characteristics of Aspergillus niger An-76 Induced by Food Processing Byproducts and Based on Integrated Functional Omics.

PubMed

Liu, Lin; Gong, Weili; Sun, Xiaomeng; Chen, Guanjun; Wang, Lushan

2018-02-07

Byproducts of food processing can be utilized for the production of high-value-added enzyme cocktails. In this study, we utilized integrated functional omics technology to analyze composition and functional characteristics of extracellular enzymes produced by Aspergillus niger grown on food processing byproducts. The results showed that oligosaccharides constituted by arabinose, xylose, and glucose in wheat bran were able to efficiently induce the production of extracellular enzymes of A. niger. Compared with other substrates, wheat bran was more effective at inducing the secretion of β-glucosidases from GH1 and GH3 families, as well as >50% of proteases from A1-family aspartic proteases. Compared with proteins induced by single wheat bran or soybean dregs, the protein yield induced by their mixture was doubled, and the time required to reach peak enzyme activity was shortened by 25%. This study provided a technical platform for the complex formulation of various substrates and functional analysis of extracellular enzymes.

Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5).

PubMed

Aspeborg, Henrik; Coutinho, Pedro M; Wang, Yang; Brumer, Harry; Henrissat, Bernard

2012-09-20

The large Glycoside Hydrolase family 5 (GH5) groups together a wide range of enzymes acting on β-linked oligo- and polysaccharides, and glycoconjugates from a large spectrum of organisms. The long and complex evolution of this family of enzymes and its broad sequence diversity limits functional prediction. With the objective of improving the differentiation of enzyme specificities in a knowledge-based context, and to obtain new evolutionary insights, we present here a new, robust subfamily classification of family GH5. About 80% of the current sequences were assigned into 51 subfamilies in a global analysis of all publicly available GH5 sequences and associated biochemical data. Examination of subfamilies with catalytically-active members revealed that one third are monospecific (containing a single enzyme activity), although new functions may be discovered with biochemical characterization in the future. Furthermore, twenty subfamilies presently have no characterization whatsoever and many others have only limited structural and biochemical data. Mapping of functional knowledge onto the GH5 phylogenetic tree revealed that the sequence space of this historical and industrially important family is far from well dispersed, highlighting targets in need of further study. The analysis also uncovered a number of GH5 proteins which have lost their catalytic machinery, indicating evolution towards novel functions. Overall, the subfamily division of GH5 provides an actively curated resource for large-scale protein sequence annotation for glycogenomics; the subfamily assignments are openly accessible via the Carbohydrate-Active Enzyme database at http://www.cazy.org/GH5.html.

Engineering of a novel tri-functional enzyme with MnSOD, catalase and cell-permeable activities.

PubMed

Luangwattananun, Piriya; Yainoy, Sakda; Eiamphungporn, Warawan; Songtawee, Napat; Bülow, Leif; Ayudhya, Chartchalerm Isarankura Na; Prachayasittikul, Virapong

2016-04-01

Cooperative function of superoxide dismutase (SOD) and catalase (CAT), in protection against oxidative stress, is known to be more effective than the action of either single enzyme. Chemical conjugation of the two enzymes resulted in molecules with higher antioxidant activity and therapeutic efficacy. However, chemical methods holds several drawbacks; e.g., loss of enzymatic activity, low homogeneity, time-consuming, and the need of chemical residues removal. Yet, the conjugated enzymes have never been proven to internalize into target cells. In this study, by employing genetic and protein engineering technologies, we reported designing and production of a bi-functional protein with SOD and CAT activities for the first time. To enable cellular internalization, cell penetrating peptide from HIV-1 Tat (TAT) was incorporated. Co-expression of CAT-MnSOD and MnSOD-TAT fusion genes allowed simultaneous self-assembly of the protein sequences into a large protein complex, which is expected to contained one tetrameric structure of CAT, four tetrameric structures of MnSOD and twelve units of TAT. The protein showed cellular internalization and superior protection against paraquat-induced cell death as compared to either complex bi-functional protein without TAT or to native enzymes fused with TAT. This study not only provided an alternative strategy to produce multifunctional protein complex, but also gained an insight into the development of therapeutic agent against oxidative stress-related conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Functional Analyses of Resurrected and Contemporary Enzymes Illuminate an Evolutionary Path for the Emergence of Exolysis in Polysaccharide Lyase Family 2.

PubMed

McLean, Richard; Hobbs, Joanne K; Suits, Michael D; Tuomivaara, Sami T; Jones, Darryl R; Boraston, Alisdair B; Abbott, D Wade

2015-08-28

Family 2 polysaccharide lyases (PL2s) preferentially catalyze the β-elimination of homogalacturonan using transition metals as catalytic cofactors. PL2 is divided into two subfamilies that have been generally associated with secretion, Mg(2+) dependence, and endolysis (subfamily 1) and with intracellular localization, Mn(2+) dependence, and exolysis (subfamily 2). When present within a genome, PL2 genes are typically found as tandem copies, which suggests that they provide complementary activities at different stages along a catabolic cascade. This relationship most likely evolved by gene duplication and functional divergence (i.e. neofunctionalization). Although the molecular basis of subfamily 1 endolytic activity is understood, the adaptations within the active site of subfamily 2 enzymes that contribute to exolysis have not been determined. In order to investigate this relationship, we have conducted a comparative enzymatic analysis of enzymes dispersed within the PL2 phylogenetic tree and elucidated the structure of VvPL2 from Vibrio vulnificus YJ016, which represents a transitional member between subfamiles 1 and 2. In addition, we have used ancestral sequence reconstruction to functionally investigate the segregated evolutionary history of PL2 progenitor enzymes and illuminate the molecular evolution of exolysis. This study highlights that ancestral sequence reconstruction in combination with the comparative analysis of contemporary and resurrected enzymes holds promise for elucidating the origins and activities of other carbohydrate active enzyme families and the biological significance of cryptic metabolic pathways, such as pectinolysis within the zoonotic marine pathogen V. vulnificus. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Cellulose fiber-enzyme composites fabricated through layer-by-layer nanoassembly.

PubMed

Xing, Qi; Eadula, Sandeep R; Lvov, Yuri M

2007-06-01

Cellulose microfibers were coated with enzymes, laccase and urease, through layer-by-layer assembly by alternate adsorption with oppositely charged polycations. The formation of organized polyelectrolyte and enzyme multilayer films of 15-20 nm thickness was demonstrated by quartz crystal microbalance, zeta-potential analysis, and confocal laser scanning microscopy. These biocomposites retained enzymatic catalytic activity, which was proportional to the number of coated enzyme layers. For laccase-fiber composites, around 50% of its initial activity was retained after 2 weeks of storage at 4 degrees C. The synthesis of calcium carbonate microparticles on urease-fiber composites confirmed urease functionality and demonstrated its possible applications. This strategy could be employed to fabricate fiber-based composites with novel biological functions.

Materials Research Center, University of Pittsburgh

DTIC Science & Technology

1994-04-29

porosities could be synthesized with active protein contained within the material. The incorporation of proteins into polyacrylates seres as a model system...enzyme with PEG and incorporate the modified enzyme into polyacrylates (Figure IV.D.3.1). The activity and stability of the functionalized enzyme have been... polyacrylate polymer. By varying the ratio of solvent (chloroform) and non-solvent (carbon tetrachloride) during the free-radical initiated

Expanding the Enzyme Universe: Accessing Non-Natural Reactions by Mechanism-Guided Directed Evolution

PubMed Central

Renata, Hans; Wang, Z. Jane

2015-01-01

High selectivities and exquisite control over reaction outcomes entice chemists to use biocatalysts in organic synthesis. However, many useful reactions are not accessible because they are not in nature’s known repertoire. We will use this review to outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progressions have been recapitulated in the laboratory starting from extant enzymes. We then examine non-native enzyme activities that have been discovered and exploited for chemical synthesis, emphasizing reactions that do not have natural counterparts. The new functions have mechanistic parallels to the native reaction mechanisms that often manifest as catalytic promiscuity and the ability to convert from one function to the other with minimal mutation. We present examples of how non-natural activities have been improved by directed evolution, mimicking the process used by nature to create new catalysts. Examples of new enzyme functions include epoxide opening reactions with non-natural nucleophiles catalyzed by a laboratory-evolved halohydrin dehalogenase, cyclopropanation and other carbene transfer reactions catalyzed by cytochrome P450 variants, and non-natural modes of cyclization by a modified terpene synthase. Lastly, we describe discoveries of non-native catalytic functions that may provide future opportunities for expanding the enzyme universe. PMID:25649694

The Lysozyme from Insect (Manduca sexta) is a Cold-Adapted Enzyme

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

Sotelo-Mundo,R.; Lopez-Zavala, A.; Garcia-Orozco, K.

Enzymatic activity is dependent on temperature, although some proteins have evolved to retain activity at low temperatures at the expense of stability. Cold adapted enzymes are present in a variety of organisms and there is ample interest in their structure-function relationships. Lysozyme (E.C. 3.2.1.17) is one of the most studied enzymes due to its antibacterial activity against Gram positive bacteria and is also a cold adapted protein. In this work the characterization of lysozyme from the insect Manduca sexta and its activity at low temperatures is presented. Both M. sexta lysozymes natural and recombinant showed a higher content of {alpha}-helixmore » secondary structure compared to that of hen egg white lysozyme and a higher specific enzymatic activity in the range of 5-30 {sup o}C. These results together with measured thermodynamic activation parameters support the designation of M. sexta lysozyme as a cold adapted enzyme. Therefore, the insect recombinant lysozyme is feasible as a model for structure-function studies for cold-adapted proteins.« less

Proteolytic Cascade for the Activation of the Insect Toll Pathway Induced by the Fungal Cell Wall Component

PubMed Central

Roh, Kyung-Baeg; Kim, Chan-Hee; Lee, Hanna; Kwon, Hyun-Mi; Park, Ji-Won; Ryu, Ji-Hwan; Kurokawa, Kenji; Ha, Nam-Chul; Lee, Won-Jae; Lemaitre, Bruno; Söderhäll, Kenneth; Lee, Bok-Luel

2009-01-01

The insect Toll signaling pathway is activated upon recognition of Gram-positive bacteria and fungi, resulting in the expression of antimicrobial peptides via NF-κB-like transcription factor. This activation is mediated by a serine protease cascade leading to the processing of Spätzle, which generates the functional ligand of the Toll receptor. Recently, we identified three serine proteases mediating Toll pathway activation induced by lysine-type peptidoglycan of Gram-positive bacteria. However, the identities of the downstream serine protease components of Gram-negative-binding protein 3 (GNBP3), a receptor for a major cell wall component β-1,3-glucan of fungi, and their order of activation have not been characterized yet. Here, we identified three serine proteases that are required for Toll activation by β-1,3-glucan in the larvae of a large beetle, Tenebrio molitor. The first one is a modular serine protease functioning immediately downstream of GNBP3 that proteolytically activates the second one, a Spätzle-processing enzyme-activating enzyme that in turn activates the third serine protease, a Spätzle-processing enzyme. The active form of Spätzle-processing enzyme then cleaves Spätzle into the processed Spätzle as Toll ligand. In addition, we show that injection of β-1,3-glucan into Tenebrio larvae induces production of two antimicrobial peptides, Tenecin 1 and Tenecin 2, which are also inducible by injection of the active form of Spätzle-processing enzyme-activating enzyme or processed Spätzle. These results demonstrate a three-step proteolytic cascade essential for the Toll pathway activation by fungal β-1,3-glucan in Tenebrio larvae, which is shared with lysine-type peptidoglycan-induced Toll pathway activation. PMID:19473968

Computational multiscale modeling in protein--ligand docking.

PubMed

Taufer, Michela; Armen, Roger; Chen, Jianhan; Teller, Patricia; Brooks, Charles

2009-01-01

In biological systems, the binding of small molecule ligands to proteins is a crucial process for almost every aspect of biochemistry and molecular biology. Enzymes are proteins that function by catalyzing specific biochemical reactions that convert reactants into products. Complex organisms are typically composed of cells in which thousands of enzymes participate in complex and interconnected biochemical pathways. Some enzymes serve as sequential steps in specific pathways (such as energy metabolism), while others function to regulate entire pathways and cellular functions [1]. Small molecule ligands can be designed to bind to a specific enzyme and inhibit the biochemical reaction. Inhibiting the activity of key enzymes may result in the entire biochemical pathways being turned on or off [2], [3]. Many small molecule drugs marketed today function in this generic way as enzyme inhibitors. If research identifies a specific enzyme as being crucial to the progress of disease, then this enzyme may be targeted with an inhibitor, which may slow down or reverse the progress of disease. In this way, enzymes are targeted from specific pathogens (e.g., virus, bacteria, fungi) for infectious diseases [4], [5], and human enzymes are targeted for noninfectious diseases such as cardiovascular disease, cancer, diabetes, and neurodegenerative diseases [6].

Lignocellulose-Degrading Microbial Communities in Landfill Sites Represent a Repository of Unexplored Biomass-Degrading Diversity.

PubMed

Ransom-Jones, Emma; McCarthy, Alan J; Haldenby, Sam; Doonan, James; McDonald, James E

2017-01-01

The microbial conversion of lignocellulosic biomass for biofuel production represents a renewable alternative to fossil fuels. However, the discovery of new microbial enzymes with high activity is critical for improving biomass conversion processes. While attempts to identify superior lignocellulose-degrading enzymes have focused predominantly on the animal gut, biomass-degrading communities in landfill sites represent an unexplored resource of hydrolytic enzymes for biomass conversion. Here, to address the paucity of information on biomass-degrading microbial diversity beyond the gastrointestinal tract, cellulose (cotton) "baits" were incubated in landfill leachate microcosms to enrich the landfill cellulolytic microbial community for taxonomic and functional characterization. Metagenome and 16S rRNA gene amplicon sequencing demonstrated the dominance of Firmicutes , Bacteroidetes , Spirochaetes , and Fibrobacteres in the landfill cellulolytic community. Functional metagenome analysis revealed 8,371 carbohydrate active enzymes (CAZymes) belonging to 244 CAZyme families. In addition to observing biomass-degrading enzymes of anaerobic bacterial "cellulosome" systems of members of the Firmicutes , we report the first detection of the Fibrobacter cellulase system and the Bacteroidetes polysaccharide utilization locus (PUL) in landfill sites. These data provide evidence for the presence of multiple mechanisms of biomass degradation in the landfill microbiome and highlight the extraordinary functional diversity of landfill microorganisms as a rich source of biomass-degrading enzymes of potential biotechnological significance. IMPORTANCE The microbial conversion of lignocellulosic biomass for biofuel production represents a renewable alternative to fossil fuels. However, the discovery of new microbial enzymes with high activity is critical for improving biomass conversion processes. While attempts to identify superior lignocellulose-degrading enzymes have focused predominantly on the animal gut, biomass-degrading communities in landfill sites represent an unexplored resource of hydrolytic enzymes for biomass conversion. Here, we identified Firmicutes , Spirochaetes , and Fibrobacteres as key phyla in the landfill cellulolytic community, detecting 8,371 carbohydrate active enzymes (CAZymes) that represent at least three of the recognized strategies for cellulose decomposition. These data highlight substantial hydrolytic enzyme diversity in landfill sites as a source of new enzymes for biomass conversion.

Lignocellulose-Degrading Microbial Communities in Landfill Sites Represent a Repository of Unexplored Biomass-Degrading Diversity

PubMed Central

Ransom-Jones, Emma; McCarthy, Alan J.; Haldenby, Sam; Doonan, James

2017-01-01

ABSTRACT The microbial conversion of lignocellulosic biomass for biofuel production represents a renewable alternative to fossil fuels. However, the discovery of new microbial enzymes with high activity is critical for improving biomass conversion processes. While attempts to identify superior lignocellulose-degrading enzymes have focused predominantly on the animal gut, biomass-degrading communities in landfill sites represent an unexplored resource of hydrolytic enzymes for biomass conversion. Here, to address the paucity of information on biomass-degrading microbial diversity beyond the gastrointestinal tract, cellulose (cotton) “baits” were incubated in landfill leachate microcosms to enrich the landfill cellulolytic microbial community for taxonomic and functional characterization. Metagenome and 16S rRNA gene amplicon sequencing demonstrated the dominance of Firmicutes, Bacteroidetes, Spirochaetes, and Fibrobacteres in the landfill cellulolytic community. Functional metagenome analysis revealed 8,371 carbohydrate active enzymes (CAZymes) belonging to 244 CAZyme families. In addition to observing biomass-degrading enzymes of anaerobic bacterial “cellulosome” systems of members of the Firmicutes, we report the first detection of the Fibrobacter cellulase system and the Bacteroidetes polysaccharide utilization locus (PUL) in landfill sites. These data provide evidence for the presence of multiple mechanisms of biomass degradation in the landfill microbiome and highlight the extraordinary functional diversity of landfill microorganisms as a rich source of biomass-degrading enzymes of potential biotechnological significance. IMPORTANCE The microbial conversion of lignocellulosic biomass for biofuel production represents a renewable alternative to fossil fuels. However, the discovery of new microbial enzymes with high activity is critical for improving biomass conversion processes. While attempts to identify superior lignocellulose-degrading enzymes have focused predominantly on the animal gut, biomass-degrading communities in landfill sites represent an unexplored resource of hydrolytic enzymes for biomass conversion. Here, we identified Firmicutes, Spirochaetes, and Fibrobacteres as key phyla in the landfill cellulolytic community, detecting 8,371 carbohydrate active enzymes (CAZymes) that represent at least three of the recognized strategies for cellulose decomposition. These data highlight substantial hydrolytic enzyme diversity in landfill sites as a source of new enzymes for biomass conversion. PMID:28776044

Using NMR spectroscopy to elucidate the role of molecular motions in enzyme function

PubMed Central

Lisi, George P.; Loria, J. Patrick

2015-01-01

Conformational motions play an essential role in enzyme function, often facilitating the formation of enzyme-substrate complexes and/or product release. Although considerable debate remains regarding the role of molecular motions in the conversion of enzymatic substrates to products, numerous examples have found motions to be crucial for optimization of enzyme scaffolds, effective substrate binding, and product dissociation. Conformational fluctuations are often rate-limiting to enzyme catalysis, primarily through product release, with the chemical reaction occurring much more quickly. As a result, the direct involvement of motions at various stages along the enzyme reaction coordinate remains largely unknown and untested. In the following review, we describe the use of solution NMR techniques designed to probe various timescales of molecular motions and detail examples in which motions play a role in propagating catalytic effects from the active site and directly participate in essential aspects of enzyme function. PMID:26952190

Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination.

PubMed

Rejón, Juan D; Zienkiewicz, Agnieszka; Rodríguez-García, María Isabel; Castro, Antonio J

2012-10-01

A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized. The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods. Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles. In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma.

Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes.

PubMed

Ramadan, Abdelaziz; Nemoto, Keiichirou; Seki, Motoaki; Shinozaki, Kazuo; Takeda, Hiroyuki; Takahashi, Hirotaka; Sawasaki, Tatsuya

2015-11-10

Protein ubiquitination is a ubiquitous mechanism in eukaryotes. In Arabidopsis, ubiquitin modification is mainly mediated by two ubiquitin activating enzymes (E1s), 37 ubiquitin conjugating enzymes (E2s), and more than 1300 predicted ubiquitin ligase enzymes (E3s), of which ~470 are RING-type E3s. A large proportion of the RING E3's gene products have yet to be characterised in vitro, likely because of the laborious work involved in large-scale cDNA cloning and protein expression, purification, and characterisation. In addition, several E2s, which might be necessary for the activity of certain E3 ligases, cannot be expressed by Escherichia coli or cultured insect cells and, therefore, remain uncharacterised. Using the RIKEN Arabidopsis full-length cDNA library (RAFL) with the 'split-primer' PCR method and a wheat germ cell-free system, we established protein libraries of Arabidopsis E2 and RING E3 enzymes. We expressed 35 Arabidopsis E2s including six enzymes that have not been previously expressed, and 204 RING proteins, most of which had not been functionally characterised. Thioester assays using dithiothreitol (DTT) showed DTT-sensitive ubiquitin thioester formation for all E2s expressed. In expression assays of RING proteins, 31 proteins showed high molecular smears, which are probably the result of their functional activity. The activities of another 27 RING proteins were evaluated with AtUBC10 and/or a group of different E2s. All the 27 RING E3s tested showed ubiquitin ligase activity, including 17 RING E3s. Their activities are reported for the first time. The wheat germ cell-free system used in our study, which is a eukaryotic expression system and more closely resembles the endogenous expression of plant proteins, is very suitable for expressing Arabidopsis E2s and RING E3s in their functional form. In addition, the protein libraries described here can be used for further understanding E2-E3 specificities and as platforms for protein-protein interaction screening.

Physiological enzymology: The next frontier in understanding protein structure and function at the cellular level.

PubMed

Lee, Irene; Berdis, Anthony J

2016-01-01

Historically, the study of proteins has relied heavily on characterizing the activity of a single purified protein isolated from other cellular components. This classic approach allowed scientists to unambiguously define the intrinsic kinetic and chemical properties of that protein. The ultimate hope was to extrapolate this information toward understanding how the enzyme or receptor behaves within its native cellular context. These types of detailed in vitro analyses were necessary to reduce the innate complexities of measuring the singular activity and biochemical properties of a specific enzyme without interference from other enzymes and potential competing substrates. However, recent developments in fields encompassing cell biology, molecular imaging, and chemical biology now provide the unique chemical tools and instrumentation to study protein structure, function, and regulation in their native cellular environment. These advancements provide the foundation for a new field, coined physiological enzymology, which quantifies the function and regulation of enzymes and proteins at the cellular level. In this Special Edition, we explore the area of Physiological Enzymology and Protein Function through a series of review articles that focus on the tools and techniques used to measure the cellular activity of proteins inside living cells. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. Copyright © 2015 Elsevier B.V. All rights reserved.

Activation and Function of the MAPKs and Their Substrates, the MAPK-Activated Protein Kinases

PubMed Central

Cargnello, Marie; Roux, Philippe P.

2011-01-01

Summary: The mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs by relaying extracellular signals to intracellular responses. In mammals, there are more than a dozen MAPK enzymes that coordinately regulate cell proliferation, differentiation, motility, and survival. The best known are the conventional MAPKs, which include the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinases 1 to 3 (JNK1 to -3), p38 (α, β, γ, and δ), and ERK5 families. There are additional, atypical MAPK enzymes, including ERK3/4, ERK7/8, and Nemo-like kinase (NLK), which have distinct regulation and functions. Together, the MAPKs regulate a large number of substrates, including members of a family of protein Ser/Thr kinases termed MAPK-activated protein kinases (MAPKAPKs). The MAPKAPKs are related enzymes that respond to extracellular stimulation through direct MAPK-dependent activation loop phosphorylation and kinase activation. There are five MAPKAPK subfamilies: the p90 ribosomal S6 kinase (RSK), the mitogen- and stress-activated kinase (MSK), the MAPK-interacting kinase (MNK), the MAPK-activated protein kinase 2/3 (MK2/3), and MK5 (also known as p38-regulated/activated protein kinase [PRAK]). These enzymes have diverse biological functions, including regulation of nucleosome and gene expression, mRNA stability and translation, and cell proliferation and survival. Here we review the mechanisms of MAPKAPK activation by the different MAPKs and discuss their physiological roles based on established substrates and recent discoveries. PMID:21372320

Structural and Functional Analysis of a Lytic Polysaccharide Monooxygenase Important for Efficient Utilization of Chitin in Cellvibrio japonicus*

PubMed Central

Forsberg, Zarah; Nelson, Cassandra E.; Dalhus, Bjørn; Mekasha, Sophanit; Loose, Jennifer S. M.; Crouch, Lucy I.; Røhr, Åsmund K.; Gardner, Jeffrey G.; Eijsink, Vincent G. H.; Vaaje-Kolstad, Gustav

2016-01-01

Cellvibrio japonicus is a Gram-negative soil bacterium that is primarily known for its ability to degrade plant cell wall polysaccharides through utilization of an extensive repertoire of carbohydrate-active enzymes. Several putative chitin-degrading enzymes are also found among these carbohydrate-active enzymes, such as chitinases, chitobiases, and lytic polysaccharide monooxygenases (LPMOs). In this study, we have characterized the chitin-active LPMO, CjLPMO10A, a tri-modular enzyme containing a catalytic family AA10 LPMO module, a family 5 chitin-binding module, and a C-terminal unclassified module of unknown function. Characterization of the latter module revealed tight and specific binding to chitin, thereby unraveling a new family of chitin-binding modules (classified as CBM73). X-ray crystallographic elucidation of the CjLPMO10A catalytic module revealed that the active site of the enzyme combines structural features previously only observed in either cellulose or chitin-active LPMO10s. Analysis of the copper-binding site by EPR showed a signal signature more similar to those observed for cellulose-cleaving LPMOs. The full-length LPMO shows no activity toward cellulose but is able to bind and cleave both α- and β-chitin. Removal of the chitin-binding modules reduced LPMO activity toward α-chitin compared with the full-length enzyme. Interestingly, the full-length enzyme and the individual catalytic LPMO module boosted the activity of an endochitinase equally well, also yielding similar amounts of oxidized products. Finally, gene deletion studies show that CjLPMO10A is needed by C. japonicus to obtain efficient growth on both purified chitin and crab shell particles. PMID:26858252

Structural and Functional Analysis of a Lytic Polysaccharide Monooxygenase Important for Efficient Utilization of Chitin in Cellvibrio japonicus.

PubMed

Forsberg, Zarah; Nelson, Cassandra E; Dalhus, Bjørn; Mekasha, Sophanit; Loose, Jennifer S M; Crouch, Lucy I; Røhr, Åsmund K; Gardner, Jeffrey G; Eijsink, Vincent G H; Vaaje-Kolstad, Gustav

2016-04-01

Cellvibrio japonicusis a Gram-negative soil bacterium that is primarily known for its ability to degrade plant cell wall polysaccharides through utilization of an extensive repertoire of carbohydrate-active enzymes. Several putative chitin-degrading enzymes are also found among these carbohydrate-active enzymes, such as chitinases, chitobiases, and lytic polysaccharide monooxygenases (LPMOs). In this study, we have characterized the chitin-active LPMO,CjLPMO10A, a tri-modular enzyme containing a catalytic family AA10 LPMO module, a family 5 chitin-binding module, and a C-terminal unclassified module of unknown function. Characterization of the latter module revealed tight and specific binding to chitin, thereby unraveling a new family of chitin-binding modules (classified as CBM73). X-ray crystallographic elucidation of theCjLPMO10A catalytic module revealed that the active site of the enzyme combines structural features previously only observed in either cellulose or chitin-active LPMO10s. Analysis of the copper-binding site by EPR showed a signal signature more similar to those observed for cellulose-cleaving LPMOs. The full-length LPMO shows no activity toward cellulose but is able to bind and cleave both α- and β-chitin. Removal of the chitin-binding modules reduced LPMO activity toward α-chitin compared with the full-length enzyme. Interestingly, the full-length enzyme and the individual catalytic LPMO module boosted the activity of an endochitinase equally well, also yielding similar amounts of oxidized products. Finally, gene deletion studies show thatCjLPMO10A is needed byC. japonicusto obtain efficient growth on both purified chitin and crab shell particles. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Changes in digestive enzyme activities during larval development of Chinese loach Paramisgurnus dabryanus (Dabry de Thiersant, 1872).

PubMed

Zhang, Yun-Long; Wu, Qiao-Wan; Hu, Wei-Hua; Wang, Fan; Zhao, Zhong-Bo; He, Hui; Shao, Wei-Han; Fan, Qi-Xue

2015-12-01

The digestive physiology of Chinese loach (Paramisgurnus dabryanus) was studied by assessing the specific and total activities of different pancreatic (trypsin, chymotrypsin, amylase and lipase), gastric (pepsin) and intestinal (alkaline phosphatase and leucine-aminopeptidase) enzymes from hatching to 40 days after hatching (DAH). Larvae were reared at 24.4 ± 0.4 °C and fed with rotifers from mouth opening (4 DAH) to 15 DAH, from 10 to 35 DAH with Cladocera and from 30 to 40 DAH with compound diet. Enzyme activities for trypsin, chymotrypsin, amylase and lipase were detected before the onset of exogenous feeding, indicating that these enzymes were genetically pre-programmed. Most of the pancreatic enzyme specific activities increased until 20 DAH and decreased thereafter. The pepsin activity of Chinese loach was firstly detected at 30 DAH, indicating the appearance of functional gastric gland. Alkaline phosphatase specific activity was detected from hatching onward, showed marked increase and reached the second peak at 20 DAH, while a gradual increase in specific leucine-aminopeptidase activity was observed until the end of the experiment. Accordingly, the larvae of Chinese loach possess a functional digestive system before the onset of exogenous feeding and the digestive capacity gradually increases as development progresses. The abrupt increase in intestinal enzyme activities between 10 and 20 DAH demonstrates onset of juvenile-like digestive mode in Chinese loach larvae. The increase in pepsin activity after 30 DAH indicates the shift from alkaline to acidic digestion in Chinese loach larvae, which may be considered as the onset of weaning.

Positron emitter labeled enzyme inhibitors

DOEpatents

Fowler, Joanna S.; MacGregor, Robert R.; Wolf, Alfred P.; Langstrom, Bengt

1990-01-01

This invention involves 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 inactivators 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.

Stability and activity of lactate dehydrogenase on biofunctional layers deposited by activated vapor silanization (AVS) and immersion silanization (IS)

NASA Astrophysics Data System (ADS)

Calvo, Jorge Nieto-Márquez; Elices, Manuel; Guinea, Gustavo V.; Pérez-Rigueiro, José; Arroyo-Hernández, María

2017-09-01

The interaction between surfaces and biological elements, in particular, proteins is critical for the performance of biomaterials and biosensors. This interaction can be controlled by modifying the surface in a process known as biofunctionalization. In this work, the enzyme lactate dehydrogenase (LDH) is used to study the stability of the interaction between a functional protein and amine-functionalized surfaces. Two different functionalization procedures were compared: Activated Vapor Silanization (AVS) and Immersion Silanization (IS). Adsorption kinetics is shown to follow the Langmuir model for AVS-functionalized samples, while IS-functionalized samples show a certain instability if immersed in an aqueous medium for several hours. In turn, the enzymatic activity of LDH is preserved for longer times by using glutaraldehyde as crosslinker between the AVS biofunctional surface and the enzyme.

Bacillus subtilis ribonucleases J1 and J2 form a complex with altered enzyme behaviour.

PubMed

Mathy, Nathalie; Hébert, Agnès; Mervelet, Peggy; Bénard, Lionel; Dorléans, Audrey; Li de la Sierra-Gallay, Inés; Noirot, Philippe; Putzer, Harald; Condon, Ciarán

2010-01-01

Ribonucleases J1 and J2 are recently discovered enzymes with dual 5'-to-3' exoribonucleolytic/endoribonucleolytic activity that plays a key role in the maturation and degradation of Bacillus subtilis RNAs. RNase J1 is essential, while its paralogue RNase J2 is not. Up to now, it had generally been assumed that the two enzymes functioned independently. Here we present evidence that RNases J1 and J2 form a complex that is likely to be the predominant form of these enzymes in wild-type cells. While both RNase J1 and the RNase J1/J2 complex have robust 5'-to-3' exoribonuclease activity in vitro, RNase J2 has at least two orders of magnitude weaker exonuclease activity, providing a possible explanation for why RNase J1 is essential. The association of the two proteins also has an effect on the endoribonucleolytic properties of RNases J1 and J2. While the individual enzymes have similar endonucleolytic cleavage activities and specificities, as a complex they behave synergistically to alter cleavage site preference and to increase cleavage efficiency at specific sites. These observations dramatically change our perception of how these ribonucleases function and provide an interesting example of enzyme subfunctionalization after gene duplication.

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

Lopez, Murielle; Kurkal-Siebert, V; Dunn, Rachel V.

Water is widely assumed to be essential for life, although the exact molecular basis of this requirement is unclear. Water facilitates protein motions, and although enzyme activity has been demonstrated at low hydrations in organic solvents, such nonaqueous solvents may allow the necessary motions for catalysis. To examine enzyme function in the absence of solvation and bypass diffusional constraints we have tested the ability of an enzyme, pig liver esterase, to catalyze alcoholysis as an anhydrous powder, in a reaction system of defined water content and where the substrates and products are gaseous. At hydrations of 3 ( 2) moleculesmore » of water per molecule of enzyme, activity is several orders-of-magnitude greater than nonenzymatic catalysis. Neutron spectroscopy indicates that the fast ( nanosecond) global anharmonic dynamics of the anhydrous functional enzyme are suppressed. This indicates that neither hydration water nor fast anharmonic dynamics are required for catalysis by this enzyme, implying that one of the biological requirements of water may lie with its role as a diffusion medium rather than any of its more specific properties.« less

An active lifestyle induces positive antioxidant enzyme modulation in peripheral blood mononuclear cells of overweight/obese postmenopausal women.

PubMed

Farinha, Juliano Boufleur; De Carvalho, Nélson Rodrigues; Steckling, Flávia Mariel; De Vargas, Liziane Da Silva; Courtes, Aline Alves; Stefanello, Sílvio Terra; Martins, Caroline Curry; Bresciani, Guilherme; Dos Santos, Daniela Lopes; Soares, Félix Alexandre Antunes

2015-01-15

The aim of this study was to investigate the effects of an active lifestyle on mitochondrial functioning, viability, bioenergetics, and redox status markers in peripheral blood mononuclear cells (PBMC) of overweight/ obese postmenopausal women. We performed a cross-sectional study with postmenopausal women aged 45–64 years and body mass index N 25 kg/m2, divided into physically active (n = 23) and sedentary (n = 12) groups. Mitochondria functioning and viability, bioenergetics and redox status parameters were assessed in PBMC with spectrophotometric and fluorometric assays. No differences were found in the enzyme activity of complexes I and II of the electron transport chain (ETC), mitochondrial superoxide dismutase (MnSOD) activity, methyl-tetrazolium reduction levels and reduced glutathione and oxidized glutathione levels between the groups. However, the physically active group presented higher levels of reactive oxygen species (ROS) (P= 0.04) and increased catalase (CAT) (P= 0.029), total (P= 0.011) and cytosolic SOD (CuZnSOD) (P= 0.009) activities. An active lifestyle that includes aerobic exercise for at least 30 min, three times per week may improve antioxidant enzyme activities in PBMC in overweight/obese postmenopausal women, without changes in the activity of the ETC enzymes. However, this low intensity physical activity is not able to induce relevant mitochondrial adaptations.

Structural and functional comparison of two human liver dihydrodiol dehydrogenases associated with 3 alpha-hydroxysteroid dehydrogenase activity.

PubMed Central

Deyashiki, Y; Taniguchi, H; Amano, T; Nakayama, T; Hara, A; Sawada, H

1992-01-01

Two monomeric dihydrodiol dehydrogenases with pI values of 5.4 and 7.6 were co-purified with androsterone dehydrogenase activity to homogeneity from human liver. The two enzymes differed from each other on peptide mapping and in their heat-stabilities; with respect to the latter the dihydrodiol dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase activities of the respective enzymes were similarly inactivated. The pI 5.4 enzyme was equally active towards trans- and cis-benzene dihydrodiols, and towards (S)- and (R)-forms of indan-1-ol and 1,2,3,4-tetrahydronaphth-1-ol and oxidized the 3 alpha-hydroxy group of C19-, C21- and C24-steroids, whereas the pI 7.6 enzyme showed high specificity for trans-benzene dihydrodiol, (S)-forms of the alicyclic alcohols and C19- and C21-steroids. Although the two enzymes reduced various xenobiotic carbonyl compounds and the 3-oxo group of C19- and C21-steroids, and were A-specific in the hydrogen transfer from NADPH, only the pI 5.4 enzyme showed reductase activity towards 7 alpha-hydroxy-5 beta-cholestan-3-one and dehydrolithocholic acid. The affinity of the two enzymes for the steroidal substrates was higher than that for the xenobiotic substrates. The two enzymes also showed different susceptibilities to the inhibition by anti-inflammatory drugs and bile acids. Whereas the pI-5.4 enzyme was highly sensitive to anti-inflammatory steroids, showing mixed-type inhibitions with respect to indan-1-ol and androsterone, the pI 7.6 enzyme was inhibited more potently by non-steroidal anti-inflammatory drugs and bile acids than by the steroidal drugs, and the inhibitions were all competitive. These structural and functional differences suggest that the two enzymes are 3 alpha-hydroxysteroid dehydrogenase isoenzymes. Images Fig. 2. PMID:1554355

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.

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.

The protein cofactor allows the sequence of an RNase P ribozyme to diversify by maintaining the catalytically active structure of the enzyme.

PubMed Central

Kim, J J; Kilani, A F; Zhan, X; Altman, S; Liu, F

1997-01-01

To study the effect proteins have on the catalysis and evolution of RNA enzymes, we simulated evolution of RNase P catalytic M1 RNA in vitro, in the presence and absence of its C5 protein cofactor. In the presence of C5, functional M1 sequence variants (not catalytically active in the absence of C5) were selected in addition to those identical to M1. C5 maintains the catalytically active structure of the variants and allows for an enhanced spectrum of M1 molecules to function in the context of a ribonucleoprotein (RNP) complex. The generation of an RNP enzyme, requiring both RNA and protein components, from a catalytically active RNA molecule has implications for how modern RNP complexes evolved from ancestral RNAs. PMID:9174096

Immobilisation and characterisation of biocatalytic co-factor recycling enzymes, glucose dehydrogenase and NADH oxidase, on aldehyde functional ReSyn™ polymer microspheres.

PubMed

Twala, Busisiwe V; Sewell, B Trevor; Jordaan, Justin

2012-05-10

The use of enzymes in industrial applications is limited by their instability, cost and difficulty in their recovery and re-use. Immobilisation is a technique which has been shown to alleviate these limitations in biocatalysis. Here we describe the immobilisation of two biocatalytically relevant co-factor recycling enzymes, glucose dehydrogenase (GDH) and NADH oxidase (NOD) on aldehyde functional ReSyn™ polymer microspheres with varying functional group densities. The successful immobilisation of the enzymes on this new high capacity microsphere technology resulted in the maintenance of activity of ∼40% for GDH and a maximum of 15.4% for NOD. The microsphere variant with highest functional group density of ∼3500 μmol g⁻¹ displayed the highest specific activity for the immobilisation of both enzymes at 33.22 U mg⁻¹ and 6.75 U mg⁻¹ for GDH and NOD with respective loading capacities of 51% (0.51 mg mg⁻¹) and 129% (1.29 mg mg⁻¹). The immobilised GDH further displayed improved activity in the acidic pH range. Both enzymes displayed improved pH and thermal stability with the most pronounced thermal stability for GDH displayed on ReSyn™ A during temperature incubation at 65 °C with a 13.59 fold increase, and NOD with a 2.25-fold improvement at 45 °C on the same microsphere variant. An important finding is the suitability of the microspheres for stabilisation of the multimeric protein GDH. Copyright © 2012 Elsevier Inc. All rights reserved.

The structure of Plasmodium falciparum serine hydroxymethyltransferase reveals a novel redox switch that regulates its activities

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

Chitnumsub, Penchit, E-mail: penchit@biotec.or.th; Ittarat, Wanwipa; Jaruwat, Aritsara

2014-06-01

The crystal structure of P. falciparum SHMT revealed snapshots of an intriguing disulfide/sulfhydryl switch controlling the functional activity. Plasmodium falciparum serine hydroxymethyltransferase (PfSHMT), an enzyme in the dTMP synthesis cycle, is an antimalarial target because inhibition of its expression or function has been shown to be lethal to the parasite. As the wild-type enzyme could not be crystallized, protein engineering of residues on the surface was carried out. The surface-engineered mutant PfSHMT-F292E was successfully crystallized and its structure was determined at 3 Å resolution. The PfSHMT-F292E structure is a good representation of PfSHMT as this variant revealed biochemical properties similarmore » to those of the wild type. Although the overall structure of PfSHMT is similar to those of other SHMTs, unique features including the presence of two loops and a distinctive cysteine pair formed by Cys125 and Cys364 in the tetrahydrofolate (THF) substrate binding pocket were identified. These structural characteristics have never been reported in other SHMTs. Biochemical characterization and mutation analysis of these two residues confirm that they act as a disulfide/sulfhydryl switch to regulate the THF-dependent catalytic function of the enzyme. This redox switch is not present in the human enzyme, in which the cysteine pair is absent. The data reported here can be further exploited as a new strategy to specifically disrupt the activity of the parasite enzyme without interfering with the function of the human enzyme.« less

Prediction of Detailed Enzyme Functions and Identification of Specificity Determining Residues by Random Forests

PubMed Central

Nagao, Chioko; Nagano, Nozomi; Mizuguchi, Kenji

2014-01-01

Determining enzyme functions is essential for a thorough understanding of cellular processes. Although many prediction methods have been developed, it remains a significant challenge to predict enzyme functions at the fourth-digit level of the Enzyme Commission numbers. Functional specificity of enzymes often changes drastically by mutations of a small number of residues and therefore, information about these critical residues can potentially help discriminate detailed functions. However, because these residues must be identified by mutagenesis experiments, the available information is limited, and the lack of experimentally verified specificity determining residues (SDRs) has hindered the development of detailed function prediction methods and computational identification of SDRs. Here we present a novel method for predicting enzyme functions by random forests, EFPrf, along with a set of putative SDRs, the random forests derived SDRs (rf-SDRs). EFPrf consists of a set of binary predictors for enzymes in each CATH superfamily and the rf-SDRs are the residue positions corresponding to the most highly contributing attributes obtained from each predictor. EFPrf showed a precision of 0.98 and a recall of 0.89 in a cross-validated benchmark assessment. The rf-SDRs included many residues, whose importance for specificity had been validated experimentally. The analysis of the rf-SDRs revealed both a general tendency that functionally diverged superfamilies tend to include more active site residues in their rf-SDRs than in less diverged superfamilies, and superfamily-specific conservation patterns of each functional residue. EFPrf and the rf-SDRs will be an effective tool for annotating enzyme functions and for understanding how enzyme functions have diverged within each superfamily. PMID:24416252

Substrate specificity characterization for eight putative nudix hydrolases. Evaluation of criteria for substrate identification within the Nudix family.

PubMed

Nguyen, Vi N; Park, Annsea; Xu, Anting; Srouji, John R; Brenner, Steven E; Kirsch, Jack F

2016-12-01

The nearly 50,000 known Nudix proteins have a diverse array of functions, of which the most extensively studied is the catalyzed hydrolysis of aberrant nucleotide triphosphates. The functions of 171 Nudix proteins have been characterized to some degree, although physiological relevance of the assayed activities has not always been conclusively demonstrated. We investigated substrate specificity for eight structurally characterized Nudix proteins, whose functions were unknown. These proteins were screened for hydrolase activity against a 74-compound library of known Nudix enzyme substrates. We found substrates for four enzymes with k cat /K m values >10,000 M -1  s -1 : Q92EH0_LISIN of Listeria innocua serovar 6a against ADP-ribose, Q5LBB1_BACFN of Bacillus fragilis against 5-Me-CTP, and Q0TTC5_CLOP1 and Q0TS82_CLOP1 of Clostridium perfringens against 8-oxo-dATP and 3'-dGTP, respectively. To ascertain whether these identified substrates were physiologically relevant, we surveyed all reported Nudix hydrolytic activities against NTPs. Twenty-two Nudix enzymes are reported to have activity against canonical NTPs. With a single exception, we find that the reported k cat /K m values exhibited against these canonical substrates are well under 10 5 M -1  s -1 . By contrast, several Nudix enzymes show much larger k cat /K m values (in the range of 10 5 to >10 7 M -1  s -1 ) against noncanonical NTPs. We therefore conclude that hydrolytic activities exhibited by these enzymes against canonical NTPs are not likely their physiological function, but rather the result of unavoidable collateral damage occasioned by the enzymes' inability to distinguish completely between similar substrate structures. Proteins 2016; 84:1810-1822. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Amyloid fibrils as a nanoscaffold for enzyme immobilization.

PubMed

Pilkington, Sarah M; Roberts, Sarah J; Meade, Susie J; Gerrard, Juliet A

2010-01-01

Amyloid fibrils are a misfolded state, formed by many proteins when subjected to denaturing conditions. Their constituent amino acids make them ideally suited as a readily functionalized nanoscaffold for enzyme immobilization and their strength, stability, and nanometer size are attractive features for exploitation in the creation of new bionanomaterials. We report successful functionalization of amyloid fibrils by conjugation to glucose oxidase (GOD) using glutaraldehyde. GOD retained activity upon attachment and successful cross-linking was determined using electrophoresis, centrifugation, sucrose gradient centrifugation, and TEM. The resulting functionalized enzyme scaffold was then incorporated into a model poly(vinyl alcohol) (PVOH) film, to create a new bionanomaterial. The antibacterial effect of the functionalized film was then tested on E. coli, the growth of which was inhibited, demonstrating the incorporation of GOD antibacterial activity into the PVOH film. The incorporation of the GOD-functionalized amyloid fibrils into PVOH provides an excellent 'proof of concept' model for the creation of a new bionanomaterial using a functionalized amyloid fibril scaffold.

Covalent enzyme immobilization onto carbon nanotubes using a membrane reactor

NASA Astrophysics Data System (ADS)

Voicu, Stefan Ioan; Nechifor, Aurelia Cristina; Gales, Ovidiu; Nechifor, Gheorghe

2011-05-01

Composite porous polysulfone-carbon nanotubes membranes were prepared by dispersing carbon nanotubes into a polysulfone solution followed by the membrane formation by phase inversion-immersion precipitation technique. The carbon nanotubes with amino groups on surface were functionalized with different enzymes (carbonic anhydrase, invertase, diastase) using cyanuric chloride as linker between enzyme and carbon nanotube. The composite membrane was used as a membrane reactor for a better dispersion of carbon nanotubes and access to reaction centers. The membrane also facilitates the transport of enzymes to active carbon nanotubes centers for functionalization (amino groups). The functionalized carbon nanotubes are isolated by dissolving the membranes after the end of reaction. Carbon nanotubes with covalent immobilized enzymes are used for biosensors fabrications. The obtained membranes were characterized by Scanning Electron Microscopy, Thermal analysis, FT-IR Spectroscopy, Nuclear Magnetic Resonance, and functionalized carbon nanotubes were characterized by FT-IR spectroscopy.

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.

SACCHARIS: an automated pipeline to streamline discovery of carbohydrate active enzyme activities within polyspecific families and de novo sequence datasets.

PubMed

Jones, Darryl R; Thomas, Dallas; Alger, Nicholas; Ghavidel, Ata; Inglis, G Douglas; Abbott, D Wade

2018-01-01

Deposition of new genetic sequences in online databases is expanding at an unprecedented rate. As a result, sequence identification continues to outpace functional characterization of carbohydrate active enzymes (CAZymes). In this paradigm, the discovery of enzymes with novel functions is often hindered by high volumes of uncharacterized sequences particularly when the enzyme sequence belongs to a family that exhibits diverse functional specificities (i.e., polyspecificity). Therefore, to direct sequence-based discovery and characterization of new enzyme activities we have developed an automated in silico pipeline entitled: Sequence Analysis and Clustering of CarboHydrate Active enzymes for Rapid Informed prediction of Specificity (SACCHARIS). This pipeline streamlines the selection of uncharacterized sequences for discovery of new CAZyme or CBM specificity from families currently maintained on the CAZy website or within user-defined datasets. SACCHARIS was used to generate a phylogenetic tree of a GH43, a CAZyme family with defined subfamily designations. This analysis confirmed that large datasets can be organized into sequence clusters of manageable sizes that possess related functions. Seeding this tree with a GH43 sequence from Bacteroides dorei DSM 17855 (BdGH43b, revealed it partitioned as a single sequence within the tree. This pattern was consistent with it possessing a unique enzyme activity for GH43 as BdGH43b is the first described α-glucanase described for this family. The capacity of SACCHARIS to extract and cluster characterized carbohydrate binding module sequences was demonstrated using family 6 CBMs (i.e., CBM6s). This CBM family displays a polyspecific ligand binding profile and contains many structurally determined members. Using SACCHARIS to identify a cluster of divergent sequences, a CBM6 sequence from a unique clade was demonstrated to bind yeast mannan, which represents the first description of an α-mannan binding CBM. Additionally, we have performed a CAZome analysis of an in-house sequenced bacterial genome and a comparative analysis of B. thetaiotaomicron VPI-5482 and B. thetaiotaomicron 7330, to demonstrate that SACCHARIS can generate "CAZome fingerprints", which differentiate between the saccharolytic potential of two related strains in silico. Establishing sequence-function and sequence-structure relationships in polyspecific CAZyme families are promising approaches for streamlining enzyme discovery. SACCHARIS facilitates this process by embedding CAZyme and CBM family trees generated from biochemically to structurally characterized sequences, with protein sequences that have unknown functions. In addition, these trees can be integrated with user-defined datasets (e.g., genomics, metagenomics, and transcriptomics) to inform experimental characterization of new CAZymes or CBMs not currently curated, and for researchers to compare differential sequence patterns between entire CAZomes. In this light, SACCHARIS provides an in silico tool that can be tailored for enzyme bioprospecting in datasets of increasing complexity and for diverse applications in glycobiotechnology.

Allosteric activation transitions in enzymes and biomolecular motors: insights from atomistic and coarse-grained simulations.

PubMed

Daily, Michael D; Yu, Haibo; Phillips, George N; Cui, Qiang

2013-01-01

The chemical step in enzymes is usually preceded by a kinetically distinct activation step that involves large-scale conformational transitions. In "simple" enzymes this step corresponds to the closure of the active site; in more complex enzymes, such as biomolecular motors, the activation step is more complex and may involve interactions with other biomolecules. These activation transitions are essential to the function of enzymes and perturbations in the scale and/or rate of these transitions are implicated in various serious human diseases; incorporating key flexibilities into engineered enzymes is also considered a major remaining challenge in rational enzyme design. Therefore it is important to understand the underlying mechanism of these transitions. This is a significant challenge to both experimental and computational studies because of the allosteric and multi-scale nature of such transitions. Using our recent studies of two enzyme systems, myosin and adenylate kinase (AK), we discuss how atomistic and coarse-grained simulations can be used to provide insights into the mechanism of activation transitions in realistic systems. Collectively, the results suggest that although many allosteric transitions can be viewed as domain displacements mediated by flexible hinges, there are additional complexities and various deviations. For example, although our studies do not find any evidence for "cracking" in AK, our results do underline the contribution of intra-domain properties (e.g., dihedral flexibility) to the rate of the transition. The study of mechanochemical coupling in myosin highlights that local changes important to chemistry require stabilization from more extensive structural changes; in this sense, more global structural transitions are needed to activate the chemistry in the active site. These discussions further emphasize the importance of better understanding factors that control the degree of co-operativity for allosteric transitions, again hinting at the intimate connection between protein stability and functional flexibility. Finally, a number of topics of considerable future interest are briefly discussed.

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.

A Novel Enzyme Portfolio for Red Algal Polysaccharide Degradation in the Marine Bacterium Paraglaciecola hydrolytica S66T Encoded in a Sizeable Polysaccharide Utilization Locus.

PubMed

Schultz-Johansen, Mikkel; Bech, Pernille K; Hennessy, Rosanna C; Glaring, Mikkel A; Barbeyron, Tristan; Czjzek, Mirjam; Stougaard, Peter

2018-01-01

Marine microbes are a rich source of enzymes for the degradation of diverse polysaccharides. Paraglaciecola hydrolytica S66 T is a marine bacterium capable of hydrolyzing polysaccharides found in the cell wall of red macroalgae. In this study, we applied an approach combining genomic mining with functional analysis to uncover the potential of this bacterium to produce enzymes for the hydrolysis of complex marine polysaccharides. A special feature of P. hydrolytica S66 T is the presence of a large genomic region harboring an array of carbohydrate-active enzymes (CAZymes) notably agarases and carrageenases. Based on a first functional characterization combined with a comparative sequence analysis, we confirmed the enzymatic activities of several enzymes required for red algal polysaccharide degradation by the bacterium. In particular, we report for the first time, the discovery of novel enzyme activities targeting furcellaran, a hybrid carrageenan containing both β-carrageenan and κ/β-carrageenan motifs. Some of these enzymes represent a new subfamily within the CAZy classification. From the combined analyses, we propose models for the complete degradation of agar and κ/β-type carrageenan by P. hydrolytica S66 T . The novel enzymes described here may find value in new bio-based industries and advance our understanding of the mechanisms responsible for recycling of red algal polysaccharides in marine ecosystems.

Modification of deoiled cumin dietary fiber with laccase and cellulase under high hydrostatic pressure.

PubMed

Ma, Mengmei; Mu, Taihua

2016-01-20

In this study, we evaluated the effects of high hydrostatic pressure (HHP) and enzyme (laccase and cellulase) treatment on the structural, physicochemical, and functional properties and antioxidant activity of deoiled cumin dietary fiber (DF). HHP-enzyme treatment increased the contents of soluble dietary fiber (SDF) (30.37 g/100g), monosaccharides (except for glucose), uronic acids, and total polyphenol. HHP-enzyme treatment altered the honey-comb structure of DF and generated new polysaccharides. DF modified by HHP-enzyme treatment exhibited improved water retention capacity (10.02 g/g), water swelling capacity (11.19 mL/g), fat and glucose absorption capacities (10.44 g/g, 22.18-63.54 mmol/g), α-amylase activity inhibition ration (37.95%), and bile acid retardation index (48.85-52.58%). The antioxidant activity of DF was mainly correlated to total polyphenol content (R=0.8742). Therefore, DF modified by HHP-enzyme treatment from deoiled cumin could be used as a fiber-rich ingredient in functional foods. Copyright © 2015. Published by Elsevier Ltd.

Effect of detergents, trypsin, and bivalent metal ions on interfacial activation and functioning of phospholipase D.

PubMed

Madyarov, Sh R

2014-07-01

The effects of detergents, trypsin, and bivalent metal ions on production of phosphatidic and lysophosphatidic acids by the action of phospholipase D (PLD) on lecithin and lysolecithin were studied. It was found that these reaction products and dodecyl sulfate ions activate PLD, whereas other anionic detergents are less effective. A protective effect of the functioning enzyme against its hydrolytic inactivation by trypsin was found. Bivalent metal ions can be arranged in the following sequence by their ability to activate PLD in the hydrolysis of lecithin and lysolecithin: Ca2+>Sr2+>Ba2+>Mg2+. These results are considered in relation to a proposed mechanism of activation and functioning of PLD with the participation of clusters of phosphatidates and lysophosphatidates. Such Me2+-induced formation of rafts or microdomains from the products of hydrolysis of phospholipids can rationalize not only PLD activation and self-regulation, but also the action of this mechanism on other components and properties of biomembranes. PLD and other lipolytic enzymes can be classified as lateral vector enzymes.

Direct interaction of beta-amyloid with Na,K-ATPase as a putative regulator of the enzyme function

NASA Astrophysics Data System (ADS)

Petrushanko, Irina Yu.; Mitkevich, Vladimir A.; Anashkina, Anastasia A.; Adzhubei, Alexei A.; Burnysheva, Ksenia M.; Lakunina, Valentina A.; Kamanina, Yulia V.; Dergousova, Elena A.; Lopina, Olga D.; Ogunshola, Omolara O.; Bogdanova, Anna Yu.; Makarov, Alexander A.

2016-06-01

By maintaining the Na+ and K+ transmembrane gradient mammalian Na,K-ATPase acts as a key regulator of neuronal electrotonic properties. Na,K-ATPase has an important role in synaptic transmission and memory formation. Accumulation of beta-amyloid (Aβ) at the early stages of Alzheimer’s disease is accompanied by reduction of Na,K-ATPase functional activity. The molecular mechanism behind this phenomenon is not known. Here we show that the monomeric Aβ(1-42) forms a tight (Kd of 3 μM), enthalpy-driven equimolar complex with α1β1 Na,K-ATPase. The complex formation results in dose-dependent inhibition of the enzyme hydrolytic activity. The binding site of Aβ(1-42) is localized in the “gap” between the alpha- and beta-subunits of Na,K-ATPase, disrupting the enzyme functionality by preventing the subunits from shifting towards each other. Interaction of Na,K-ATPase with exogenous Aβ(1-42) leads to a pronounced decrease of the enzyme transport and hydrolytic activity and Src-kinase activation in neuroblastoma cells SH-SY5Y. This interaction allows regulation of Na,K-ATPase activity by short-term increase of the Aβ(1-42) level. However prolonged increase of Aβ(1-42) level under pathological conditions could lead to chronical inhibition of Na,K-ATPase and disruption of neuronal function. Taken together, our data suggest the role of beta-amyloid as a novel physiological regulator of Na,K-ATPase.

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.

Identification of novel biomass-degrading enzymes from genomic dark matter: Populating genomic sequence space with functional annotation.

PubMed

Piao, Hailan; Froula, Jeff; Du, Changbin; Kim, Tae-Wan; Hawley, Erik R; Bauer, Stefan; Wang, Zhong; Ivanova, Nathalia; Clark, Douglas S; Klenk, Hans-Peter; Hess, Matthias

2014-08-01

Although recent nucleotide sequencing technologies have significantly enhanced our understanding of microbial genomes, the function of ∼35% of genes identified in a genome currently remains unknown. To improve the understanding of microbial genomes and consequently of microbial processes it will be crucial to assign a function to this "genomic dark matter." Due to the urgent need for additional carbohydrate-active enzymes for improved production of transportation fuels from lignocellulosic biomass, we screened the genomes of more than 5,500 microorganisms for hypothetical proteins that are located in the proximity of already known cellulases. We identified, synthesized and expressed a total of 17 putative cellulase genes with insufficient sequence similarity to currently known cellulases to be identified as such using traditional sequence annotation techniques that rely on significant sequence similarity. The recombinant proteins of the newly identified putative cellulases were subjected to enzymatic activity assays to verify their hydrolytic activity towards cellulose and lignocellulosic biomass. Eleven (65%) of the tested enzymes had significant activity towards at least one of the substrates. This high success rate highlights that a gene context-based approach can be used to assign function to genes that are otherwise categorized as "genomic dark matter" and to identify biomass-degrading enzymes that have little sequence similarity to already known cellulases. The ability to assign function to genes that have no related sequence representatives with functional annotation will be important to enhance our understanding of microbial processes and to identify microbial proteins for a wide range of applications. © 2014 Wiley Periodicals, Inc.

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.

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.

Quantitative Analysis of the Effective Functional Structure in Yeast Glycolysis

PubMed Central

De la Fuente, Ildefonso M.; Cortes, Jesus M.

2012-01-01

The understanding of the effective functionality that governs the enzymatic self-organized processes in cellular conditions is a crucial topic in the post-genomic era. In recent studies, Transfer Entropy has been proposed as a rigorous, robust and self-consistent method for the causal quantification of the functional information flow among nonlinear processes. Here, in order to quantify the functional connectivity for the glycolytic enzymes in dissipative conditions we have analyzed different catalytic patterns using the technique of Transfer Entropy. The data were obtained by means of a yeast glycolytic model formed by three delay differential equations where the enzymatic rate equations of the irreversible stages have been explicitly considered. These enzymatic activity functions were previously modeled and tested experimentally by other different groups. The results show the emergence of a new kind of dynamical functional structure, characterized by changing connectivity flows and a metabolic invariant that constrains the activity of the irreversible enzymes. In addition to the classical topological structure characterized by the specific location of enzymes, substrates, products and feedback-regulatory metabolites, an effective functional structure emerges in the modeled glycolytic system, which is dynamical and characterized by notable variations of the functional interactions. The dynamical structure also exhibits a metabolic invariant which constrains the functional attributes of the enzymes. Finally, in accordance with the classical biochemical studies, our numerical analysis reveals in a quantitative manner that the enzyme phosphofructokinase is the key-core of the metabolic system, behaving for all conditions as the main source of the effective causal flows in yeast glycolysis. PMID:22393350

Immobilization of lipase and keratinase on functionalized SBA-15 nanostructured materials

NASA Astrophysics Data System (ADS)

Le, Hy G.; Vu, Tuan A.; Tran, Hoa T. K.; Dang, Phuong T.

2013-12-01

SBA-15 nanostructured materials were synthesized via hydrothermal treatment and were functionalized with 3- aminopropyltriethoxysilane (APTES). The obtained samples were characterized by different techniques such as XRD, BET, TEM, IR and DTA. After functionalization, it showed that these nanostrucrured materials still maintained the hexagonal pore structure of the parent SBA-15. The model enzyms chosen in this study were lipase and keratinase. Lipase was a biocatalyst for hydrolyzation of long chain triglycerides or methyl esters of long chain alcohols and fatty acids; keratinase is a proteolytic enzyme that catalyzes the cleavage of keratin. The functionalized SBA-15 materials were used to immobilize lipase and keratinase, exhibiting higher activity than that of the unfunctionalized pure silica SBA-15 ones. This might be due to the enhancing of surface hydrophobicity upon functionalization. The surface functionalization of the nanostructured silicas with organic groups can favor the interaction between enzyme and the supports and consequently increasing the operational stability of the immobilized enzymes. The loading of lipase on functionalized SBA-15 materials was higher than that of keratinase. This might be rationalized by the difference in size of enzyms.

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 involves 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 inactivators 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 andmore » 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.« less

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

Conservation of Dynamics Associated with Biological Function in an Enzyme Superfamily.

PubMed

Narayanan, Chitra; Bernard, David N; Bafna, Khushboo; Gagné, Donald; Chennubhotla, Chakra S; Doucet, Nicolas; Agarwal, Pratul K

2018-03-06

Enzyme superfamily members that share common chemical and/or biological functions also share common features. While the role of structure is well characterized, the link between enzyme function and dynamics is not well understood. We present a systematic characterization of intrinsic dynamics of over 20 members of the pancreatic-type RNase superfamily, which share a common structural fold. This study is motivated by the fact that the range of chemical activity as well as molecular motions of RNase homologs spans over 10 5 folds. Dynamics was characterized using a combination of nuclear magnetic resonance experiments and computer simulations. Phylogenetic clustering led to the grouping of sequences into functionally distinct subfamilies. Detailed characterization of the diverse RNases showed conserved dynamical traits for enzymes within subfamilies. These results suggest that selective pressure for the conservation of dynamical behavior, among other factors, may be linked to the distinct chemical and biological functions in an enzyme superfamily. Copyright © 2018 Elsevier Ltd. All rights reserved.

Differences in the Activities of Eight Enzymes from Ten Soil Fungi and Their Possible Influences on the Surface Structure, Functional Groups, and Element Composition of Soil Colloids

PubMed Central

Wang, Wenjie; Li, Yanhong; Wang, Huimei; Zu, Yuangang

2014-01-01

How soil fungi function in soil carbon and nutrient cycling is not well understood by using fungal enzymatic differences and their interactions with soil colloids. Eight extracellular enzymes, EEAs (chitinase, carboxymethyl cellulase, β-glucosidase, protease, acid phosphatase, polyphenol oxidase, laccase, and guaiacol oxidase) secreted by ten fungi were compared, and then the fungi that showed low and high enzymatic activity were co-cultured with soil colloids for the purpose of finding fungi-soil interactions. Some fungi (Gomphidius rutilus, Russula integra, Pholiota adiposa, and Geastrum mammosum) secreted 3–4 enzymes with weak activities, while others (Cyathus striatus, Suillus granulate, Phallus impudicus, Collybia dryophila, Agaricus sylvicola, and Lactarius deliciosus) could secret over 5 enzymes with high activities. The differences in these fungi contributed to the alterations of functional groups (stretching bands of O-H, N-H, C-H, C = O, COO- decreased by 11–60%, while P = O, C-O stretching, O-H bending and Si-O-Si stretching increased 9–22%), surface appearance (disappearance of adhesive organic materials), and elemental compositions (11–49% decreases in C1s) in soil colloids. Moreover, more evident changes were generally in high enzymatic fungi (C. striatus) compared with low enzymatic fungi (G. rutilus). Our findings indicate that inter-fungi differences in EEA types and activities might be responsible for physical and chemical changes in soil colloids (the most active component of soil matrix), highlighting the important roles of soil fungi in soil nutrient cycling and functional maintenance. PMID:25398013

Differences in the activities of eight enzymes from ten soil fungi and their possible influences on the surface structure, functional groups, and element composition of soil colloids.

PubMed

Wang, Wenjie; Li, Yanhong; Wang, Huimei; Zu, Yuangang

2014-01-01

How soil fungi function in soil carbon and nutrient cycling is not well understood by using fungal enzymatic differences and their interactions with soil colloids. Eight extracellular enzymes, EEAs (chitinase, carboxymethyl cellulase, β-glucosidase, protease, acid phosphatase, polyphenol oxidase, laccase, and guaiacol oxidase) secreted by ten fungi were compared, and then the fungi that showed low and high enzymatic activity were co-cultured with soil colloids for the purpose of finding fungi-soil interactions. Some fungi (Gomphidius rutilus, Russula integra, Pholiota adiposa, and Geastrum mammosum) secreted 3-4 enzymes with weak activities, while others (Cyathus striatus, Suillus granulate, Phallus impudicus, Collybia dryophila, Agaricus sylvicola, and Lactarius deliciosus) could secret over 5 enzymes with high activities. The differences in these fungi contributed to the alterations of functional groups (stretching bands of O-H, N-H, C-H, C = O, COO- decreased by 11-60%, while P = O, C-O stretching, O-H bending and Si-O-Si stretching increased 9-22%), surface appearance (disappearance of adhesive organic materials), and elemental compositions (11-49% decreases in C1s) in soil colloids. Moreover, more evident changes were generally in high enzymatic fungi (C. striatus) compared with low enzymatic fungi (G. rutilus). Our findings indicate that inter-fungi differences in EEA types and activities might be responsible for physical and chemical changes in soil colloids (the most active component of soil matrix), highlighting the important roles of soil fungi in soil nutrient cycling and functional maintenance.

The active centre of triose phosphate isomerase

PubMed Central

Burton, Pamela M.; Waley, S. G.

1966-01-01

The molecular weight and amino acid composition of triose phosphate isomerase have been determined. The molecular weight (43000) is lower and the molecular activity (500000) higher than those of most other glycolytic enzymes. Reaction with iodoacetate (studied with radioactive reagent) takes place in two phases: in the first phase, at pH6·3, cysteine and methionine groups react and enzymic activity is unimpaired; in the second phase, histidine reacts and enzymic activity is lost. Photo-oxidation leads to inactivation, with loss of cysteine, of histidine and of tryptophan, but little loss of tyrosine. The mechanism postulated for the action of the enzyme demands the intervention of a group functioning as a base, and the results obtained are consistent with histidine's being the basic group in the active centre. PMID:5969283

Structural Diversity in the Dandelion (Taraxacum officinale) Polyphenol Oxidase Family Results in Different Responses to Model Substrates

PubMed Central

Dirks-Hofmeister, Mareike E.; Singh, Ratna; Leufken, Christine M.; Inlow, Jennifer K.; Moerschbacher, Bruno M.

2014-01-01

Polyphenol oxidases (PPOs) are ubiquitous type-3 copper enzymes that catalyze the oxygen-dependent conversion of o-diphenols to the corresponding quinones. In most plants, PPOs are present as multiple isoenzymes that probably serve distinct functions, although the precise relationship between sequence, structure and function has not been addressed in detail. We therefore compared the characteristics and activities of recombinant dandelion PPOs to gain insight into the structure–function relationships within the plant PPO family. Phylogenetic analysis resolved the 11 isoenzymes of dandelion into two evolutionary groups. More detailed in silico and in vitro analyses of four representative PPOs covering both phylogenetic groups were performed. Molecular modeling and docking predicted differences in enzyme-substrate interactions, providing a structure-based explanation for grouping. One amino acid side chain positioned at the entrance to the active site (position HB2+1) potentially acts as a “selector” for substrate binding. In vitro activity measurements with the recombinant, purified enzymes also revealed group-specific differences in kinetic parameters when the selected PPOs were presented with five model substrates. The combination of our enzyme kinetic measurements and the in silico docking studies therefore indicate that the physiological functions of individual PPOs might be defined by their specific interactions with different natural substrates. PMID:24918587

Structural diversity in the dandelion (Taraxacum officinale) polyphenol oxidase family results in different responses to model substrates.

PubMed

Dirks-Hofmeister, Mareike E; Singh, Ratna; Leufken, Christine M; Inlow, Jennifer K; Moerschbacher, Bruno M

2014-01-01

Polyphenol oxidases (PPOs) are ubiquitous type-3 copper enzymes that catalyze the oxygen-dependent conversion of o-diphenols to the corresponding quinones. In most plants, PPOs are present as multiple isoenzymes that probably serve distinct functions, although the precise relationship between sequence, structure and function has not been addressed in detail. We therefore compared the characteristics and activities of recombinant dandelion PPOs to gain insight into the structure-function relationships within the plant PPO family. Phylogenetic analysis resolved the 11 isoenzymes of dandelion into two evolutionary groups. More detailed in silico and in vitro analyses of four representative PPOs covering both phylogenetic groups were performed. Molecular modeling and docking predicted differences in enzyme-substrate interactions, providing a structure-based explanation for grouping. One amino acid side chain positioned at the entrance to the active site (position HB2+1) potentially acts as a "selector" for substrate binding. In vitro activity measurements with the recombinant, purified enzymes also revealed group-specific differences in kinetic parameters when the selected PPOs were presented with five model substrates. The combination of our enzyme kinetic measurements and the in silico docking studies therefore indicate that the physiological functions of individual PPOs might be defined by their specific interactions with different natural substrates.

Using NMR spectroscopy to elucidate the role of molecular motions in enzyme function.

PubMed

Lisi, George P; Loria, J Patrick

2016-02-01

Conformational motions play an essential role in enzyme function, often facilitating the formation of enzyme-substrate complexes and/or product release. Although considerable debate remains regarding the role of molecular motions in the conversion of enzymatic substrates to products, numerous examples have found motions to be crucial for optimization of enzyme scaffolds, effective substrate binding, and product dissociation. Conformational fluctuations are often rate-limiting to enzyme catalysis, primarily through product release, with the chemical reaction occurring much more quickly. As a result, the direct involvement of motions at various stages along the enzyme reaction coordinate remains largely unknown and untested. In the following review, we describe the use of solution NMR techniques designed to probe various timescales of molecular motions and detail examples in which motions play a role in propagating catalytic effects from the active site and directly participate in essential aspects of enzyme function. Copyright © 2015 Elsevier B.V. All rights reserved.

In silico studies on tryparedoxin peroxidase of Leishmania infantum: structural aspects.

PubMed

Singh, Bishal Kumar; Dubey, Vikash Kumar

2009-09-01

Tryparedoxin peroxidase (TryP) is a key enzyme of the trypanothione-dependent metabolism for removal of oxidative stress in leishmania. These enzymes function as antioxidants through their peroxidase and peroxynitrite reductase activities. Inhibitors of this enzyme are presumed to be antilesihmania drugs and structural studies are prerequisite of rational drug design. We have constructed three dimensional structure of TryP of Leishmania infantum using comparative modeling. Structural analysis reveals several interesting features. Moreover, it shows remarkable structural difference with human host glutathione peroxidase, an enzyme involved in similar function and TryP from Leishmania major.

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

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

PubMed

Baltar, Federico

2017-01-01

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

Laccase-Functionalized Graphene Oxide Assemblies as Efficient Nanobiocatalysts for Oxidation Reactions

PubMed Central

Patila, Michaela; Kouloumpis, Antonios; Gournis, Dimitrios; Rudolf, Petra; Stamatis, Haralambos

2016-01-01

Multi-layer graphene oxide-enzyme nanoassemblies were prepared through the multi-point covalent immobilization of laccase from Trametes versicolor (TvL) on functionalized graphene oxide (fGO). The catalytic properties of the fGO-TvL nanoassemblies were found to depend on the number of the graphene oxide-enzyme layers present in the nanostructure. The fGO-TvL nanoassemblies exhibit an enhanced thermal stability at 60 °C, as demonstrated by a 4.7-fold higher activity as compared to the free enzyme. The multi-layer graphene oxide-enzyme nanoassemblies can efficiently catalyze the oxidation of anthracene, as well as the decolorization of an industrial dye, pinacyanol chloride. These materials retained almost completely their decolorization activity after five reaction cycles, proving their potential as efficient nano- biocatalysts for various applications. PMID:26927109

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.

Functionalization of a membrane sublayer using reverse filtration of enzymes and dopamine coating.

PubMed

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

2014-12-24

High permeability, high enzyme loading, and strong antifouling ability are the desired features for a biocatalytic membrane to be used in an enzymatic membrane reactor (EMR). To achieve these goals, the membrane sublayer was enriched with laccase by reverse filtration in this case, and the resulting enzyme-loaded sublayer was covered with a dopamine coating. After membrane reversal, the virgin membrane skin layer was facing the feed and the enzymes were entrapped by a polydopamine network in the membrane sublayer. Thus, the membrane sublayer was functionalized as a catalytically active layer. The effects of the original membrane properties (i.e., materials, pore size, and structure), enzyme type (i.e., laccase and alcohol dehydrogenase), and coating conditions (i.e., time and pH) on the resulting biocatalytic membrane permeability, enzyme loading, and activity were investigated. Using a RC10 kDa membrane with sponge-like sublayer to immobilize laccase with dopamine coating, the trade-off between permeability and enzyme loading was broken, and enzyme loading reached 44.5% without any permeability loss. After 85 days of storage and reuse 14 times, more than 80% of the immobilized laccase activity was retained for the membrane with a dopamine coating, while the relative activity was less than 40% without the coating. The resistance to high temperature and acidic/alkaline pH was also improved by the dopamine coating for the immobilized laccase. Moreover, this biocatalytic membrane could resist mild hydrodynamic cleaning (e.g., back-flushing), but the catalytic ability was reduced by chemical cleaning at extreme pH (e.g., 1.5 and 11.5). Since the immobilized enzyme is not directly facing the bulk of EMRs and the substrate can be specifically selected by the separation skin layer, this biocatalytic membrane is promising for cascade catalytic reactions.

A Quaternary Mechanism Enables the Complex Biological Functions of Octameric Human UDP-glucose Pyrophosphorylase, a Key Enzyme in Cell Metabolism

PubMed Central

Führing, Jana Indra; Cramer, Johannes Thomas; Schneider, Julia; Baruch, Petra; Gerardy-Schahn, Rita; Fedorov, Roman

2015-01-01

In mammals, UDP-glucose pyrophosphorylase (UGP) is the only enzyme capable of activating glucose-1-phosphate (Glc-1-P) to UDP-glucose (UDP-Glc), a metabolite located at the intersection of virtually all metabolic pathways in the mammalian cell. Despite the essential role of its product, the molecular basis of UGP function is poorly understood. Here we report the crystal structure of human UGP in complex with its product UDP-Glc. Beyond providing first insight into the active site architecture, we describe the substrate binding mode and intermolecular interactions in the octameric enzyme that are crucial to its activity. Importantly, the quaternary mechanism identified for human UGP in this study may be common for oligomeric sugar-activating nucleotidyltransferases. Elucidating such mechanisms is essential for understanding nucleotide sugar metabolism and opens the perspective for the development of drugs that specifically inhibit simpler organized nucleotidyltransferases in pathogens. PMID:25860585

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.

Halophiles and their enzymes: negativity put to good use.

PubMed

DasSarma, Shiladitya; DasSarma, Priya

2015-06-01

Halophilic microorganisms possess stable enzymes that function in very high salinity, an extreme condition that leads to denaturation, aggregation, and precipitation of most other proteins. Genomic and structural analyses have established that the enzymes of halophilic Archaea and many halophilic Bacteria are negatively charged due to an excess of acidic over basic residues, and altered hydrophobicity, which enhance solubility and promote function in low water activity conditions. Here, we provide an update on recent bioinformatic analysis of predicted halophilic proteomes as well as experimental molecular studies on individual halophilic enzymes. Recent efforts on discovery and utilization of halophiles and their enzymes for biotechnology, including biofuel applications are also considered. Copyright © 2015 Elsevier Ltd. All rights reserved.

Halophiles and their enzymes: Negativity put to good use

PubMed Central

DasSarma, Shiladitya; DasSarma, Priya

2015-01-01

Halophilic microorganisms possess stable enzymes that function in very high salinity, an extreme condition that leads to denaturation, aggregation, and precipitation of most other proteins. Genomic and structural analyses have established that the enzymes of halophilic Archaea and many halophilic Bacteria are negatively charged due to an excess of acidic over basic residues, and altered hydrophobicity, which enhance solubility and promote function in low water activity conditions. Here, we provide an update on recent bioinformatic analysis of predicted halophilic proteomes as well as experimental molecular studies on individual halophilic enzymes. On-going efforts on discovery and utilization of halophiles and their enzymes for biotechnology, including biofuel applications are also considered. PMID:26066288

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.

Bridging Enzymatic Structure Function via Mechanics: A Coarse-Grain Approach.

PubMed

Sacquin-Mora, S

2016-01-01

Flexibility is a central aspect of protein function, and ligand binding in enzymes involves a wide range of structural changes, ranging from large-scale domain movements to small loop or side-chain rearrangements. In order to understand how the mechanical properties of enzymes, and the mechanical variations that are induced by ligand binding, relate to enzymatic activity, we carried out coarse-grain Brownian dynamics simulations on a set of enzymes whose structures in the unbound and ligand-bound forms are available in the Protein Data Bank. Our results show that enzymes are remarkably heterogeneous objects from a mechanical point of view and that the local rigidity of individual residues is tightly connected to their part in the protein's overall structure and function. The systematic comparison of the rigidity of enzymes in their unbound and bound forms highlights the fact that small conformational changes can induce large mechanical effects, leading to either more or less flexibility depending on the enzyme's architecture and the location of its ligand-biding site. These mechanical variations target a limited number of specific residues that occupy key locations for enzymatic activity, and our approach thus offers a mean to detect perturbation-sensitive sites in enzymes, where the addition or removal of a few interactions will lead to important changes in the proteins internal dynamics. © 2016 Elsevier Inc. All rights reserved.

Electrochemical Nanoparticle-Enzyme Sensors for Screening Bacterial Contamination in Drinking Water

PubMed Central

Chen, Juhong; Jiang, Ziwen; Ackerman, Jonathan D.; Yazdani, Mahdieh; Hou, Singyuk

2015-01-01

Traditional plating and culturing methods used to quantify bacteria commonly require hours to days from sampling to results. We present here a simple, sensitive and rapid electrochemical method for bacteria detection in drinking water based on gold nanoparticle-enzyme complexes. The gold nanoparticles were functionalized with positively charged quaternary amine headgroups that could bind to enzymes through electrostatic interactions, resulting in inhibition of enzymatic activity. In the presence of bacteria, the nanoparticles released from the enzymes and preferentially bound to the bacteria, resulting in an increase in enzyme activity, releasing a redox-active phenol from the substrate. We employed this strategy for the electrochemical sensing of Escherichia coli and Staphylococcus aureus, resulting in a rapid detection (<1h) with high sensitivity (102 CFU·mL−1). PMID:26042607

Pericyclic reactions catalyzed by chorismate-utilizing enzymes

PubMed Central

Lamb, Audrey L.

2011-01-01

One of the fundamental questions of enzymology is how catalytic power is derived. This review focuses on recent developments in the structure-function relationships of chorismate-utilizing enzymes involved in siderophore biosynthesis to provide insight into the biocatalysis of pericyclic reactions. Specifically, salicylate synthesis by the two-enzyme pathway in Pseudomonas aeruginosa is examined. The isochorismate-pyruvate lyase is discussed in the context of its homologues, the chorismate mutases, and the isochorismate synthase is compared to its homologues in the MST-family (menaquinone, siderophore or tryptophan biosynthesis) of enzymes. The tentative conclusion is that the activities observed cannot be reconciled by inspection of the active site participants alone. Instead, individual activities must arise from unique dynamic properties of each enzyme that are tuned to promote specific chemistries. PMID:21823653

Ubiquitin C-terminal electrophiles are activity-based probes for identification and mechanistic study of ubiquitin conjugating machinery.

PubMed

Love, Kerry Routenberg; Pandya, Renuka K; Spooner, Eric; Ploegh, Hidde L

2009-04-17

Protein modification by ubiquitin (Ub) and ubiquitin-like modifiers (Ubl) requires the action of activating (E1), conjugating (E2), and ligating (E3) enzymes and is a key step in the specific destruction of proteins. Deubiquitinating enzymes (DUBs) deconjugate substrates modified with Ub/Ubl's and recycle Ub inside the cell. Genome mining based on sequence homology to proteins with known function has assigned many enzymes to this pathway without confirmation of either conjugating or DUB activity. Function-dependent methodologies are still the most useful for rapid identification or assessment of biological activity of expressed proteins from cells. Activity-based protein profiling uses chemical probes that are active-site-directed for the classification of protein activities in complex mixtures. Here we show that the design and use of an expanded set of Ub-based electrophilic probes allowed us to recover and identify members of each enzyme class in the ubiquitin-proteasome system, including E3 ligases and DUBs with previously unverified activity. We show that epitope-tagged Ub-electrophilic probes can be used as activity-based probes for E3 ligase identification by in vitro labeling and activity studies of purified enzymes identified from complex mixtures in cell lysate. Furthermore, the reactivity of our probe with the HECT domain of the E3 Ub ligase ARF-BP1 suggests that multiple cysteines may be in the vicinity of the E2-binding site and are capable of the transfer of Ub to self or to a substrate protein.

Efficient magnetic recycling of covalently attached enzymes on carbon-coated metallic nanomagnets.

PubMed

Zlateski, Vladimir; Fuhrer, Roland; Koehler, Fabian M; Wharry, Scott; Zeltner, Martin; Stark, Wendelin J; Moody, Thomas S; Grass, Robert N

2014-04-16

In the pursuit of robust and reusable biocatalysts for industrial synthetic chemistry, nanobiotechnology is currently taking a significant part. Recently, enzymes have been immobilized on different nanoscaffold supports. Carbon coated metallic nanoparticles were found to be a practically useful support for enzyme immobilization due to their large surface area, high magnetic saturation, and manipulatable surface chemistry. In this study carbon coated cobalt nanoparticles were chemically functionalized (diazonium chemistry), activated for bioconjugation (N,N-disuccinimidyl carbonate), and subsequently used in enzyme immobilization. Three enzymes, β-glucosidase, α-chymotrypsin, and lipase B were successfully covalently immobilized on the magnetic nonsupport. The enzyme-particle conjugates formed retained their activity and stability after immobilization and were efficiently recycled from milliliter to liter scales in short recycle times.

The deubiquitinating enzyme USP36 controls selective autophagy activation by ubiquitinated proteins.

PubMed

Taillebourg, Emmanuel; Gregoire, Isabel; Viargues, Perrine; Jacomin, Anne-Claire; Thevenon, Dominique; Faure, Mathias; Fauvarque, Marie-Odile

2012-05-01

Initially described as a nonspecific degradation process induced upon starvation, autophagy is now known also to be involved in the degradation of specific ubiquitinated substrates such as mitochondria, bacteria and aggregated proteins, ensuring crucial functions in cell physiology and immunity. We report here that the deubiquitinating enzyme USP36 controls selective autophagy activation in Drosophila and in human cells. We show that dUsp36 loss of function autonomously inhibits cell growth while activating autophagy. Despite the phenotypic similarity, dUSP36 is not part of the TOR signaling pathway. Autophagy induced by dUsp36 loss of function depends on p62/SQSTM1, an adaptor for delivering cargo marked by polyubiquitin to autophagosomes. Consistent with p62 requirement, dUsp36 mutant cells display nuclear aggregates of ubiquitinated proteins, including Histone H2B, and cytoplasmic ubiquitinated proteins; the latter are eliminated by autophagy. Importantly, USP36 function in p62-dependent selective autophagy is conserved in human cells. Our work identifies a novel, crucial role for a deubiquitinating enzyme in selective autophagy.

A novel lactone-forming carboxylesterase: molecular identification of a tuliposide A-converting enzyme in tulip.

PubMed

Nomura, Taiji; Ogita, Shinjiro; Kato, Yasuo

2012-06-01

Tuliposides, the glucose esters of 4-hydroxy-2-methylenebutanoate and 3,4-dihydroxy-2-methylenebutanoate, are major secondary metabolites in tulip (Tulipa gesneriana). Their lactonized aglycons, tulipalins, function as defensive chemicals due to their biological activities. We recently found that tuliposide-converting enzyme (TCE) purified from tulip bulbs catalyzed the conversion of tuliposides to tulipalins, but the possibility of the presence of several TCE isozymes was raised: TCE in tissues other than bulbs is different from bulb TCE. Here, to prove this hypothesis, TCE was purified from petals, which have the second highest TCE activity after bulbs. The purified enzyme, like the bulb enzyme, preferentially accepted tuliposides as substrates, with 6-tuliposide A the best substrate, which allowed naming the enzyme tuliposide A-converting enzyme (TCEA), but specific activity and molecular mass differed between the petal and bulb enzymes. After peptide sequencing, a novel cDNA (TgTCEA) encoding petal TCEA was isolated, and the functional characterization of the recombinant enzyme verified that TgTCEA catalyzes the conversion of 6-tuliposide A to tulipalin A. TgTCEA was transcribed in all tulip tissues but not in bulbs, indicating the presence of a bulb-specific TgTCEA, as suggested by the distinct enzymatic characters between the petal and bulb enzymes. Plastidial localization of TgTCEA enzyme was revealed, which allowed proposing a cytological mechanism of TgTCE-mediated tulipalin formation in the tulip defensive strategy. Site-directed mutagenesis of TgTCEA suggested that the oxyanion hole and catalytic triad characteristic of typical carboxylesterases are essential for the catalytic process of TgTCEA enzyme. To our knowledge, TgTCEA is the first identified member of the lactone-forming carboxylesterases, specifically catalyzing intramolecular transesterification.

A Novel Lactone-Forming Carboxylesterase: Molecular Identification of a Tuliposide A-Converting Enzyme in Tulip1[W

PubMed Central

Nomura, Taiji; Ogita, Shinjiro; Kato, Yasuo

2012-01-01

Tuliposides, the glucose esters of 4-hydroxy-2-methylenebutanoate and 3,4-dihydroxy-2-methylenebutanoate, are major secondary metabolites in tulip (Tulipa gesneriana). Their lactonized aglycons, tulipalins, function as defensive chemicals due to their biological activities. We recently found that tuliposide-converting enzyme (TCE) purified from tulip bulbs catalyzed the conversion of tuliposides to tulipalins, but the possibility of the presence of several TCE isozymes was raised: TCE in tissues other than bulbs is different from bulb TCE. Here, to prove this hypothesis, TCE was purified from petals, which have the second highest TCE activity after bulbs. The purified enzyme, like the bulb enzyme, preferentially accepted tuliposides as substrates, with 6-tuliposide A the best substrate, which allowed naming the enzyme tuliposide A-converting enzyme (TCEA), but specific activity and molecular mass differed between the petal and bulb enzymes. After peptide sequencing, a novel cDNA (TgTCEA) encoding petal TCEA was isolated, and the functional characterization of the recombinant enzyme verified that TgTCEA catalyzes the conversion of 6-tuliposide A to tulipalin A. TgTCEA was transcribed in all tulip tissues but not in bulbs, indicating the presence of a bulb-specific TgTCEA, as suggested by the distinct enzymatic characters between the petal and bulb enzymes. Plastidial localization of TgTCEA enzyme was revealed, which allowed proposing a cytological mechanism of TgTCE-mediated tulipalin formation in the tulip defensive strategy. Site-directed mutagenesis of TgTCEA suggested that the oxyanion hole and catalytic triad characteristic of typical carboxylesterases are essential for the catalytic process of TgTCEA enzyme. To our knowledge, TgTCEA is the first identified member of the lactone-forming carboxylesterases, specifically catalyzing intramolecular transesterification. PMID:22474185

Contribution of the mu loop to the structure and function of rat glutathione transferase M1-1

PubMed Central

Hearne, Jennifer L.; Colman, Roberta F.

2006-01-01

The “mu loop,” an 11-residue loop spanning amino acid residues 33–43, is a characteristic structural feature of the mu class of glutathione transferases. To assess the contribution of the mu loop to the structure and function of rat GST M1-1, amino acid residues 35–44 (35GDAPDYDRSQ44) were excised by deletion mutagenesis, resulting in the “Deletion Enzyme.” Kinetic studies reveal that the Km values of the Deletion Enzyme are markedly increased compared with those of the wild-type enzyme: 32-fold for 1-chloro-2,4-dinitrobenzene, 99-fold for glutathione, and 880-fold for monobromobimane, while the Vmax value for each substrate is increased only modestly. Results from experiments probing the structure of the Deletion Enzyme, in comparison with that of the wild-type enzyme, suggest that the secondary and quaternary structures have not been appreciably perturbed. Thermostability studies indicate that the Deletion Enzyme is as stable as the wild-type enzyme at 4°C and 10°C, but it rapidly loses activity at 25°C, unlike the wild-type enzyme. In the temperature range of 4°C through 25°C, the loss of activity of the Deletion Enzyme is not the result of a change in its structure, as determined by circular dichroism spectroscopy and sedimentation equilibrium centrifugation. Collectively, these results indicate that the mu loop is not essential for GST M1-1 to maintain its structure nor is it required for the enzyme to retain some catalytic activity. However, it is an important determinant of the enzyme's affinity for its substrates. PMID:16672236

Scaling of oxidative and glycolytic enzymes in mammals.

PubMed

Emmett, B; Hochachka, P W

1981-09-01

The catalytic activities of several oxidative and glycolytic enzymes were determined in the gastrocnemius muscle of 10 mammalian species differing in body weight by nearly 6 orders of magnitude. When expressed in terms of units gm-1, the activities of enzymes functioning in oxidative metabolism (citrate synthase, beta-hydroxybutyrylCoA dehydrogenase, and malate dehydrogenase) decrease as body weight increases. Log-log plots (activity gm-1 vs body mass) yield straight lines with negative slopes that are less than the allometric exponent (-0.25) typically observed for basal metabolic rates. Since the amount of power a muscle can generate depends upon the catalytic potential of its enzyme machinery (the higher the catalytic potential the higher the maximum rate of energy generation), these data predict that the scope for aerobic activity in large mammals should be greater than in small mammals if nothing else becomes limiting, a result in fact recently obtained by Taylor et al. (Respir. Physiol., 1981). In contrast to the scaling of oxidative enzymes, the activities of enzymes functioning in anaerobic glycogenolysis (glycogen phosphorylase, pyruvate kinase, and lactate dehydrogenase) increase as body size increases. Log-log plots (activity gm-1 vs body mass) display a positive slope indicating that the larger the animal the higher the glycolytic potential of its skeletal muscles. This unexpected result may indicate higher relative power costs for burst type locomotion in larger mammals, which is in fact observed in within-species studies of man. However, the scaling of anaerobic muscle power has not been closely assessed in between-species comparisons of mammals varying greatly in body size.

Sustained gastrointestinal activity of dendronized polymer-enzyme conjugates

NASA Astrophysics Data System (ADS)

Fuhrmann, Gregor; Grotzky, Andrea; Lukić, Ružica; Matoori, Simon; Luciani, Paola; Yu, Hao; Zhang, Baozhong; Walde, Peter; Schlüter, A. Dieter; Gauthier, Marc A.; Leroux, Jean-Christophe

2013-07-01

Methods to stabilize and retain enzyme activity in the gastrointestinal tract are investigated rarely because of the difficulty of protecting proteins from an environment that has evolved to promote their digestion. Preventing the degradation of enzymes under these conditions, however, is critical for the development of new protein-based oral therapies. Here we show that covalent conjugation to polymers can stabilize orally administered therapeutic enzymes at different locations in the gastrointestinal tract. Architecturally and functionally diverse polymers are used to protect enzymes sterically from inactivation and to promote interactions with mucin on the stomach wall. Using this approach the in vivo activity of enzymes can be sustained for several hours in the stomach and/or in the small intestine. These findings provide new insight and a firm basis for the development of new therapeutic and imaging strategies based on orally administered proteins using a simple and accessible technology.

Lactate racemase is a nickel-dependent enzyme activated by a widespread maturation system

PubMed Central

Desguin, Benoît; Goffin, Philippe; Viaene, Eric; Kleerebezem, Michiel; Martin-Diaconescu, Vlad; Maroney, Michael J; Declercq, Jean-Paul; Soumillion, Patrice; Hols, Pascal

2014-01-01

Racemases catalyze the inversion of stereochemistry in biological molecules, giving the organism the ability to use both isomers. Among them, lactate racemase remains unexplored due to its intrinsic instability and lack of molecular characterization. Here we determine the genetic basis of lactate racemization in Lactobacillus plantarum. We show that, unexpectedly, the racemase is a nickel-dependent enzyme with a novel α/β fold. In addition, we decipher the process leading to an active enzyme, which involves the activation of the apo-enzyme by a single nickel-containing maturation protein that requires preactivation by two other accessory proteins. Genomic investigations reveal the wide distribution of the lactate racemase system among prokaryotes, showing the high significance of both lactate enantiomers in carbon metabolism. The even broader distribution of the nickel-based maturation system suggests a function beyond activation of the lactate racemase and possibly linked with other undiscovered nickel-dependent enzymes. PMID:24710389

Bundle-sheath thylakoids from NADP-malic enzyme-type C4 plants require an exogenous electron donor for enzyme light activation.

PubMed

Lavergne, D; Droux, M; Jacquot, J P; Miginiac-Maslow, M; Champigny, M L; Gadal, P

1985-10-01

Light activation of either NADP-malate dehydrogenase (EC 1.1.1.82) or fructose-1,6-bisphosphate phosphatase (EC 3.1.3.11) was assayed in a reconstituted chloroplastic, system comprising the isolated proteins of the ferredoxin-thioredoxin light-activation system and thylakoids from either mesophyll or bundle-sheath tissues of different C4 plants. While C4-plant thylakoids functionned almost equally well with C3-or C4-plant proteins, the photosyntem-II-deficient bundle-sheath thylakoids from the NADP-malic enzyme type, were unable to perform enzyme photoactivation unless supplemented with an electron donor to photosystem I. Bundle-sheath thylakoids isolated from plants showing no photosystem-II deficiency did not require such an addition. The results are discussed with respect to a possible requirement for a physiological reductant of ferredoxin for enzyme light activation in bundle-sheath, tissues.

Making the Rate: Enzyme Dynamics

ERIC Educational Resources Information Center

Ragsdale, Frances R.

2004-01-01

An enzyme exercise to address the problem of students inability to visualize chemical reaction at the molecular level is described. This exercise is designed as a dry lab exercise but can be modified into a classroom activity then can be augmented by a wet lab procedure, thereby providing students with a practical exposure to enzyme function.

Malabsorption syndrome observed in the common octopus Octopus vulgaris infected with Aggregata octopiana (Protista: Apicomplexa).

PubMed

Gestal, C; Páez de la Cadena, M; Pascual, S

2002-08-15

Octopus vulgaris infected with Aggregata octopiana were collected from an open-water culture system in the Ría of Aldán (NW Spain). Digestive tract infection values were determined with the use of a Neubauer chamber by counting the number of A. octopiana sporocysts. After determining enzyme activity values by the colorimetric Api-Zym system Biomerieux, one representative enzyme of glycosidases, peptid hydrolases and phosphoric hydrolases showing high activity was spectrophotometrically analysed. The enzymes were maltase and leucine-aminopeptidase (LAP) involved in the absorption process, and acid phosphatase, a lysosomic enzyme, respectively. Enzymatic activity of maltase and LAP decreased significantly, with increased sporocyst counts. However, acid phosphatase activity increased with severity of infection, indicating the presence of degradative enzymes from phagocytic cells in the infected area. A detrimental effect on gastrointestinal function may result from a decrease or malfunction of absorption enzymes. The results suggest a malabsorption syndrome resulting from parasitic infection.

Structure-guided mutational analysis reveals the functional requirements for product specificity of DOT1 enzymes.

PubMed

Dindar, Gülcin; Anger, Andreas M; Mehlhorn, Christine; Hake, Sandra B; Janzen, Christian J

2014-11-12

DOT1 enzymes are conserved methyltransferases that catalyse the methylation of lysine 79 on histone H3 (H3K79). Most eukaryotes contain one DOT1 enzyme, whereas African trypanosomes have two homologues, DOT1A and DOT1B, with different enzymatic activities. DOT1A mediates mono- and dimethylation of H3K76, the homologue of H3K79 in other organisms, whereas DOT1B additionally catalyses H3K76 trimethylation. However, it is unclear how these different enzymatic activities are achieved. Here we employ a trypanosomal nucleosome reconstitution system and structure-guided homology modelling to identify critical residues within and outside the catalytic centre that modulate product specificity. Exchange of these residues transfers the product specificity from one enzyme to the other, and reveals the existence of distinct regulatory domains adjacent to the catalytic centre. Our study provides the first evidence that a few crucial residues in DOT1 enzymes are sufficient to catalyse methyl-state-specific reactions. These results might also have far-reaching consequences for the functional understanding of homologous enzymes in higher eukaryotes.

Adjustment of Conformational Flexibility is a Key Event in the Thermal Adaptation of Proteins

NASA Astrophysics Data System (ADS)

Zavodszky, Peter; Kardos, Jozsef; Svingor, Adam; Petsko, Gregory A.

1998-06-01

3-Isopropylmalate dehydrogenase (IPMDH, E.C. 1.1.1.85) from the thermophilic bacterium Thermus thermophilus HB8 is homologous to IPMDH from the mesophilic Escherichia coli, but has an approximately 17 degrees C higher melting temperature. Its temperature optimum is 22-25 degrees C higher than that of the E. coli enzyme; however, it is hardly active at room temperature. The increased conformational rigidity required to stabilize the thermophilic enzyme against heat denaturation might explain its different temperature-activity profile. Hydrogen/deuterium exchange studies were performed on this thermophilic-mesophilic enzyme pair to compare their conformational flexibilities. It was found that Th. thermophilus IPMDH is significantly more rigid at room temperature than E. coli IPMDH, whereas the enzymes have nearly identical flexibilities under their respective optimal working conditions, suggesting that evolutionary adaptation tends to maintain a ``corresponding state'' regarding conformational flexibility. These observations confirm that conformational fluctuations necessary for catalytic function are restricted at room temperature in the thermophilic enzyme, suggesting a close relationship between conformational flexibility and enzyme function.

Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm

NASA Astrophysics Data System (ADS)

Fu, Jinglin; Yang, Yuhe Renee; Johnson-Buck, Alexander; Liu, Minghui; Liu, Yan; Walter, Nils G.; Woodbury, Neal W.; Yan, Hao

2014-07-01

Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein-DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.

The 2-oxoacid dehydrogenase multi-enzyme complex of the archaeon Thermoplasma acidophilum - recombinant expression, assembly and characterization.

PubMed

Heath, Caroline; Posner, Mareike G; Aass, Hans C; Upadhyay, Abhishek; Scott, David J; Hough, David W; Danson, Michael J

2007-10-01

The aerobic archaea possess four closely spaced, adjacent genes that encode proteins showing significant sequence identities with the bacterial and eukaryal components comprising the 2-oxoacid dehydrogenase multi-enzyme complexes. However, catalytic activities of such complexes have never been detected in the archaea, although 2-oxoacid ferredoxin oxidoreductases that catalyze the equivalent metabolic reactions are present. In the current paper, we clone and express the four genes from the thermophilic archaeon, Thermoplasma acidophilum, and demonstrate that the recombinant enzymes are active and assemble into a large (M(r) = 5 x 10(6)) multi-enzyme complex. The post-translational incorporation of lipoic acid into the transacylase component of the complex is demonstrated, as is the assembly of this enzyme into a 24-mer core to which the other components bind to give the functional multi-enzyme system. This assembled complex is shown to catalyze the oxidative decarboxylation of branched-chain 2-oxoacids and pyruvate to their corresponding acyl-CoA derivatives. Our data constitute the first proof that the archaea possess a functional 2-oxoacid dehydrogenase complex.

Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm.

PubMed

Fu, Jinglin; Yang, Yuhe Renee; Johnson-Buck, Alexander; Liu, Minghui; Liu, Yan; Walter, Nils G; Woodbury, Neal W; Yan, Hao

2014-07-01

Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein-DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.

Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S -Transferases

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

Stoddard, Ethan G.; Killinger, Bryan J.; Nair, Reji N.

Glutathione S-transferases (GSTs) comprise a highly diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione to various endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured by colorimetric assays, measurement of the individual contribution from specific isoforms and their contribution to the detoxification of xenobiotics in complex biological samples has not been possible. For this reason, we have developed two activity-based probes that characterize active glutathione transferases in mammalian tissues. The GST active site is comprised of a glutathione binding “G site” and a distinct substrate binding “Hmore » site”. Therefore, we developed (1) a glutathione-based photoaffinity probe (GSH-ABP) to target the “G site”, and (2) a probe designed to mimic a substrate molecule and show “H site” activity (GST-ABP). The GSH-ABP features a photoreactive moiety for UV-induced covalent binding to GSTs and glutathione-binding enzymes. The GST-ABP is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and “G” and “H” site specificity was carried out using a series of competitors in liver homogenates. Herein, we present robust tools for the novel characterization of enzyme- and active site-specific GST activity in mammalian model systems.« less

Expanding the enzyme universe: accessing non-natural reactions by mechanism-guided directed evolution.

PubMed

Renata, Hans; Wang, Z Jane; Arnold, Frances H

2015-03-09

High selectivity and exquisite control over the outcome of reactions entice chemists to use biocatalysts in organic synthesis. However, many useful reactions are not accessible because they are not in nature's known repertoire. In this Review, we outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progression has been recapitulated in the laboratory starting from extant enzymes. We then examine non-native enzyme activities that have been exploited for chemical synthesis, with an emphasis on reactions that do not have natural counterparts. Non-natural activities can be improved by directed evolution, thus mimicking the process used by nature to create new catalysts. Finally, we describe the discovery of non-native catalytic functions that may provide future opportunities for the expansion of the enzyme universe. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enzymatic Conversion of CO2 to Bicarbonate in Functionalized Mesoporous Silica

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

Yu, Yuehua; Chen, Baowei; Qi, Wen N.

2012-05-01

We report here that carbonic anhydrase (CA), the fastest enzyme that can covert carbon dioxide to bicarbonate, can be spontaneously entrapped in functionalized mesoporous silica (FMS) with super-high loading density (up to 0.5 mg of protein/mg of FMS) due to the dominant electrostatic interaction. The binding of CA to HOOC-FMS can result in the protein’s conformational change comparing to the enzyme free in solution, but can be overcome with increased protein loading density. The higher the protein loading density, the less conformational change, hence the higher enzymatic activity and the higher enzyme immobilization efficiency. The electrostatically bound CA can bemore » released by changing pH. The released enzyme still displayed the native conformational structure and the same high enzymatic activity as that prior to the enzyme entrapment. This work opens up a new approach converting carbon dioxide to biocarbonate in a biomimetic nanoconfiguration that can be integrated with the other part of biosynthesis process for the assimilation of carbon dioxide.« less

Optical observation of correlated motions in dihydrofolate reductase

NASA Astrophysics Data System (ADS)

Xu, Mengyang; Niessen, Katherine; Pace, James; Cody, Vivian; Markelz, Andrea

2015-03-01

Enzyme function relies on its structural flexibility to make conformational changes for substrate binding and product release. An example of a metabolic enzyme where such structural changes are vital is dihydrofolate reductase (DHFR). DHFR is essential in both prokaryotes and eukaryotes for the nucleotide biosynthesis by catalyzing the reduction of dihydrofolate to tetrahydrofolate. NMR dynamical measurements found large amplitude fast dynamics that could indicate rigid-body, twisting-hinge motion for ecDHFR that may mediate flux. The role of such long-range correlated motions in function was suggested by the observed sharp decrease in enzyme activity for the single point mutation G121V, which is remote from active sites. This decrease in activity may be caused by the mutation interfering with the long-range intramolecular vibrations necessary for rapid access to functional configurations. We use our new technique of crystal anisotropy terahertz microscopy (CATM), to observe correlated motions in ecDHFR crystals with the bonding of NADPH and methotrexate. We compare the measured intramolecular vibrational spectrum with calculations using normal mode analysis.

Evolutionary analysis of the TPP-dependent enzyme family.

PubMed

Costelloe, Seán J; Ward, John M; Dalby, Paul A

2008-01-01

The evolutionary relationships of the thiamine pyrophosphate (TPP)-dependent family of enzymes was investigated by generation of a neighbor joining phylogenetic tree using sequences from the conserved pyrophosphate (PP) and pyrimidine (Pyr) binding domains of 17 TPP-dependent enzymes. This represents the most comprehensive analysis of TPP-dependent enzyme evolution to date. The phylogeny was shown to be robust by comparison with maximum likelihood trees generated for each individual enzyme and also broadly confirms the evolutionary history proposed recently from structural comparisons alone (Duggleby 2006). The phylogeny is most parsimonious with the TPP enzymes having arisen from a homotetramer which subsequently diverged into an alpha(2)beta(2) heterotetramer. The relationship between the PP- and Pyr-domains and the recruitment of additional protein domains was examined using the transketolase C-terminal (TKC)-domain as an example. This domain has been recruited by several members of the family and yet forms no part of the active site and has unknown function. Removal of the TKC-domain was found to increase activity toward beta-hydroxypyruvate and glycolaldehyde. Further truncations of the Pyr-domain yielded several variants with retained activity. This suggests that the influence of TKC-domain recruitment on the evolution of the mechanism and specificity of transketolase (TK) has been minor, and that the smallest functioning unit of TK comprises the PP- and Pyr-domains, whose evolutionary histories extend to all TPP-dependent enzymes.

Redox regulation and reaction mechanism of human cystathionine-beta-synthase: a PLP-dependent hemesensor protein.

PubMed

Banerjee, Ruma; Zou, Cheng-Gang

2005-01-01

Cystathionine beta-synthase in mammals lies at a pivotal crossroad in methionine metabolism directing flux toward cysteine synthesis and catabolism. The enzyme exhibits a modular organization and complex regulation. It catalyzes the beta-replacement of the hydroxyl group of serine with the thiolate of homocysteine and is unique in being the only known pyridoxal phosphate-dependent enzyme that also contains heme b as a cofactor. The heme functions as a sensor and modulates enzyme activity in response to redox change and to CO binding. Mutations in this enzyme are the single most common cause of hereditary hyperhomocysteinemia. Elucidation of the crystal structure of a truncated and highly active form of the human enzyme containing the heme- and pyridoxal phosphate binding domains has afforded a structural perspective on mechanistic and mutation analysis studies. The C-terminal regulatory domain containing two CBS motifs exerts intrasteric regulation and binds the allosteric activator, S-adenosylmethionine. Studies with mammalian cells in culture as well as with animal models have unraveled multiple layers of regulation of cystathionine beta-synthase in response to redox perturbations and reveal the important role of this enzyme in glutathione-dependent redox homestasis. This review discusses the recent advances in our understanding of the structure, mechanism, and regulation of cystathionine beta-synthase from the perspective of its physiological function, focusing on the clinically relevant human enzyme.

Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination

PubMed Central

Rejón, Juan D.; Zienkiewicz, Agnieszka; Rodríguez-García, María Isabel; Castro, Antonio J.

2012-01-01

Background and Aims A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized. Methods The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods. Key Results Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles. Conclusions In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma. PMID:22922586

Changes in the structure and function of soil ecosystems in soils contaminated with heavy metals

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

Kuperman, R.; Parmelee, R.; Carreiro, M.

1995-09-01

The structure and function of soil communities in an area with a wide range of concentrations of heavy metals was studied in portions of the U.S. Army`s Aberdeen Proving Ground, Maryland. The study included survey of soil macro- and microinvertebrate communities, soil microorganisms, enzyme activities and the rates of nutrient dynamics in soil. Soil macroinvertebrate communities showed significant reductions in the adundance of several taxonomic and functional groups in contaminated areas. The total numbers of nematodes and numbers of fungivore, bacterivore and omnivore-predator nematodes were lower in the more contaminated areas. The numbers of active bacteria and fungi were lowermore » in areas of soil contamination. Significant reduction in the activities of all enzymes closely paralleled the increase in heavy metal concentrations. Ten-to-fifty fold reductions in enzyme activities were observed as heavy metal concentrations increased. These results suggest that soil contamination with heavy metals may have detrimental effects on soil biota and the rates of organic matter degradation and subsequent release of nutrients to aboveground communities in the area.« less

Changes in the structure and function of soil ecosystems in soils contaminated with heavy metals

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

Kuperman, R.; Parmelee, R.; Carreiro, M.

1995-06-01

The structure and function of soil communities in an area with a wide range of concentrations of heavy metals was studied in portions of the U.S. Army`s Aberdeen Proving Ground, Maryland. The study included survey of soil macro- and microinvertebrate communities, soil microorganisms, enzyme activities and the rates of nutrient dynamics in soil. Soil macroinvertebrate communities showed significant reductions in the abundance of several taxonomic and functional groups in contaminated areas. The total numbers of nematodes and numbers of fungivore, bacterivore and omnivore-predator nematodes were lower in the more contaminated areas. The numbers of active bacteria and fungi were lowermore » in areas of soil contamination. Significant reduction in the activities of all enzymes closely paralleled the increase in heavy metal concentrations. Ten-to-fifty fold reductions in enzyme activities were observed as heavy metal concentrations increased. These results suggest that soil contamination with heavy metals may have detrimental effects on soil biota and the rates of organic matter degradation and subsequent release of nutrients to aboveground communities in the area.« less

Functional enzyme-based modeling approach for dynamic simulation of denitrification process in hyporheic zone sediments: Genetically structured microbial community model

NASA Astrophysics Data System (ADS)

Song, H. S.; Li, M.; Qian, W.; Song, X.; Chen, X.; Scheibe, T. D.; Fredrickson, J.; Zachara, J. M.; Liu, C.

2016-12-01

Modeling environmental microbial communities at individual organism level is currently intractable due to overwhelming structural complexity. Functional guild-based approaches alleviate this problem by lumping microorganisms into fewer groups based on their functional similarities. This reduction may become ineffective, however, when individual species perform multiple functions as environmental conditions vary. In contrast, the functional enzyme-based modeling approach we present here describes microbial community dynamics based on identified functional enzymes (rather than individual species or their groups). Previous studies in the literature along this line used biomass or functional genes as surrogate measures of enzymes due to the lack of analytical methods for quantifying enzymes in environmental samples. Leveraging our recent development of a signature peptide-based technique enabling sensitive quantification of functional enzymes in environmental samples, we developed a genetically structured microbial community model (GSMCM) to incorporate enzyme concentrations and various other omics measurements (if available) as key modeling input. We formulated the GSMCM based on the cybernetic metabolic modeling framework to rationally account for cellular regulation without relying on empirical inhibition kinetics. In the case study of modeling denitrification process in Columbia River hyporheic zone sediments collected from the Hanford Reach, our GSMCM provided a quantitative fit to complex experimental data in denitrification, including the delayed response of enzyme activation to the change in substrate concentration. Our future goal is to extend the modeling scope to the prediction of carbon and nitrogen cycles and contaminant fate. Integration of a simpler version of the GSMCM with PFLOTRAN for multi-scale field simulations is in progress.

Muscle enzyme release does not predict muscle function impairment after triathlon.

PubMed

Margaritis, I; Tessier, F; Verdera, F; Bermon, S; Marconnet, P

1999-06-01

We sought to determine the effects of a long distance triathlon (4 km swim, 120 km bike-ride, and 30 km run) on the four-day kinetics of the biochemical markers of muscle damage, and whether they were quantitatively linked with muscle function impairment and soreness. Data were collected from 2 days before until 4 days after the completion of the race. Twelve triathletes performed the triathlon and five did not. Maximal voluntary contraction (MVC), muscle soreness (DOMS) and total serum CK, CK-MB, LDH, AST and ALT activities were assessed. Significant changes after triathlon completion were found for all muscle damage indirect markers over time (p < 0.0001). MVC of the knee extensor and flexor muscles decreased over time (p < 0.05). There is disparity in the time point at which peak values where reached for DOMS, MVC and enzyme leakage. There is no correlation between serum enzyme leakage, DOMS and MVC impairment which occur after triathlon. Long distance triathlon race caused muscle damage, but extent, as well as muscle recovery cannot be evaluated by the magnitude of changes in serum enzyme activities. Muscle enzyme release cannot be used to predict the magnitude of the muscle function impairment caused by muscle damage.

Armored Enzyme-Nanohybrids and Their Catalytic Function Under Challenging Conditions.

PubMed

Zore, Omkar V; Kasi, Rajeswari M; Kumar, Challa V

2017-01-01

Synthesis and characterization of highly stable and functional bienzyme-polymer triads assembled on layered graphene oxide (GO) are described here. Glucose oxidase (GOx) and horseradish peroxidase (HRP) were used as model enzymes and polyacrylic acid (PAA) as model polymer to armor the enzymes. PAA-armored GOx and HRP covalent conjugates were further protected from denaturation by adsorption onto GO nanosheets. Structure and morphology of this enzyme-polymer-nanosheet hybrid biocatalyst (GOx-HRP-PAA/GO) were confirmed by agarose gel electrophoresis, zeta potential, circular dichroism, and transmission electron microscopy. The armored biocatalysts retained full enzymatic activities under challenging conditions of pH (2.5-7.4), warm temperatures (65°C), and presence of chemical denaturants, 4mM sodium dodecyl sulfate, while GOx/HRP physical mixtures without the armor had very little activity under the same conditions. Therefore, this novel combination of two orthogonal approaches, enzyme conjugation with PAA and subsequent physical adsorption onto GO nanosheets, resulted in super stable hybrid biocatalysts that function under harsh conditions. Therefore, this general and powerful approach may be used to design environmentally friendly, green, biocompatible, and biodegradable biocatalysts for energy production in biofuel cell or biobattery applications. © 2017 Elsevier Inc. All rights reserved.

Introduction of unnatural amino acids into chalcone isomerase.

PubMed

Bednar, R A; McCaffrey, C; Shan, K

1991-01-01

The active site cysteine residue of chalcone isomerase was rapidly and selectively modified under denaturing conditions with a variety of electrophilic reagents. These denatured and modified enzyme were renatured to produce enzyme derivatives containing a series of unnatural amino acids in the active site. Addition of methyl, ethyl, butyl, heptyl, and benzyl groups to the cysteine sulfur does not abolish catalytic activity, although the activity decreases as the steric bulk of the amino acid side-chain increases. Modification of the cysteine to introduce a charged homoglutamate or a neutral homoglutamine analogue results in retention of 22% of the catalytic activity. Addition of a methylthio group (SMe) to the cysteine residue of native chalcone isomerase preserves 85% of the catalytic activity measured with 2',4',4-trihydroxychalcone, 2',4',6',4-tetrahydroxychalcone, or 2'-hydroxy-4-methoxychalcone as substrates. The competitive inhibition constant for 4',4-dihydroxychalcone, the substrate inhibition constant for 2',4',4-trihydroxychalcone, and other steady-state kinetic parameters for the methanethiolated enzyme are very similar to those of the native enzyme. The strong binding of 4',4-dihydroxychalcone to the methanethiolated enzyme shows that there is no steric repulsion between this modified amino acid residue and the substrate analogue. This structure-activity study clearly demonstrates that the active site cysteine residue does not function as an acid-base or nucleophilic group in producing the catalysis or substrate inhibition observed with chalcone isomerase. The method presented in this paper allows for the rapid introduction of a series of unnatural amino acids into the active site as a means of probing the structure-function relationship.

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.

Enzymes and Enzyme Activity Encoded by Nonenveloped Viruses.

PubMed

Azad, Kimi; Banerjee, Manidipa; Johnson, John E

2017-09-29

Viruses are obligate intracellular parasites that rely on host cell machineries for their replication and survival. Although viruses tend to make optimal use of the host cell protein repertoire, they need to encode essential enzymatic or effector functions that may not be available or accessible in the host cellular milieu. The enzymes encoded by nonenveloped viruses-a group of viruses that lack any lipid coating or envelope-play vital roles in all the stages of the viral life cycle. This review summarizes the structural, biochemical, and mechanistic information available for several classes of enzymes and autocatalytic activity encoded by nonenveloped viruses. Advances in research and development of antiviral inhibitors targeting specific viral enzymes are also highlighted.

Inhibitors of enzymes catalyzing modifications to histone lysine residues: structure, function and activity.

PubMed

Lillico, Ryan; Stesco, Nicholas; Khorshid Amhad, Tina; Cortes, Claudia; Namaka, Mike P; Lakowski, Ted M

2016-05-01

Gene expression is partly controlled by epigenetic mechanisms including histone-modifying enzymes. Some diseases are caused by changes in gene expression that can be mitigated by inhibiting histone-modifying enzymes. This review covers the enzyme inhibitors targeting histone lysine modifications. We summarize the enzymatic mechanisms of histone lysine acetylation, deacetylation, methylation and demethylation and discuss the biochemical roles of these modifications in gene expression and in disease. We discuss inhibitors of lysine acetylation, deacetylation, methylation and demethylation defining their structure-activity relationships and their potential mechanisms. We show that there are potentially indiscriminant off-target effects on gene expression even with the use of selective epigenetic enzyme inhibitors.

Maintaining Genetic Integrity Under Extreme Conditions: Novel DNA Damage Repair Biology in the Archaea

DTIC Science & Technology

2013-11-23

Genetic analysis of Nre DNA repair function A4 Conclusions B. Widening the net in the search for new DNA-directed enzyme activities C. New tools for H...Figure 1) were hypothesised to be novel DNA repair enzymes . The stated aims of the proposal were to use a combination of genetic, biochemical and...in E.coli Almost all proteins that interact directly with PCNA are enzymes possessing DNA-directed activities such as nucleases, glycosylases

Network Analysis of Enzyme Activities and Metabolite Levels and Their Relationship to Biomass in a Large Panel of Arabidopsis Accessions[C][W][OA

PubMed Central

Sulpice, Ronan; Trenkamp, Sandra; Steinfath, Matthias; Usadel, Bjorn; Gibon, Yves; Witucka-Wall, Hanna; Pyl, Eva-Theresa; Tschoep, Hendrik; Steinhauser, Marie Caroline; Guenther, Manuela; Hoehne, Melanie; Rohwer, Johann M.; Altmann, Thomas; Fernie, Alisdair R.; Stitt, Mark

2010-01-01

Natural genetic diversity provides a powerful resource to investigate how networks respond to multiple simultaneous changes. In this work, we profile maximum catalytic activities of 37 enzymes from central metabolism and generate a matrix to investigate species-wide connectivity between metabolites, enzymes, and biomass. Most enzyme activities change in a highly coordinated manner, especially those in the Calvin-Benson cycle. Metabolites show coordinated changes in defined sectors of metabolism. Little connectivity was observed between maximum enzyme activities and metabolites, even after applying multivariate analysis methods. Measurements of posttranscriptional regulation will be required to relate these two functional levels. Individual enzyme activities correlate only weakly with biomass. However, when they are used to estimate protein abundances, and the latter are summed and expressed as a fraction of total protein, a significant positive correlation to biomass is observed. The correlation is additive to that obtained between starch and biomass. Thus, biomass is predicted by two independent integrative metabolic biomarkers: preferential investment in photosynthetic machinery and optimization of carbon use. PMID:20699391

High sensibility to reactivation by acidic lipids of the recombinant human plasma membrane Ca2+-ATPase isoform 4xb purified from Saccharomyces cerevisiae.

PubMed

Cura, Carolina I; Corradi, Gerardo R; Rinaldi, Débora E; Adamo, Hugo P

2008-12-01

The human plasma membrane Ca2+ pump (isoform 4xb) was expressed in Saccharomyces cerevisiae and purified by calmodulin-affinity chromatography. Under optimal conditions the recombinant enzyme (yPMCA) hydrolyzed ATP in a Ca2+ dependent manner at a rate of 15 micromol/mg/min. The properties of yPMCA were compared to those of the PMCA purified from human red cells (ePMCA). The mobility of yPMCA in SDS-PAGE was the expected for the hPMCA4xb protein but slightly lower than that of ePMCA. Both enzymes achieved maximal activity when supplemented with acidic phospholipids. However, while ePMCA in mixed micelles of phosphatidylcholine-detergent had 30% of its maximal activity, the yPMCA enzyme was nearly inactive. Increasing the phosphatidylcholine content of the micelles did not increase the activity of yPMCA but the activity in the presence of phosphatidylcholine improved by partially removing the detergent. The reactivation of the detergent solubilized yPMCA required specifically acidic lipids and, as judged by the increase in the level of phosphoenzyme, it involved the increase in the amount of active enzyme. These results indicate that the function of yPMCA is highly sensitive to delipidation and the restitution of acidic lipids is needed for a functional enzyme.

Functional Characterization of the Vitamin K2 Biosynthetic Enzyme UBIAD1

PubMed Central

Hirota, Yoshihisa; Nakagawa, Kimie; Sawada, Natsumi; Okuda, Naoko; Suhara, Yoshitomo; Uchino, Yuri; Kimoto, Takashi; Funahashi, Nobuaki; Kamao, Maya; Tsugawa, Naoko; Okano, Toshio

2015-01-01

UbiA prenyltransferase domain-containing protein 1 (UBIAD1) plays a significant role in vitamin K2 (MK-4) synthesis. We investigated the enzymological properties of UBIAD1 using microsomal fractions from Sf9 cells expressing UBIAD1 by analysing MK-4 biosynthetic activity. With regard to UBIAD1 enzyme reaction conditions, highest MK-4 synthetic activity was demonstrated under basic conditions at a pH between 8.5 and 9.0, with a DTT ≥0.1 mM. In addition, we found that geranyl pyrophosphate and farnesyl pyrophosphate were also recognized as a side-chain source and served as a substrate for prenylation. Furthermore, lipophilic statins were found to directly inhibit the enzymatic activity of UBIAD1. We analysed the aminoacid sequences homologies across the menA and UbiA families to identify conserved structural features of UBIAD1 proteins and focused on four highly conserved domains. We prepared protein mutants deficient in the four conserved domains to evaluate enzyme activity. Because no enzyme activity was detected in the mutants deficient in the UBIAD1 conserved domains, these four domains were considered to play an essential role in enzymatic activity. We also measured enzyme activities using point mutants of the highly conserved aminoacids in these domains to elucidate their respective functions. We found that the conserved domain I is a substrate recognition site that undergoes a structural change after substrate binding. The conserved domain II is a redox domain site containing a CxxC motif. The conserved domain III is a hinge region important as a catalytic site for the UBIAD1 enzyme. The conserved domain IV is a binding site for Mg2+/isoprenyl side-chain. In this study, we provide a molecular mapping of the enzymological properties of UBIAD1. PMID:25874989

Effects of Recurring Droughts on Extracellular Enzyme Activity in Mountain Grassland

NASA Astrophysics Data System (ADS)

Fuchslueger, L.; Bahn, M.; Kienzl, S.; Hofhansl, F.; Schnecker, J.; Richter, A.

2015-12-01

Water availability is a key factor for biogeochemical processes and determines microbial activity and functioning, and thereby organic matter decomposition in soils by affecting the osmotic potential, soil pore connectivity, substrate diffusion and nutrient availability. Low water availability during drought periods therefore directly affects microbial activity. Recurring drought periods likely induce shifts in microbial structure that might be reflected in altered responses of microbial turnover of organic matter by extracellular enzymes. To study this we measured a set of potential extracellular enzyme activity rates (cellobiohydrolase CBH; leucine-amino-peptidase LAP; phosphatase PHOS; phenoloxidase POX), in grassland soils that were exposed to extreme experimental droughts during the growing seasons of up to five subsequent years. During the first drought period after eight weeks of rain exclusion all measured potential enzyme activities were significantly decreased. In parallel, soil extractable organic carbon and nitrogen concentrations increased and microbial community structure, determined by phospholipid fatty acid analysis, changed. In soils that were exposed to two and three drought periods only PHOS decreased. After four years of drought again CBH, PHOS and POX decreased, while LAP was unaffected; after five years of drought PHOS and POX decreased and CBH and LAP remained stable. Thus, our results suggest that recurring extreme drought events can cause different responses of extracellular enzyme activities and that the responses change over time. We will discuss whether and to what degree these changes were related to shifts in microbial community composition. However, independent of whether a solitary or a recurrent drought was imposed, in cases when enzyme activity rates were altered during drought, they quickly recovered after rewetting. Overall, our data suggest that microbial functioning in mountain grassland is sensitive to drought, but highly resilient even after five years of drought.

pH-sensitive pHluorins as a molecular sensor for in situ monitoring of enzyme-catalyzed prodrug activation.

PubMed

Liu, Hui; Cao, Xiaodan; Wang, Ping; Ma, Xingyuan

2017-07-01

This work examines the feasibility of using a pH-sensitive fluorescent protein as a molecular reporter for enzyme-catalyzed prodrug activation reaction. Specifically, a ratiometric pHluorins was examined for detection of the activity of horseradish peroxidase (HRP) for the activation of indole-3-acetic acid. The pHluorins and HRP were conjugated chemically, forming a biocatalyst with a self-reporting function. Results showed that the characteristic fluorescence intensity ratio of the conjugate shifted from 1.47 to 1.40 corresponding to the progress of the prodrug activation reaction. The effectiveness of applying the conjugate for inhibition of the growth of Bcap-37 cells was also demonstrated simultaneously with reaction monitoring. The results reveal a very promising approach to realizing in situ monitoring of enzyme activities based on pH shifting for enzyme-based prodrug therapy applications. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

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.

Structural and Functional Consequences of Circular Permutation on the Active Site of Old Yellow Enzyme

DOE PAGES

Daugherty, Ashley B.; Horton, John R.; Cheng, Xiaodong; ...

2014-12-09

Circular permutation of the NADPH-dependent oxidoreductase Old Yellow Enzyme from Saccharomyces pastorianus (OYE1) can significantly enhance the enzyme’s catalytic performance. Termini relocation into four regions of the protein (sectors I–IV) near the active site has proven effective in altering enzyme function. To better understand the structural consequences and rationalize the observed functional gains in these OYE1 variants, we selected representatives from sectors I–III for further characterization by biophysical methods and X-ray crystallography. These investigations not only show trends in enzyme stability and quaternary structure as a function of termini location but also provide a possible explanation for the catalytic gainsmore » in our top-performing OYE variant (new N-terminus at residue 303; sector III). Crystallographic analysis indicates that termini relocation into sector III affects the loop β6 region (amino acid positions: 290–310) of OYE1, which forms a lid over the active site. Peptide backbone cleavage greatly enhances local flexibility, effectively converting the loop into a tether and consequently increasing the environmental exposure of the active site. Interestingly, such an active site remodeling does not negatively impact the enzyme’s activity and stereoselectivity; neither does it perturb the conformation of other key active site residues with the exception of Y375. These observations were confirmed in truncation experiments, deleting all residues of the loop β6 region in our OYE variant. Intrigued by the finding that circular permutation leaves most of the key catalytic residues unchanged, we also tested OYE permutants for possible additive or synergistic effects of amino acid substitutions. Distinct functional changes in these OYE variants were detected upon mutations at W116, known in native OYE1 to cause inversion of diastereoselectivity for (S)-carvone reduction. In conclusion, our findings demonstrate the contribution of loop β6 toward determining the stereoselectivity of OYE1, an important insight for future OYE engineering efforts.« less

Microbial Enzyme Activities of Wetland Soils as Indicators of Nutrient Condition: A Test in Wetlands of Gulf of Mexico Coastal Watershed

EPA Science Inventory

Microbial enzyme activities measured from wetland soils are being tested as indicators of wetland nutrient function and human disturbance. This is part of an assessment of condition of wetlands being conducted by the U.S. EPA Gulf Ecology Division in coastal watersheds along the...

Solvent effects on enzymes - Implications for extraterrestrial life.

NASA Technical Reports Server (NTRS)

Heinrich, M. R.

1972-01-01

Review of several studies on the alterations taking place in the structure, catalytic activity, specificity, and stability of an enzyme when some or all of the water in the medium is replaced by another solvent. These studies show the utility of solvents as a tool for probing enzyme function. They also suggest that solvents other than water should be investigated as media for controlling and directing enzyme reactions.

Structural and Functional Mechanisms of Adaptations of WrbA in Extremophilic Organisms

DTIC Science & Technology

2010-05-11

organisms adapt to high temperature. A model of the thermophilic enzyme was constructed based on the crystal Structure of the mesophilie counterpart to...binding for the thermophilic enzyme was independent of ligand concentration. Comparison of enzyme activities between the two proteins with a variety of...extremophilic organisms adapt to high temperature. A model of the thermophilic enzyme was constructed based on the crystal structure of the mesophilic

Inhibition of Monometalated Methionine Aminopeptidase: Inhibitor Discovery and Crystallographic Analysis†

PubMed Central

Huang, Min; Xie, Sheng-Xue; Ma, Ze-Qiang; Huang, Qing-Qing; Nan, Fa-Jun; Ye, Qi-Zhuang

2008-01-01

Two divalent metal ions are commonly seen in the active site cavity of methionine aminopeptidase, and at least one of the metal ions is directly involved in catalysis. Although ample structural and functional information is available for dimetalated enzyme, methionine aminopeptidase likely functions as a monometalated enzyme under physiological conditions. Information on structure, as well as catalysis and inhibition, of the monometalated enzyme is lacking. By improving conditions of high throughput screening, we identified a unique inhibitor with specificity toward the monometalated enzyme. Kinetic characterization indicates a mutual exclusivity in binding between the inhibitor and the second metal ion at the active site. This is confirmed by X-ray structure, and this inhibitor coordinates with the first metal ion and occupies the space normally occupied by the second metal ion. Kinetic and structural analyses of the inhibition by this and other inhibitors provide insight in designing effective inhibitors of methionine aminopeptidase. PMID:17948983

Computational design of enzyme-ligand binding using a combined energy function and deterministic sequence optimization algorithm.

PubMed

Tian, Ye; Huang, Xiaoqiang; Zhu, Yushan

2015-08-01

Enzyme amino-acid sequences at ligand-binding interfaces are evolutionarily optimized for reactions, and the natural conformation of an enzyme-ligand complex must have a low free energy relative to alternative conformations in native-like or non-native sequences. Based on this assumption, a combined energy function was developed for enzyme design and then evaluated by recapitulating native enzyme sequences at ligand-binding interfaces for 10 enzyme-ligand complexes. In this energy function, the electrostatic interaction between polar or charged atoms at buried interfaces is described by an explicitly orientation-dependent hydrogen-bonding potential and a pairwise-decomposable generalized Born model based on the general side chain in the protein design framework. The energy function is augmented with a pairwise surface-area based hydrophobic contribution for nonpolar atom burial. Using this function, on average, 78% of the amino acids at ligand-binding sites were predicted correctly in the minimum-energy sequences, whereas 84% were predicted correctly in the most-similar sequences, which were selected from the top 20 sequences for each enzyme-ligand complex. Hydrogen bonds at the enzyme-ligand binding interfaces in the 10 complexes were usually recovered with the correct geometries. The binding energies calculated using the combined energy function helped to discriminate the active sequences from a pool of alternative sequences that were generated by repeatedly solving a series of mixed-integer linear programming problems for sequence selection with increasing integer cuts.

Transmutation of human glutathione transferase A2-2 with peroxidase activity into an efficient steroid isomerase.

PubMed

Pettersson, Par L; Johansson, Ann-Sofie; Mannervik, Bengt

2002-08-16

A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.

Reconciling Apparent Variability in Effects of Biochar Amendment on Soil Enzyme Activities by Assay Optimization

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

Bailey, Vanessa L.; Fansler, Sarah J.; Smith, Jeffery L.

2011-02-01

Applying biochar to soils as an ameliorative substance and mechanism for C sequestration has received a great deal of interest in light of the sustained fertility observed in the Terra Preta soils of Brazil. The effects of synthetic biochars on biochemical processes needs to be better understood in order to determine if this is a reasonable practice in managed systems. The biochar studied was formed from the fast-pyrolysis of a switchgrass feedstock. Four soil enzymes were studied: β-glucosidase, β-N-acetylglucosaminidase, lipase, and leucine aminopeptidase. Both colorimetric and fluorescent assays were used for β-glucosidase and β-N-acetylglucosaminidase. Seven days after biochar was addedmore » to microcosms of a Palouse silt loam, the fluorescence-based assays indicated increased activities of the four enzymes, compared to non-amended soil. To clarify the mechanisms of the observed effects,in the absence of soil, purified enzymes or substrates were briefly exposed to biochar and then assayed. Except for β-N-acetylglucosaminidase, the exposure of substrate to biochar reduced the apparent activity of the remaining three enzymes in vitro, suggesting that sorption reactions between the substrate and biochar either removed the substrate from the assays or impeded the enzyme binding. The activity of purified β-N-acetylglucosaminidase increased significantly following biochar exposure, suggesting a chemical stimulation of enzyme functioning. We conclude that biochar added to soil acts as a substrate that can stimulate the soil microbial biomass and its activity. Our in vitro study suggests that biochar is not biochemically inert. Biochar amendments are likely to have effects that are currently difficult to predict, and that could impact overall soil function.« less

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.

Magnetic Fe3O4@MCM-41 core-shell nanoparticles functionalized with thiol silane for efficient l-asparaginase immobilization.

PubMed

Ulu, Ahmet; Noma, Samir Abbas Ali; Koytepe, Suleyman; Ates, Burhan

2018-06-06

l-Asparaginase (l-ASNase) is a vital enzyme for medical treatment and food industry. Here, we assessed the use of Fe 3 O 4 @Mobil Composition of Matter No. 41 (MCM-41) magnetic nanoparticles as carrier matrix for l-ASNase immobilization. In addition, surface of Fe 3 O 4 @MCM-41 magnetic nanoparticles was functionalized with 3-mercaptopropyltrimethoxysilane (MPTMS) to enhance stability of l-ASNase. The chemical structure, thermal properties, magnetic profile and morphology of the thiol-functionalized Fe 3 O 4 @MCM-41 magnetic nanoparticles were characterized with Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), differential thermal analysis (DTA), differential scanning calorimetry (DSC), vibrating sample magnetometer (VSM), scanning electron microscope (SEM), energy dispersive X-ray (EDX) spectroscopy and zeta-potential measurement. l-ASNase was covalently immobilized onto the thiol-functionalized Fe 3 O 4 @MCM-41 magnetic nanoparticles. The properties of the immobilized enzyme, including optimum pH, temperature, kinetic parameters, thermal stability, reusability and storage stability were investigated and compared to free one. Immobilized enzyme was found to be stable over a wide range of pH and temperature range than free enzyme. The immobilized l-ASNase also showed higher thermal stability after 180 min incubation at 50 °C. The immobilized enzyme still retained 63% of its original activity after 16 times of reuse. The Km value for the immobilized enzyme was 1.15-fold lower than the free enzyme, which indicates increased affinity for the substrate. Additionally, the immobilized enzyme was active over 65% and 53% after 30 days of storage at 4 °C and room temperature (∼25 °C), respectively. Thereby, the results confirmed that thiol-functionalized Fe 3 O 4 @MCM-41 magnetic nanoparticles had high efficiency for l-ASNase immobilization and improved stability of L-ASNase.

A major cathepsin B protease from the liver fluke Fasciola hepatica has atypical active site features and a potential role in the digestive tract of newly excysted juvenile parasites

PubMed Central

Beckham, Simone A.; Piedrafita, David; Phillips, Carolyn I.; Samarawickrema, Nirma; Law, Ruby H.P.; Smooker, Peter M.; Quinsey, Noelene S.; Irving, James A.; Greenwood, Deanne; Verhelst, Steven H. L.; Bogyo, Matthew; Turk, Boris; Coetzer, Theresa H.; Wijeyewickrema, Lakshmi C.; Spithill, Terry W.; Pike, Robert N.

2012-01-01

The newly excysted juvenile (NEJ) stage of the Fasciola hepatica lifecycle occurs just prior to invasion into the wall of the gut of the host, rendering it an important target for drug development. The cathepsin B enzymes from NEJ flukes have recently been demonstrated to be crucial to invasion and migration by the parasite. Here we characterize one of the cathepsin B enzymes (recombinant FhcatB1) from NEJ flukes. FhcatB1 has biochemical properties distinct from mammalian cathepsin B enzymes, with an atypical preference for Ile over Leu or Arg residues at the P2 substrate position and an inability to act as an exopeptidase. FhcatB1 was active across a broad pH range (optimal activity at pH 5.5–7.0) and resistant to inhibition by cystatin family inhibitors from sheep and humans, suggesting that this enzyme would be able to function in extracellular environments in its mammalian hosts. It appears, however, that the FhcatB1 protease functions largely as a digestive enzyme in the gut of the parasite, due to the localization of a specific, fluorescently labeled inhibitor with an Ile at the P2 position. Molecular modelling and dynamics were used to predict the basis for the unusual substrate specificity: a P2 Ile residue positions the substrate optimally for interaction with catalytic residues of the enzyme, and the enzyme lacks an occluding loop His residue crucial for exopeptidase activity. The unique features of the enzyme, particularly with regard to its specificity and likely importance to a vital stage of the parasite’s life cycle, make it an excellent target for therapeutic inhibitors or vaccination. PMID:19401154

Large-scale filament formation inhibits the activity of CTP synthetase

PubMed Central

Barry, Rachael M; Bitbol, Anne-Florence; Lorestani, Alexander; Charles, Emeric J; Habrian, Chris H; Hansen, Jesse M; Li, Hsin-Jung; Baldwin, Enoch P; Wingreen, Ned S; Kollman, Justin M; Gitai, Zemer

2014-01-01

CTP Synthetase (CtpS) is a universally conserved and essential metabolic enzyme. While many enzymes form small oligomers, CtpS forms large-scale filamentous structures of unknown function in prokaryotes and eukaryotes. By simultaneously monitoring CtpS polymerization and enzymatic activity, we show that polymerization inhibits activity, and CtpS's product, CTP, induces assembly. To understand how assembly inhibits activity, we used electron microscopy to define the structure of CtpS polymers. This structure suggests that polymerization sterically hinders a conformational change necessary for CtpS activity. Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation. This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels. We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable. DOI: http://dx.doi.org/10.7554/eLife.03638.001 PMID:25030911

Mining for hemicellulases in the fungus-growing termite Pseudacanthotermes militaris using functional metagenomics.

PubMed

Bastien, Géraldine; Arnal, Grégory; Bozonnet, Sophie; Laguerre, Sandrine; Ferreira, Fernando; Fauré, Régis; Henrissat, Bernard; Lefèvre, Fabrice; Robe, Patrick; Bouchez, Olivier; Noirot, Céline; Dumon, Claire; O'Donohue, Michael

2013-05-14

The metagenomic analysis of gut microbiomes has emerged as a powerful strategy for the identification of biomass-degrading enzymes, which will be no doubt useful for the development of advanced biorefining processes. In the present study, we have performed a functional metagenomic analysis on comb and gut microbiomes associated with the fungus-growing termite, Pseudacanthotermes militaris. Using whole termite abdomens and fungal-comb material respectively, two fosmid-based metagenomic libraries were created and screened for the presence of xylan-degrading enzymes. This revealed 101 positive clones, corresponding to an extremely high global hit rate of 0.49%. Many clones displayed either β-d-xylosidase (EC 3.2.1.37) or α-l-arabinofuranosidase (EC 3.2.1.55) activity, while others displayed the ability to degrade AZCL-xylan or AZCL-β-(1,3)-β-(1,4)-glucan. Using secondary screening it was possible to pinpoint clones of interest that were used to prepare fosmid DNA. Sequencing of fosmid DNA generated 1.46 Mbp of sequence data, and bioinformatics analysis revealed 63 sequences encoding putative carbohydrate-active enzymes, with many of these forming parts of sequence clusters, probably having carbohydrate degradation and metabolic functions. Taxonomic assignment of the different sequences revealed that Firmicutes and Bacteroidetes were predominant phyla in the gut sample, while microbial diversity in the comb sample resembled that of typical soil samples. Cloning and expression in E. coli of six enzyme candidates identified in the libraries provided access to individual enzyme activities, which all proved to be coherent with the primary and secondary functional screens. This study shows that the gut microbiome of P. militaris possesses the potential to degrade biomass components, such as arabinoxylans and arabinans. Moreover, the data presented suggests that prokaryotic microorganisms present in the comb could also play a part in the degradation of biomass within the termite mound, although further investigation will be needed to clarify the complex synergies that might exist between the different microbiomes that constitute the termitosphere of fungus-growing termites. This study exemplifies the power of functional metagenomics for the discovery of biomass-active enzymes and has provided a collection of potentially interesting biocatalysts for further study.

Reconstruction of Cysteine Biosynthesis Using Engineered Cysteine-Free and Methionine-Free Enzymes

NASA Technical Reports Server (NTRS)

Wang, Kendrick; Fujishima, Kosuke; Abe, Nozomi; Nakahigashi, Kenji; Endy, Drew; Rothschild, Lynn J.

2016-01-01

Ten of the proteinogenic amino acids can be generated abiotically while the remaining thirteen require biology for their synthesis. Paradoxically, the biosynthesis pathways observed in nature require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine. Here, we substituted alternate amino acids for cysteine and also methionine, which is biosynthesized from cysteine, in serine acetyl transferase (CysE) and O-acetylserine sulfhydrylase (CysM). CysE function was rescued by cysteine-and-methionine-free enzymes and CysM function was rescued by cysteine-free enzymes. Structural modeling suggests that methionine stabilizes CysM and is present in the active site of CysM. Cysteine is not conserved among CysE and CysM protein orthologs, suggesting that cysteine is not functionally important for its own synthesis. Engineering biosynthetic enzymes that lack the amino acids being synthesized provides insights into the evolution of amino acid biosynthesis and pathways for bioengineering.

The nature of chemical innovation: new enzymes by evolution.

PubMed

Arnold, Frances H

2015-11-01

I describe how we direct the evolution of non-natural enzyme activities, using chemical intuition and information on structure and mechanism to guide us to the most promising reaction/enzyme systems. With synthetic reagents to generate new reactive intermediates and just a few amino acid substitutions to tune the active site, a cytochrome P450 can catalyze a variety of carbene and nitrene transfer reactions. The cyclopropanation, N-H insertion, C-H amination, sulfimidation, and aziridination reactions now demonstrated are all well known in chemical catalysis but have no counterparts in nature. The new enzymes are fully genetically encoded, assemble and function inside of cells, and can be optimized for different substrates, activities, and selectivities. We are learning how to use nature's innovation mechanisms to marry some of the synthetic chemists' favorite transformations with the exquisite selectivity and tunability of enzymes.

Dioxygen Binding, Activation, and Reduction to H2O by Cu Enzymes.

PubMed

Solomon, Edward I

2016-07-05

Oxygen intermediates in copper enzymes exhibit unique spectroscopic features that reflect novel geometric and electronic structures that are key to reactivity. This perspective will describe: (1) the bonding origin of the unique spectroscopic features of the coupled binuclear copper enzymes and how this overcomes the spin forbiddenness of O2 binding and activates monooxygenase activity, (2) how the difference in exchange coupling in the non-coupled binuclear Cu enzymes controls the reaction mechanism, and (3) how the trinuclear Cu cluster present in the multicopper oxidases leads to a major structure/function difference in enabling the irreversible reductive cleavage of the O-O bond with little overpotential and generating a fully oxidized intermediate, different from the resting enzyme studied by crystallography, that is key in enabling fast PCET in the reductive half of the catalytic cycle.

Enzyme Engineering for In Situ Immobilization.

PubMed

Rehm, Fabian B H; Chen, Shuxiong; Rehm, Bernd H A

2016-10-14

Enzymes are used as biocatalysts in a vast range of industrial applications. Immobilization of enzymes to solid supports or their self-assembly into insoluble particles enhances their applicability by strongly improving properties such as stability in changing environments, re-usability and applicability in continuous biocatalytic processes. The possibility of co-immobilizing various functionally related enzymes involved in multistep synthesis, conversion or degradation reactions enables the design of multifunctional biocatalyst with enhanced performance compared to their soluble counterparts. This review provides a brief overview of up-to-date in vitro immobilization strategies while focusing on recent advances in enzyme engineering towards in situ self-assembly into insoluble particles. In situ self-assembly approaches include the bioengineering of bacteria to abundantly form enzymatically active inclusion bodies such as enzyme inclusions or enzyme-coated polyhydroxyalkanoate granules. These one-step production strategies for immobilized enzymes avoid prefabrication of the carrier as well as chemical cross-linking or attachment to a support material while the controlled oriented display strongly enhances the fraction of accessible catalytic sites and hence functional enzymes.

Investigating the effect of gallium curcumin and gallium diacetylcurcumin complexes on the structure, function and oxidative stability of the peroxidase enzyme and their anticancer and antibacterial activities.

PubMed

Jahangoshaei, Parisa; Hassani, Leila; Mohammadi, Fakhrossadat; Hamidi, Akram; Mohammadi, Khosro

2015-10-01

Curcumin has a wide spectrum of biological and pharmacological activities including anti-inflammatory, antioxidant, antiproliferative, antimicrobial and anticancer activities. Complexation of curcumin with metals has gained attention in recent years for improvement of its stability. In this study, the effect of gallium curcumin and gallium diacetylcurcumin on the structure, function and oxidative stability of horseradish peroxidase (HRP) enzyme were evaluated by spectroscopic techniques. In addition to the enzymatic investigation, the cytotoxic effect of the complexes was assessed on bladder, MCF-7 breast cancer and LNCaP prostate carcinoma cell lines by MTT assay. Furthermore, antibacterial activity of the complexes against S. aureus and E. coli was explored by dilution test method. The results showed that the complexes improve activity of HRP and also increase its tolerance against the oxidative condition. After addition of the complexes, affinity of HRP for hydrogen peroxide substrate decreases, while the affinity increases for phenol substrate. Circular dichroism, intrinsic and synchronous fluorescence spectra showed that the enzyme structure around the catalytic heme group becomes less compact and also the distance between the heme group and tryptophan residues increases due to binding of the complexes to HRP. On the whole, it can be concluded that the change in the enzyme structure upon binding to the gallium curcumin and gallium diacetylcurcumin complexes results in an increase in the antioxidant efficiency and activity of the peroxidise enzyme. The result of anticancer and antibacterial activities suggested that the complexes exhibit the potential for cancer treatment, but they have no significant antibacterial activity.

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.

Comparative structural modeling of six old yellow enzymes (OYEs) from the necrotrophic fungus Ascochyta rabiei: insight into novel OYE classes with differences in cofactor binding, organization of active site residues and stereopreferences.

PubMed

Nizam, Shadab; Gazara, Rajesh Kumar; Verma, Sandhya; Singh, Kunal; Verma, Praveen Kumar

2014-01-01

Old Yellow Enzyme (OYE1) was the first flavin-dependent enzyme identified and characterized in detail by the entire range of physical techniques. Irrespective of this scrutiny, true physiological role of the enzyme remains a mystery. In a recent study, we systematically identified OYE proteins from various fungi and classified them into three classes viz. Class I, II and III. However, there is no information about the structural organization of Class III OYEs, eukaryotic Class II OYEs and Class I OYEs of filamentous fungi. Ascochyta rabiei, a filamentous phytopathogen which causes Ascochyta blight (AB) in chickpea possesses six OYEs (ArOYE1-6) belonging to the three OYE classes. Here we carried out comparative homology modeling of six ArOYEs representing all the three classes to get an in depth idea of structural and functional aspects of fungal OYEs. The predicted 3D structures of A. rabiei OYEs were refined and evaluated using various validation tools for their structural integrity. Analysis of FMN binding environment of Class III OYE revealed novel residues involved in interaction. The ligand para-hydroxybenzaldehyde (PHB) was docked into the active site of the enzymes and interacting residues were analyzed. We observed a unique active site organization of Class III OYE in comparison to Class I and II OYEs. Subsequently, analysis of stereopreference through structural features of ArOYEs was carried out, suggesting differences in R/S selectivity of these proteins. Therefore, our comparative modeling study provides insights into the FMN binding, active site organization and stereopreference of different classes of ArOYEs and indicates towards functional differences of these enzymes. This study provides the basis for future investigations towards the biochemical and functional characterization of these enigmatic enzymes.

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

PubMed Central

Baltar, Federico

2018-01-01

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

A Multifunctional Surface That Simultaneously Balances Hydrophilic Enzyme Catalysis and Hydrophobic Water Repellency.

PubMed

Lawton, Timothy J; Uzarski, Joshua R; Filocamo, Shaun F

2016-08-16

The compatibility of multiple functions at a single interface is difficult to achieve, but is even more challenging when the functions directly counteract one another. This study provides insight into the creation of a simultaneously multifunctional surface formed by balancing two orthogonal functions; water repellency and enzyme catalysis. A partially fluorinated thiol is used to impart bulk hydrophobicity on the surface, and an N-hydroxysuccinimide ester-terminated thiol provides a specific anchoring sites for the covalent enzyme attachment. Different ratios of the two thiols are mixed together to form amphiphilic self-assembled monolayers, which are characterized with polarization-modulation infrared reflection-absorption spectroscopy and contact angle goniometry. The enzyme activity is measured by a fluorescence assay. With the results collected here, specific surface compositions are identified at which the orthogonal functions of water repellency and enzyme catalysis are balanced and exist simultaneously. An understanding of how to effectively balance orthogonal functions at surfaces can be extended to a number of higher-scale applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tree species, tree genotypes and tree genotypic diversity levels affect microbe-mediated soil ecosystem functions in a subtropical forest.

PubMed

Purahong, Witoon; Durka, Walter; Fischer, Markus; Dommert, Sven; Schöps, Ricardo; Buscot, François; Wubet, Tesfaye

2016-11-18

Tree species identity and tree genotypes contribute to the shaping of soil microbial communities. However, knowledge about how these two factors influence soil ecosystem functions is still lacking. Furthermore, in forest ecosystems tree genotypes co-occur and interact with each other, thus the effects of tree genotypic diversity on soil ecosystem functions merit attention. Here we investigated the effects of tree species, tree genotypes and genotypic diversity levels, alongside soil physicochemical properties, on the overall and specific soil enzyme activity patterns. Our results indicate that tree species identity, tree genotypes and genotypic diversity level have significant influences on overall and specific soil enzyme activity patterns. These three factors influence soil enzyme patterns partly through effects on soil physicochemical properties and substrate quality. Variance partitioning showed that tree species identity, genotypic diversity level, pH and water content all together explained ~30% variations in the overall patterns of soil enzymes. However, we also found that the responses of soil ecosystem functions to tree genotypes and genotypic diversity are complex, being dependent on tree species identity and controlled by multiple factors. Our study highlights the important of inter- and intra-specific variations in tree species in shaping soil ecosystem functions in a subtropical forest.

Tree species, tree genotypes and tree genotypic diversity levels affect microbe-mediated soil ecosystem functions in a subtropical forest

PubMed Central

Purahong, Witoon; Durka, Walter; Fischer, Markus; Dommert, Sven; Schöps, Ricardo; Buscot, François; Wubet, Tesfaye

2016-01-01

Tree species identity and tree genotypes contribute to the shaping of soil microbial communities. However, knowledge about how these two factors influence soil ecosystem functions is still lacking. Furthermore, in forest ecosystems tree genotypes co-occur and interact with each other, thus the effects of tree genotypic diversity on soil ecosystem functions merit attention. Here we investigated the effects of tree species, tree genotypes and genotypic diversity levels, alongside soil physicochemical properties, on the overall and specific soil enzyme activity patterns. Our results indicate that tree species identity, tree genotypes and genotypic diversity level have significant influences on overall and specific soil enzyme activity patterns. These three factors influence soil enzyme patterns partly through effects on soil physicochemical properties and substrate quality. Variance partitioning showed that tree species identity, genotypic diversity level, pH and water content all together explained ~30% variations in the overall patterns of soil enzymes. However, we also found that the responses of soil ecosystem functions to tree genotypes and genotypic diversity are complex, being dependent on tree species identity and controlled by multiple factors. Our study highlights the important of inter- and intra-specific variations in tree species in shaping soil ecosystem functions in a subtropical forest. PMID:27857198

The nature and function of microbial enzymes in subsurface marine sediments

NASA Astrophysics Data System (ADS)

Steen, A. D.; Schmidt, J.

2016-02-01

Isotopic and genomic evidence indicates that marine sediments contain populations of active heterotrophic microorganisms which appear to metabolize old, detrital, apparently recalcitrant organic matter. In surface communities, heterotrophs use extracellular enzymes to access complex organic matter. In subsurface sediments, in which microbial doubling times can be on the order of hundreds or thousands of years, it is not clear whether extracellular enzymes could remain stable and active long enough to constitute a 'profitable' stragtegy for accessing complex organic carbon. Here we present evidence that a wide range of extracellular enzyme are active in subsurface sediments from two different environments: the White Oak River, NC, and deep (up to 80 m) sediments of the Baltic Sea Basin recovered from IODP Expedition 347. In the White Oak River, enzymes from deeper sediments appear to be better-adapted to highly-degraded organic matter than enzymes from surface sediments. In the Baltic Sea, preliminary data suggest that enzymes related to nitrogen acquisition are preferentially expressed. By characterizing the extracellular enzymes present in marine sediments, we hope to achieve a better understanding of the mechanisms that control sedimentary organic matter remineralization and preservation.

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

PubMed Central

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

2015-01-01

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

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

DOE PAGES

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

2015-04-22

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

Electrochemistry of metalloproteins: protein film electrochemistry for the study of E. coli [NiFe]-hydrogenase-1.

PubMed

Evans, Rhiannon M; Armstrong, Fraser A

2014-01-01

Protein film electrochemistry is a technique which allows the direct control of redox-active enzymes, providing particularly detailed information on their catalytic properties. The enzyme is deposited onto a working electrode tip, and through control of the applied potential the enzyme activity is monitored as electrical current, allowing for direct study of inherent activity as electrons are transferred to and from the enzyme redox center(s). No mediators are used. Because the only enzyme present in the experiment is bound at the electrode surface, gaseous and liquid phase inhibitors can be introduced and removed whilst the enzyme remains in situ. Potential control means that kinetics and thermodynamics are explored simultaneously; the kinetics of a reaction can be studied as a function of potential. Steady-state catalytic rates are observed directly as current (for a given potential) and non-steady-state rates (such as interconversions between different forms of the enzyme) are observed from the change in current with time. The more active the enzyme, the higher the current and the better the signal-to-noise. In this chapter we outline the practical aspects of PFE for studying electroactive enzymes, using the Escherichia coli [NiFe]-hydrogenase 1 (Hyd-1) as an example.

Life-history evolution and the microevolution of intermediary metabolism: activities of lipid-metabolizing enzymes in life-history morphs of a wing-dimorphic cricket.

PubMed

Zera, Anthony J; Zhao, Zhangwu

2003-03-01

Although a considerable amount of information is available on the ecology, genetics, and physiology of life-history traits, much more limited data are available on the biochemical and genetic correlates of life-history variation within species. Specific activities of five enzymes of lipid biosynthesis and two enzymes of amino acid catabolism were compared among lines selected for flight-capable (LW[f]) versus flightless (SW) morphs of the cricket Gryllus firmus. These morphs, which exist in natural populations, differ genetically in ovarian growth (100-400% higher in SW) and aspects of flight capability including the size of wings and flight muscles, and the concentration of triglyceride flight fuel (40% greater in LW[f]). Consistently higher activity of each enzyme in LW(f) versus SW-selected lines, and strong co-segregation between morph and enzyme activity, demonstrated genetically based co-variance between wing morph and enzyme activity. Developmental profiles of enzyme activities strongly paralleled profiles of triglyceride accumulation during adulthood and previous measures of in vivo lipid biosynthesis. These data strongly imply that genetically based elevation in activities of lipogenic enzymes, and enzymes controlling the conversion of amino acids into lipids, is an important cause underlying the elevated accumulation of triglyceride in the LW(f) morph, a key biochemical component of the trade-off between elevated early fecundity and flight capability. Global changes in lipid and amino-acid metabolism appear to have resulted from microevolutionary alteration of regulators of metabolism. Finally, strong genotype x environment (diet) interactions were observed for most enzyme activities. Future progress in understanding the functional causes of life-history evolution requires a more detailed synthesis of the fields of life-history evolution and metabolic biochemistry. Wing polymorphism is a powerful experimental model in such integrative studies.

Structure, function and regulation of the enzymes in the starch biosynthetic pathway.

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

Geiger, Jim

Starch is the major reserve polysaccharide in nature and accounts for the majority of the caloric intact of humans. It is also gaining importance as a renewable and biodegradable industrial material. There is burgeoning interest in increasing the amount and altering the properties of the plant starches by plant genetic modification. A rational approach to this effort will require a detailed, atomic-level understanding of the enzymatic processes that produce the starch granule. The starch granule is a complex particle made up of alternating layers of crystalline and amorphous lamellae. It consists of two types of polymer, amylose, a polymer ofmore » relatively long chains of α-1,4-linked glucans that contain virtually no branches, and amylopectin, which is highly branched and contains much shorter chains. This complex structure is synthesized by the coordinate activities of the starch synthases (SS), which elongate the polysaccharide chain by addition of glucose units via α-1,4 linkages using ADP- glucose as a donor, and branching enzymes (BE), which branch the polysaccharide chain by cleavage of α₋1,4 linkages and subsequent re-attachment via α₋1,6 linkages. Several isoforms of both starch synthase (SS) and branching enzyme (BE) are found in plants, including SSI, SSII, SSIII and granule- bound SS (GBSS), and SBEI, SBEIIa and SBEIIb. These isoforms have different activities and substrate and product specificities and play different roles in creating the granule and determining the properties of the resulting starch. The overarching goal of this proposal is to begin to understand the regulation and specificities of these enzymes at the atomic level. High-resolution X-ray structures of these enzymes bound to substrates and products will be determined to visualize the molecular interactions responsible for the properties of the enzymes. Hypotheses regarding these issues will then be tested using mutagenesis and enzyme assays. To date, we have determined the structure of ADP- Glucose pyrophosphorylase from potato in its inhibited conformation, and bound to both ATP and ADP-glucose. In addition, we have determined the first structure of glycogen synthase in its "closed", catalytically active conformation bound to ADP-glucose. We also determined the structure of glycogen synthase bound to malto-oligosaccharides, showing for the first time that an enzyme in the starch biosynthetic pathway recognizes glucans not just in its active site but on binding sites on the surface of the enzyme ten’s of Angstroms from the active site. In addition our structure of a glycogen branching enzyme bound to malto-oligosaccharides identified seven distinct binding sites distributed about the surface of the enzyme. We will now determine the function of these sites to get a molecular-level picture of exactly how these enzymes interact with their polymeric substrates and confer specificity leading to the complex structure of the starch granule. We will extend our studies to other isoforms of the enzymes, to understand how their structures give rise to their distinct function. Our goal is to understand what accounts for the various functional differences between SS and SBE isoforms at a molecular level.« less

Structure and function studies on enzymes with a catalytic carboxyl group(s): from ribonuclease T1 to carboxyl peptidases

PubMed Central

TAKAHASHI, Kenji

2013-01-01

A group of enzymes, mostly hydrolases or certain transferases, utilize one or a few side-chain carboxyl groups of Asp and/or Glu as part of the catalytic machinery at their active sites. This review follows mainly the trail of studies performed by the author and his colleagues on the structure and function of such enzymes, starting from ribonuclease T1, then extending to three major types of carboxyl peptidases including aspartic peptidases, glutamic peptidases and serine-carboxyl peptidases. PMID:23759941

Pathophysiological implications of neurovascular P450 in brain disorders

PubMed Central

Ghosh, Chaitali; Hossain, Mohammed; Solanki, Jesal; Dadas, Aaron; Marchi, Nicola; Janigro, Damir

2016-01-01

Over the past decades, the significance of cytochrome P450 (CYP) enzymes has expanded beyond their role as peripheral drug metabolizers in the liver and gut. CYP enzymes are also functionally active at the neurovascular interface. CYP expression is modulated by disease states, impacting cellular functions, detoxification, and reactivity to toxic stimuli and brain drug biotransformation. Unveiling the physiological and molecular complexity of brain P450 enzymes will improve our understanding of the mechanisms underlying brain drug availability, pharmacological efficacy, and neurotoxic adverse effects from pharmacotherapy targeting brain disorders. PMID:27312874

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.

Divergence of dominant factors in soil microbial communities and functions in forest ecosystems along a climatic gradient

NASA Astrophysics Data System (ADS)

Xu, Zhiwei; Yu, Guirui; Zhang, Xinyu; He, Nianpeng; Wang, Qiufeng; Wang, Shengzhong; Xu, Xiaofeng; Wang, Ruili; Zhao, Ning

2018-03-01

Soil microorganisms play an important role in regulating nutrient cycling in terrestrial ecosystems. Most of the studies conducted thus far have been confined to a single forest biome or have focused on one or two controlling factors, and few have dealt with the integrated effects of climate, vegetation, and soil substrate availability on soil microbial communities and functions among different forests. In this study, we used phospholipid-derived fatty acid (PLFA) analysis to investigate soil microbial community structure and extracellular enzymatic activities to evaluate the functional potential of soil microbes of different types of forests in three different climatic zones along the north-south transect in eastern China (NSTEC). Both climate and forest type had significant effects on soil enzyme activities and microbial communities with considerable interactive effects. Except for soil acid phosphatase (AP), the other three enzyme activities were much higher in the warm temperate zone than in the temperate and the subtropical climate zones. The soil total PLFAs and bacteria were much higher in the temperate zone than in the warm temperate and the subtropical zones. The soil β-glucosidase (BG) and N-acetylglucosaminidase (NAG) activities were highest in the coniferous forest. Except for the soil fungi and fungi-bacteria (F/B), the different groups of microbial PLFAs were much higher in the conifer broad-leaved mixed forests than in the coniferous forests and the broad-leaved forests. In general, soil enzyme activities and microbial PLFAs were higher in primary forests than in secondary forests in temperate and warm temperate regions. In the subtropical region, soil enzyme activities were lower in the primary forests than in the secondary forests and microbial PLFAs did not differ significantly between primary and secondary forests. Different compositions of the tree species may cause variations in soil microbial communities and enzyme activities. Our results showed that the main controls on soil microbes and functions vary in different climatic zones and that the effects of soil moisture content, soil temperature, clay content, and the soil N / P ratio were considerable. This information will add value to the modeling of microbial processes and will contribute to carbon cycling in large-scale carbon models.

Gold core/Ceria shell-based redox active nanozyme mimicking the biological multienzyme complex phenomenon

DOE PAGES

Bhagat, Stuti; Srikanth Vallabani, NV; Shutthanandan, Vaithiyalingam; ...

2017-12-02

Catalytically active individual gold (Au) and cerium oxide (CeO 2) nanoparticles (NPs) are well known to exhibit specific enzyme-like activities, such as natural catalase, oxidase, superoxide dismutase, and peroxidase enzymes. Our activities have been maneuvered to design several biological applications such as immunoassays, glucose detection, radiation and free radical protection and tissue engineering. In biological systems, multienzyme complexes are involved in catalyzing important reactions of essential metabolic processes such as respiration, biomolecule synthesis, and photosynthesis. It is well known that metabolic processes linked with multienzyme complexes offer several advantages over reactions catalyzed by individual enzymes. A functional nanozyme depicting multienzymemore » like properties has eluded the researchers in the nanoscience community for the past few decades. Here, we have designed a functional multienzyme in the form of Gold (core)-CeO 2 (shell) nanoparticles (Au/CeO 2 CSNPs) exhibiting excellent peroxidase, catalase, and superoxide dismutase enzyme-like activities that are controlled simply by tuning the pH. The reaction kinetic parameters reveal that the peroxidase-like activity of this core-shell nanozyme is comparable to natural horseradish peroxidase (HRP) enzyme. Unlike peroxidase-like activity exhibited by other nanomaterials, Au/CeO 2 CSNPs showed a decrease in hydroxyl radical formation, suggesting that the biocatalytic reactions are performed by efficient electron transfers. A significant enzyme-like activity of this core-shell nanoparticle was conserved at extreme pH (2 – 11) and temperatures (up to 90 °C), clearly suggesting the superiority over natural enzymes. Further, the utility of peroxidase-like activity of this core-shell nanoparticles was extended for the detection of glucose, which showed a linear range of detection between (100 µM – 1 mM). It is hypothesized that the proximity of the redox potentials of Au+/Au and Ce (III)/Ce (IV) may result in a redox couple promoting the multienzyme activity of core-shell nanoparticles. Au/CeO 2 CSNPs may open new directions for development of single platform sensors in multiple biosensing applications.« less

Gold core/Ceria shell-based redox active nanozyme mimicking the biological multienzyme complex phenomenon

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

Bhagat, Stuti; Srikanth Vallabani, NV; Shutthanandan, Vaithiyalingam

Catalytically active individual gold (Au) and cerium oxide (CeO 2) nanoparticles (NPs) are well known to exhibit specific enzyme-like activities, such as natural catalase, oxidase, superoxide dismutase, and peroxidase enzymes. Our activities have been maneuvered to design several biological applications such as immunoassays, glucose detection, radiation and free radical protection and tissue engineering. In biological systems, multienzyme complexes are involved in catalyzing important reactions of essential metabolic processes such as respiration, biomolecule synthesis, and photosynthesis. It is well known that metabolic processes linked with multienzyme complexes offer several advantages over reactions catalyzed by individual enzymes. A functional nanozyme depicting multienzymemore » like properties has eluded the researchers in the nanoscience community for the past few decades. Here, we have designed a functional multienzyme in the form of Gold (core)-CeO 2 (shell) nanoparticles (Au/CeO 2 CSNPs) exhibiting excellent peroxidase, catalase, and superoxide dismutase enzyme-like activities that are controlled simply by tuning the pH. The reaction kinetic parameters reveal that the peroxidase-like activity of this core-shell nanozyme is comparable to natural horseradish peroxidase (HRP) enzyme. Unlike peroxidase-like activity exhibited by other nanomaterials, Au/CeO 2 CSNPs showed a decrease in hydroxyl radical formation, suggesting that the biocatalytic reactions are performed by efficient electron transfers. A significant enzyme-like activity of this core-shell nanoparticle was conserved at extreme pH (2 – 11) and temperatures (up to 90 °C), clearly suggesting the superiority over natural enzymes. Further, the utility of peroxidase-like activity of this core-shell nanoparticles was extended for the detection of glucose, which showed a linear range of detection between (100 µM – 1 mM). It is hypothesized that the proximity of the redox potentials of Au+/Au and Ce (III)/Ce (IV) may result in a redox couple promoting the multienzyme activity of core-shell nanoparticles. Au/CeO 2 CSNPs may open new directions for development of single platform sensors in multiple biosensing applications.« less

Isolation of cold-active, acidic endocellulase from Ladakh soil by functional metagenomics.

PubMed

Bhat, Archana; Riyaz-Ul-Hassan, Syed; Ahmad, Nasier; Srivastava, Nidhi; Johri, Sarojini

2013-03-01

Mining of soil sample from cold desert of Ladakh by functional metagenomics led to the isolation of cold-adapted endocellulase (CEL8M) that hydrolyses carboxymethyl cellulose (CMC). Mature CEL8M, a 347-residue polypeptide with a molecular mass of 38.9 kDa showed similarity to β-1,3-1,4 D-glucanase from Klebsiella sp. The enzyme contains the catalytic module of glycosyl hydrolase family 8 but does not possess a carbohydrate-binding domain. 3D structural model of the enzyme built by homology modeling showed an architecture of (α/α)6-barrel fold. The purified enzyme was found to be active against CMC, xylan, colloidal chitosan and lichenan but not active against avicel. Glucose was not among the initial hydrolysis products, indicating an endo mode of action. CEL8M displayed maximal activity at pH 4.5 and remained significantly active (~28 %) when the temperature decreased to 10 °C. Cold-active endocellulase CEL8M may find applications in textile industry at low temperature which can result in energy savings.

Hypoglycemic and Hypolipidemic Effects of the Cracked-Cap Medicinal Mushroom Phellinus rimosus (Higher Basidiomycetes) in Streptozotocin-Induced Diabetic Rats.

PubMed

Rony, Kuttikkadan A; Ajith, Thekkuttuparambil A; Janardhanan, Kainoor K

2015-01-01

Phellinus rimosus is a parasitic host specific polypore mushroom with profound antioxidant, antihepatotoxic, anti-inflammatory, antitumor, and antimutagenic activities. This study investigated the hypoglycemic and hypolipidemic activities of the wood-inhabiting polypore mushroom Ph. Rimosus in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced by single intraperitoneal injection of STZ (45 mg/kg) to Wistar rats. The effects of 30 days treatment with Ph. Rimosus (50 and 250 mg/ kg) and glibenclamide (0.65 mg/kg) on blood glucose level, serum insulin, serum lipid profile, liver glycogen, liver function enzymes, and non-enzymic and enzymic antioxidants activities in pancreas, liver, and kidney were evaluated in STZ-induced diabetic rats. Oral administration of Ph. Rimosus extract exhibited a significant reduction in blood glucose, triacylglycerol, total cholesterol, LDL-cholesterol, and liver function enzymes, and increased serum insulin, liver glycogen, and HDL-cholesterol levels in STZ-induced diabetic rats. Furthermore, Ph. Rimosus treatment increased antioxidant status in pancreas, liver, and kidney tissues with concomitant decreases in levels of thiobarbituric acid- reactive substances. Results of this study indicated that Ph. Rimosus possessed significant hypoglycemic and hypolipidemic activities and this effect may be related to its insulinogenic and antioxidant effect.

Versatile de novo enzyme activity in capsid proteins from an engineered M13 bacteriophage library.

PubMed

Casey, John P; Barbero, Roberto J; Heldman, Nimrod; Belcher, Angela M

2014-11-26

Biocatalysis has grown rapidly in recent decades as a solution to the evolving demands of industrial chemical processes. Mounting environmental pressures and shifting supply chains underscore the need for novel chemical activities, while rapid biotechnological progress has greatly increased the utility of enzymatic methods. Enzymes, though capable of high catalytic efficiency and remarkable reaction selectivity, still suffer from relative instability, high costs of scaling, and functional inflexibility. Herein, we developed a biochemical platform for engineering de novo semisynthetic enzymes, functionally modular and widely stable, based on the M13 bacteriophage. The hydrolytic bacteriophage described in this paper catalyzes a range of carboxylic esters, is active from 25 to 80 °C, and demonstrates greater efficiency in DMSO than in water. The platform complements biocatalysts with characteristics of heterogeneous catalysis, yielding high-surface area, thermostable biochemical structures readily adaptable to reactions in myriad solvents. As the viral structure ensures semisynthetic enzymes remain linked to the genetic sequences responsible for catalysis, future work will tailor the biocatalysts to high-demand synthetic processes by evolving new activities, utilizing high-throughput screening technology and harnessing M13's multifunctionality.

Structural Basis for Flip-Flop Action of Thiamin-Dependent Enzymes Revealed by Crystal Structure of Human Pyruvate Dehydrogenase

NASA Technical Reports Server (NTRS)

Ciszak, Ewa; Korotchkina, Lioubov G.; Dominiak, Paulina M.; Sidhu, Sukdeep; Patel, Mulchand S.

2003-01-01

The biologically active derivative of vitamin B1; thiamin pyrophosphate; is used as cofactor by many enzymes that perform a wide range of catalytic functions in the pathways of energy production. In alpha2beta2-heterotetrameric human pyruvate dehydrogenase, the first catalytic component enzyme of human pyruvate dehydrogenase complex, this cofactor is used to cleave the C(sup alpha)-C(=0) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase, the second catalytic component of the complex. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites have puzzled researchers from earlier functional studies of this enzyme. In order to gain insight into the mechanism of action of this enzyme, we determined the crystal structure of the holoform of human pyruvate dehydrogenase at 1.958, resolution. We propose a kinetic model for the flip-flop action of this enzyme through the concerted approx. 2A, shuttle-like motion of the heterodimers. The similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase and other functionally related enzymes suggests this newly defined mechanism of shuttle-like motion of domains to be common for the family of thiamin pyrophosphate-dependent enzymes.

Transition-State Ensembles Navigate the Pathways of Enzyme Catalysis.

PubMed

Mickert, Matthias J; Gorris, Hans H

2018-06-07

Transition-state theory (TST) provides an important framework for analyzing and explaining the reaction rates of enzymes. TST, however, needs to account for protein dynamic effects and heterogeneities in enzyme catalysis. We have analyzed the reaction rates of β-galactosidase and β-glucuronidase at the single molecule level by using large arrays of femtoliter-sized chambers. Heterogeneities in individual reaction rates yield information on the intrinsic distribution of the free energy of activation (Δ G ‡ ) in an enzyme ensemble. The broader distribution of Δ G ‡ in β-galactosidase compared to β-glucuronidase is attributed to β-galactosidase's multiple catalytic functions as a hydrolase and a transglycosylase. Based on the catalytic mechanism of β-galactosidase, we show that transition-state ensembles do not only contribute to enzyme catalysis but can also channel the catalytic pathway to the formation of different products. We conclude that β-galactosidase is an example of natural evolution, where a new catalytic pathway branches off from an established enzyme function. The functional division of work between enzymatic substates explains why the conformational space represented by the enzyme ensemble is larger than the conformational space that can be sampled by any given enzyme molecule during catalysis.

Identification of novel esterase-active enzymes from hot environments by use of the host bacterium Thermus thermophilus.

PubMed

Leis, Benedikt; Angelov, Angel; Mientus, Markus; Li, Haijuan; Pham, Vu T T; Lauinger, Benjamin; Bongen, Patrick; Pietruszka, Jörg; Gonçalves, Luís G; Santos, Helena; Liebl, Wolfgang

2015-01-01

Functional metagenomic screening strategies, which are independent of known sequence information, can lead to the identification of truly novel genes and enzymes. Since E. coli has been used exhaustively for this purpose as a host, it is important to establish alternative expression hosts and to use them for functional metagenomic screening for new enzymes. In this study we show that Thermus thermophilus HB27 is an excellent screening host and can be used as an alternative provider of truly novel biocatalysts. In a previous study we constructed mutant strain BL03 with multiple markerless deletions in genes for major extra- and intracellular lipolytic activities. This esterase-diminished strain was no longer able to grow on defined minimal medium supplemented with tributyrin as the sole carbon source and could be used as a host to screen for metagenomic DNA fragments that could complement growth on tributyrin. Several thousand single fosmid clones from thermophilic metagenomic libraries from heated compost and hot spring water samples were subjected to a comparative screening for esterase activity in both T. thermophilus strain BL03 and E. coli EPI300. We scored a greater number of active esterase clones in the thermophilic bacterium than in the mesophilic E. coli. From several thousand functionally screened clones only two thermostable α/β-fold hydrolase enzymes with high amino acid sequence similarity to already characterized enzymes were identifiable in E. coli. In contrast, five further fosmids were found that conferred lipolytic activities in T. thermophilus only. Four open reading frames (ORFs) were found which did not share significant similarity to known esterase enzymes but contained the conserved GXSXG motif regularly found in lipolytic enzymes. Two of the genes were expressed in both hosts and the novel thermophilic esterases, which based on their primary structures could not be assigned to known esterase or lipase families, were purified and preliminarily characterized. Our work underscores the benefit of using additional screening hosts other than E. coli for the identification of novel biocatalysts with industrial relevance.

Functional assignment of gene AAC16202.1 from Rhodobacter capsulatus SB1003: new insights into the bacterial SDR sorbitol dehydrogenases family.

PubMed

Sola-Carvajal, Agustín; García-García, María Inmaculada; Sánchez-Carrón, Guiomar; García-Carmona, Francisco; Sánchez-Ferrer, Alvaro

2012-11-01

Short-chain dehydrogenases/reductases (SDR) constitute one of the largest enzyme superfamilies with over 60,000 non-redundant sequences in the database, many of which need a correct functional assignment. Among them, the gene AAC16202.1 (NCBI) from Rhodobacter capsulatus SB1003 has been assigned in Uniprot both as a sorbitol dehydrogenase (#D5AUY1) and, as an N-acetyl-d-mannosamine dehydrogenase (#O66112), both enzymes being of biotechnological interest. When the gene was overexpressed in Escherichia coli Rosetta (DE3)pLys, the purified enzyme was not active toward N-acetyl-d-mannosamine, whereas it was active toward d-sorbitol and d-fructose. However, the relative activities toward xylitol and l-iditol (0.45 and 6.9%, respectively) were low compared with that toward d-sorbitol. Thus, the enzyme could be considered sorbitol dehydrogenase (SDH) with very low activity toward xylitol, which could increase its biotechnological interest for determining sorbitol without the unspecific cross-determination of added xylitol in food and pharma compositions. The tetrameric enzyme (120 kDa) showed similar catalytic efficiency (2.2 × 10(3) M(-1) s(-1)) to other sorbitol dehydrogenases for d-sorbitol, with an optimum pH of 9.0 and an optimum temperature of 37 °C. The enzyme was also more thermostable than other reported SDH, ammonium sulfate being the best stabilizer in this respect, increasing the melting temperature (T(m)) up to 52.9 °C. The enzyme can also be considered as a new member of the Zn(2+) independent SDH family since no effect on activity was detected in the presence of divalent cations or chelating agents. Finally, its in silico analysis enabled the specific conserved sequence blocks that are the fingerprints of bacterial sorbitol dehydrogenases and mainly located at C-terminal of the protein, to be determined for the first time. This knowledge will facilitate future data curation of present databases and a better functional assignment of newly described sequences. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Unification of [FeFe]-hydrogenases into three structural and functional groups.

PubMed

Poudel, Saroj; Tokmina-Lukaszewska, Monika; Colman, Daniel R; Refai, Mohammed; Schut, Gerrit J; King, Paul W; Maness, Pin-Ching; Adams, Michael W W; Peters, John W; Bothner, Brian; Boyd, Eric S

2016-09-01

[FeFe]-hydrogenases (Hyd) are structurally diverse enzymes that catalyze the reversible oxidation of hydrogen (H2). Recent biochemical data demonstrate new functional roles for these enzymes, including those that function in electron bifurcation where an exergonic reaction is coupled with an endergonic reaction to drive the reversible oxidation/production of H2. To identify the structural determinants that underpin differences in enzyme functionality, a total of 714 homologous sequences of the catalytic subunit, HydA, were compiled. Bioinformatics approaches informed by biochemical data were then used to characterize differences in inferred quaternary structure, HydA active site protein environment, accessory iron-sulfur clusters in HydA, and regulatory proteins encoded in HydA gene neighborhoods. HydA homologs were clustered into one of three classification groups, Group 1 (G1), Group 2 (G2), and Group 3 (G3). G1 enzymes were predicted to be monomeric while those in G2 and G3 were predicted to be multimeric and include HydB, HydC (G2/G3) and HydD (G3) subunits. Variation in the HydA active site and accessory iron-sulfur clusters did not vary by group type. Group-specific regulatory genes were identified in the gene neighborhoods of both G2 and G3 Hyd. Analyses of purified G2 and G3 enzymes by mass spectrometry strongly suggest that they are post-translationally modified by phosphorylation. These results suggest that bifurcation capability is dictated primarily by the presence of both HydB and HydC in Hyd complexes, rather than by variation in HydA. This classification scheme provides a framework for future biochemical and mutagenesis studies to elucidate the functional role of Hyd enzymes. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Spätzle-Processing Enzyme-independent Activation of the Toll Pathway in Drosophila Innate Immunity.

PubMed

Yamamoto-Hino, Miki; Goto, Satoshi

2016-05-07

The Toll pathway regulates innate immunity in insects and vertebrates. The Drosophila Toll receptor is activated by a processed form of a ligand, Spätzle. Spätzle-processing enzyme (SPE) is the only enzyme identified to date that functions in converting Spätzle to an active form during the immune response. In the present study, Toll activation induced by immune challenge was almost suppressed in spätzle mutant larvae and adults, whereas it was present in SPE mutant larvae challenged with Micrococcus luteus and adults challenged with Bacillus subtilis. Our data suggest that an unidentified protease besides SPE processes Spätzle under conditions of microbial challenge.

Expression and characterization of thermostable glycogen branching enzyme from Geobacillus mahadia Geo-05.

PubMed

Mohtar, Nur Syazwani; Abdul Rahman, Mohd Basyaruddin; Raja Abd Rahman, Raja Noor Zaliha; Leow, Thean Chor; Salleh, Abu Bakar; Mat Isa, Mohd Noor

2016-01-01

The glycogen branching enzyme (EC 2.4.1.18), which catalyses the formation of α -1,6-glycosidic branch points in glycogen structure, is often used to enhance the nutritional value and quality of food and beverages. In order to be applicable in industries, enzymes that are stable and active at high temperature are much desired. Using genome mining, the nucleotide sequence of the branching enzyme gene ( glgB ) was extracted from the Geobacillus mahadia Geo-05 genome sequence provided by the Malaysia Genome Institute. The size of the gene is 2013 bp, and the theoretical molecular weight of the protein is 78.43 kDa. The gene sequence was then used to predict the thermostability, function and the three dimensional structure of the enzyme. The gene was cloned and overexpressed in E. coli to verify the predicted result experimentally. The purified enzyme was used to study the effect of temperature and pH on enzyme activity and stability, and the inhibitory effect by metal ion on enzyme activity. This thermostable glycogen branching enzyme was found to be most active at 55 °C, and the half-life at 60 °C and 70 °C was 24 h and 5 h, respectively. From this research, a thermostable glycogen branching enzyme was successfully isolated from Geobacillus mahadia Geo-05 by genome mining together with molecular biology technique.

Rapid bursts and slow declines: on the possible evolutionary trajectories of enzymes

PubMed Central

Newton, Matilda S.; Arcus, Vickery L.; Patrick, Wayne M.

2015-01-01

The evolution of enzymes is often viewed as following a smooth and steady trajectory, from barely functional primordial catalysts to the highly active and specific enzymes that we observe today. In this review, we summarize experimental data that suggest a different reality. Modern examples, such as the emergence of enzymes that hydrolyse human-made pesticides, demonstrate that evolution can be extraordinarily rapid. Experiments to infer and resurrect ancient sequences suggest that some of the first organisms present on the Earth are likely to have possessed highly active enzymes. Reconciling these observations, we argue that rapid bursts of strong selection for increased catalytic efficiency are interspersed with much longer periods in which the catalytic power of an enzyme erodes, through neutral drift and selection for other properties such as cellular energy efficiency or regulation. Thus, many enzymes may have already passed their catalytic peaks. PMID:25926697

Ancient Regulatory Role of Lysine Acetylation in Central Metabolism

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

Nakayasu, Ernesto S.; Burnet, Meagan C.; Walukiewicz, Hanna E.

ABSTRACT Lysine acetylation is a common protein post-translational modification in bacteria and eukaryotes. Unlike phosphorylation, whose functional role in signaling has been established, it is unclear what regulatory mechanism acetylation plays and whether it is conserved across evolution. By performing a proteomic analysis of 48 phylogenetically distant bacteria, we discovered conserved acetylation sites on catalytically essential lysine residues that are invariant throughout evolution. Lysine acetylation removes the residue’s charge and changes the shape of the pocket required for substrate or cofactor binding. Two-thirds of glycolytic and tricarboxylic acid (TCA) cycle enzymes are acetylated at these critical sites. Our data suggestmore » that acetylation may play a direct role in metabolic regulation by switching off enzyme activity. We propose that protein acetylation is an ancient and widespread mechanism of protein activity regulation. IMPORTANCEPost-translational modifications can regulate the activity and localization of proteins inside the cell. Similar to phosphorylation, lysine acetylation is present in both eukaryotes and prokaryotes and modifies hundreds to thousands of proteins in cells. However, how lysine acetylation regulates protein function and whether such a mechanism is evolutionarily conserved is still poorly understood. Here, we investigated evolutionary and functional aspects of lysine acetylation by searching for acetylated lysines in a comprehensive proteomic data set from 48 phylogenetically distant bacteria. We found that lysine acetylation occurs in evolutionarily conserved lysine residues in catalytic sites of enzymes involved in central carbon metabolism. Moreover, this modification inhibits enzymatic activity. Our observations suggest that lysine acetylation is an evolutionarily conserved mechanism of controlling central metabolic activity by directly blocking enzyme active sites.« less

Ancient Regulatory Role of Lysine Acetylation in Central Metabolism

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

Nakayasu, Ernesto S.; Burnet, Meagan C.; Walukiewicz, Hanna E.

ABSTRACT Lysine acetylation is a common protein post-translational modification in bacteria and eukaryotes. Unlike phosphorylation, whose functional role in signaling has been established, it is unclear what regulatory mechanism acetylation plays and whether it is conserved across evolution. By performing a proteomic analysis of 48 phylogenetically distant bacteria, we discovered conserved acetylation sites on catalytically essential lysine residues that are invariant throughout evolution. Lysine acetylation removes the residue’s charge and changes the shape of the pocket required for substrate or cofactor binding. Two-thirds of glycolytic and tricarboxylic acid (TCA) cycle enzymes are acetylated at these critical sites. Our data suggestmore » that acetylation may play a direct role in metabolic regulation by switching off enzyme activity. We propose that protein acetylation is an ancient and widespread mechanism of protein activity regulation. IMPORTANCE Post-translational modifications can regulate the activity and localization of proteins inside the cell. Similar to phosphorylation, lysine acetylation is present in both eukaryotes and prokaryotes and modifies hundreds to thousands of proteins in cells. However, how lysine acetylation regulates protein function and whether such a mechanism is evolutionarily conserved is still poorly understood. Here, we investigated evolutionary and functional aspects of lysine acetylation by searching for acetylated lysines in a comprehensive proteomic data set from 48 phylogenetically distant bacteria. We found that lysine acetylation occurs in evolutionarily conserved lysine residues in catalytic sites of enzymes involved in central carbon metabolism. Moreover, this modification inhibits enzymatic activity. Our observations suggest that lysine acetylation is an evolutionarily conserved mechanism of controlling central metabolic activity by directly blocking enzyme active sites.« less

Ancient Regulatory Role of Lysine Acetylation in Central Metabolism

DOE PAGES

Nakayasu, Ernesto S.; Burnet, Meagan C.; Walukiewicz, Hanna E.; ...

2017-11-28

ABSTRACT Lysine acetylation is a common protein post-translational modification in bacteria and eukaryotes. Unlike phosphorylation, whose functional role in signaling has been established, it is unclear what regulatory mechanism acetylation plays and whether it is conserved across evolution. By performing a proteomic analysis of 48 phylogenetically distant bacteria, we discovered conserved acetylation sites on catalytically essential lysine residues that are invariant throughout evolution. Lysine acetylation removes the residue’s charge and changes the shape of the pocket required for substrate or cofactor binding. Two-thirds of glycolytic and tricarboxylic acid (TCA) cycle enzymes are acetylated at these critical sites. Our data suggestmore » that acetylation may play a direct role in metabolic regulation by switching off enzyme activity. We propose that protein acetylation is an ancient and widespread mechanism of protein activity regulation. IMPORTANCE Post-translational modifications can regulate the activity and localization of proteins inside the cell. Similar to phosphorylation, lysine acetylation is present in both eukaryotes and prokaryotes and modifies hundreds to thousands of proteins in cells. However, how lysine acetylation regulates protein function and whether such a mechanism is evolutionarily conserved is still poorly understood. Here, we investigated evolutionary and functional aspects of lysine acetylation by searching for acetylated lysines in a comprehensive proteomic data set from 48 phylogenetically distant bacteria. We found that lysine acetylation occurs in evolutionarily conserved lysine residues in catalytic sites of enzymes involved in central carbon metabolism. Moreover, this modification inhibits enzymatic activity. Our observations suggest that lysine acetylation is an evolutionarily conserved mechanism of controlling central metabolic activity by directly blocking enzyme active sites.« less

Whole-Cell Biocatalysis for Producing Ginsenoside Rd from Rb1 Using Lactobacillus rhamnosus GG.

PubMed

Ku, Seockmo; You, Hyun Ju; Park, Myeong Soo; Ji, Geun Eog

2016-07-28

Ginsenosides are the major active ingredients in ginseng used for human therapeutic plant medicines. One of the most well-known probiotic bacteria among the various strains on the functional food market is Lactobacillus rhamnosus GG. Biocatalytic methods using probiotic enzymes for producing deglycosylated ginsenosides such as Rd have a growing significance in the functional food industry. The addition of 2% cellobiose (w/v) to glucose-free de Man-Rogosa-Sharpe broths notably induced β-glucosidase production from L. rhamnosus GG. Enzyme production and activity were optimized at a pH, temperature, and cellobiose concentration of 6.0, 40°C, and 2% (w/v), respectively. Under these controlled conditions, β-glucosidase production in L. rhamnosus GG was enhanced by 25-fold. Additionally, whole-cell homogenates showed the highest β-glucosidase activity when compared with disrupted cell suspensions; the cell disruption step significantly decreased the β-glucosidase activity. Based on the optimized enzyme conditions, whole-cell L. rhamnosus GG was successfully used to convert ginsenoside Rb1 into Rd.

Structural and functional analysis of the ASM p.Ala359Asp mutant that causes acid sphingomyelinase deficiency.

PubMed

Acuña, Mariana; Castro-Fernández, Víctor; Latorre, Mauricio; Castro, Juan; Schuchman, Edward H; Guixé, Victoria; González, Mauricio; Zanlungo, Silvana

2016-10-21

Niemann-Pick disease (NPD) type A and B are recessive hereditary disorders caused by deficiency in acid sphingomyelinase (ASM). The p.Ala359Asp mutation has been described in several patients but its functional and structural effects in the protein are unknown. In order to characterize this mutation, we modeled the three-dimensional ASM structure using the recent available crystal of the mammalian ASM as a template. We found that the p.Ala359Asp mutation is localized in the hydrophobic core and far from the sphingomyelin binding site. However, energy function calculations using statistical potentials indicate that the mutation causes a decrease in ASM stability. Therefore, we investigated the functional effect of the p.Ala359Asp mutation in ASM expression, secretion, localization and activity in human fibroblasts. We found a 3.8% residual ASM activity compared to the wild-type enzyme, without changes in the other parameters evaluated. These results support the hypothesis that the p.Ala359Asp mutation causes structural alterations in the hydrophobic environment where ASM is located, decreasing its enzymatic activity. A similar effect was observed in other previously described NPDB mutations located outside the active site of the enzyme. This work shows the first full size ASM mutant model describe at date, providing a complete analysis of the structural and functional effects of the p.Ala359Asp mutation over the stability and activity of the enzyme. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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.

Simultaneous measurement of two enzyme activities using infrared spectroscopy: A comparative evaluation of PARAFAC, TUCKER and N-PLS modeling.

PubMed

Baum, Andreas; Hansen, Per Waaben; Meyer, Anne S; Mikkelsen, Jørn Dalgaard

2013-08-06

Enzymes are used in many processes to release fermentable sugars for green production of biofuel, or the refinery of biomass for extraction of functional food ingredients such as pectin or prebiotic oligosaccharides. The complex biomasses may, however, require a multitude of specific enzymes which are active on specific substrates generating a multitude of products. In this paper we use the plant polymer, pectin, to present a method to quantify enzyme activity of two pectolytic enzymes by monitoring their superimposed spectral evolutions simultaneously. The data is analyzed by three chemometric multiway methods, namely PARAFAC, TUCKER3 and N-PLS, to establish simultaneous enzyme activity assays for pectin lyase and pectin methyl esterase. Correlation coefficients Rpred(2) for prediction test sets are 0.48, 0.96 and 0.96 for pectin lyase and 0.70, 0.89 and 0.89 for pectin methyl esterase, respectively. The retrieved models are compared and prediction test sets show that especially TUCKER3 performs well, even in comparison to the supervised regression method N-PLS. Copyright © 2013 Elsevier B.V. All rights reserved.

Endophytic fungus Phomopsis liquidambari and different doses of N-fertilizer alter microbial community structure and function in rhizosphere of rice

PubMed Central

Siddikee, Md. Ashaduzzaman; Zereen, Mst Israt; Li, Cai-Feng; Dai, Chuan-Chao

2016-01-01

Microbial community structure and functions of rhizosphere soil of rice were investigated after applying low and high doses of nitrogenous fertilizer and Phomopsis liquidambari. Average well color development, substrate richness, catabolic diversity and soil enzymes activities varied after applying N-fertilizer and P. liquidambari and were greater in P. liquidambari treated soil than only N-fertilization. Multivariate analysis distinctly separated the catabolic and enzymes activity profile which statistically proved alteration of microbial functional diversity. Nitrogen fertilizer altered microbial community structure revealed by the increased content of total PLFAs, specific subgroup marker PLFAs except fungal PLFAs and by the decreased ratio of G+/G−, sat/monunsat, iso/anteiso, F/B except trans/cis while P. liquidambari inoculation enhanced N-fertilization effect except increased fungal PLFA and decreased trans/cis. PCA using identified marker PLFAs revealed definite discrimination among the treatments which further statistically confirmed structural changed of microbial community. Nitrogenase activity representative of N-fixing community decreased in N-fertilizer treatment while P. liquidambari inoculation increased. In short, application of P. liquidambari with low doses of N-fertilizer improved rice growth and reduced N-fertilizer requirement by increasing enzymes activities involved in C, N and P cycling, structural and functional diversity of microbes, nitrogenase activity involved in N2 fixation and accumulation of total-N. PMID:27596935

Demystifying O-GlcNAcylation: hints from peptide substrates.

PubMed

Shi, Jie; Ruijtenbeek, Rob; Pieters, Roland J

2018-03-22

O-GlcNAcylation, analogous to phosphorylation, is an essential post-translational modification of proteins at Ser/Thr residues with a single β-N-acetylglucosamine moiety. This dynamic protein modification regulates many fundamental cellular processes and its deregulation has been linked to chronic diseases such as cancer, diabetes and neurodegenerative disorders. Reversible attachment and removal of O-GlcNAc is governed only by O-GlcNAc transferase and O-GlcNAcase, respectively. Peptide substrates, derived from natural O-GlcNAcylation targets, function in the catalytic cores of these two enzymes by maintaining interactions between enzyme and substrate, which makes them ideal models for the study of O-GlcNAcylation and deglycosylation. These peptides provide valuable tools for a deeper understanding of O-GlcNAc processing enzymes. By taking advantage of peptide chemistry, recent progress in the study of activity and regulatory mechanisms of these two enzymes has advanced our understanding of their fundamental specificities as well as their potential as therapeutic targets. Hence, this review summarizes the recent achievements on this modification studied at the peptide level, focusing on enzyme activity, enzyme specificity, direct function, site-specific antibodies and peptide substrate-inspired inhibitors.

Homology analyses of the protein sequences of fatty acid synthases from chicken liver, rat mammary gland, and yeast

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

Chang, Soo-Ik; Hammes, G.G.

1989-11-01

Homology analyses of the protein sequences of chicken liver and rat mammary gland fatty acid synthases were carried out. The amino acid sequences of the chicken and rat enzymes are 67% identical. If conservative substitutions are allowed, 78% of the amino acids are matched. A region of low homologies exists between the functional domains, in particular around amino acid residues 1059-1264 of the chicken enzyme. Homologies between the active sites of chicken and rat and of chicken and yeast enzymes have been analyzed by an alignment method. A high degree of homology exists between the active sites of the chickenmore » and rat enzymes. However, the chicken and yeast enzymes show a lower degree of homology. The DADPH-binding dinucleotide folds of the {beta}-ketoacyl reductase and the enoyl reductase sites were identified by comparison with a known consensus sequence for the DADP- and FAD-binding dinucleotide folds. The active sites of all of the enzymes are primarily in hydrophobic regions of the protein. This study suggests that the genes for the functional domains of fatty acid synthase were originally separated, and these genes were connected to each other by using different connecting nucleotide sequences in different species. An alternative explanation for the differences in rat and chicken is a common ancestry and mutations in the joining regions during evolution.« less

Protective Effect of Free and Bound Polyphenol Extracts from Ginger (Zingiber officinale Roscoe) on the Hepatic Antioxidant and Some Carbohydrate Metabolizing Enzymes of Streptozotocin-Induced Diabetic Rats

PubMed Central

Kazeem, Mutiu Idowu; Akanji, Musbau Adewunmi; Yakubu, Musa Toyin; Ashafa, Anofi Omotayo Tom

2013-01-01

This study investigated the hepatoprotective effects of polyphenols from Zingiber officinale on streptozotocin-induced diabetic rats by assessing liver antioxidant enzymes, carbohydrate-metabolizing enzymes and liver function indices. Initial oral glucose tolerance test was conducted using 125 mg/kg, 250 mg/kg, and 500 mg/kg body weight of both free and bound polyphenols from Z. officinale. 28 day daily oral administration of 500 mg/kg body weight of free and bound polyphenols from Z. officinale to streptozotocin-induced (50 mg/kg) diabetic rats significantly reduced (P < 0.05) the fasting blood glucose compared to control groups. There was significant increase (P < 0.05) in the antioxidant enzymes activities in the animals treated with both polyphenols. Similarly, the polyphenols normalised the activities of some carbohydrate metabolic enzymes (hexokinase and phosphofructokinase) in the liver of the rats treated with it and significantly reduced (P < 0.05) the activities of liver function enzymes. The results from the present study have shown that both free and bound polyphenols from Z. officinale especially the free polyphenol could ameliorate liver disorders caused by diabetes mellitus in rats. This further validates the use of this species as medicinal herb and spice by the larger population of Nigerians. PMID:24367390

Protective Effect of Free and Bound Polyphenol Extracts from Ginger (Zingiber officinale Roscoe) on the Hepatic Antioxidant and Some Carbohydrate Metabolizing Enzymes of Streptozotocin-Induced Diabetic Rats.

PubMed

Kazeem, Mutiu Idowu; Akanji, Musbau Adewunmi; Yakubu, Musa Toyin; Ashafa, Anofi Omotayo Tom

2013-01-01

This study investigated the hepatoprotective effects of polyphenols from Zingiber officinale on streptozotocin-induced diabetic rats by assessing liver antioxidant enzymes, carbohydrate-metabolizing enzymes and liver function indices. Initial oral glucose tolerance test was conducted using 125 mg/kg, 250 mg/kg, and 500 mg/kg body weight of both free and bound polyphenols from Z. officinale. 28 day daily oral administration of 500 mg/kg body weight of free and bound polyphenols from Z. officinale to streptozotocin-induced (50 mg/kg) diabetic rats significantly reduced (P < 0.05) the fasting blood glucose compared to control groups. There was significant increase (P < 0.05) in the antioxidant enzymes activities in the animals treated with both polyphenols. Similarly, the polyphenols normalised the activities of some carbohydrate metabolic enzymes (hexokinase and phosphofructokinase) in the liver of the rats treated with it and significantly reduced (P < 0.05) the activities of liver function enzymes. The results from the present study have shown that both free and bound polyphenols from Z. officinale especially the free polyphenol could ameliorate liver disorders caused by diabetes mellitus in rats. This further validates the use of this species as medicinal herb and spice by the larger population of Nigerians.

Increased Diels-Alderase activity through backbone remodeling guided by Foldit players.

PubMed

Eiben, Christopher B; Siegel, Justin B; Bale, Jacob B; Cooper, Seth; Khatib, Firas; Shen, Betty W; Players, Foldit; Stoddard, Barry L; Popovic, Zoran; Baker, David

2012-01-22

Computational enzyme design holds promise for the production of renewable fuels, drugs and chemicals. De novo enzyme design has generated catalysts for several reactions, but with lower catalytic efficiencies than naturally occurring enzymes. Here we report the use of game-driven crowdsourcing to enhance the activity of a computationally designed enzyme through the functional remodeling of its structure. Players of the online game Foldit were challenged to remodel the backbone of a computationally designed bimolecular Diels-Alderase to enable additional interactions with substrates. Several iterations of design and characterization generated a 24-residue helix-turn-helix motif, including a 13-residue insertion, that increased enzyme activity >18-fold. X-ray crystallography showed that the large insertion adopts a helix-turn-helix structure positioned as in the Foldit model. These results demonstrate that human creativity can extend beyond the macroscopic challenges encountered in everyday life to molecular-scale design problems.

In Vitro Characterization and Concerted Function of Three Core Enzymes of a Glycyl Radical Enzyme - Associated Bacterial Microcompartment.

PubMed

Zarzycki, Jan; Sutter, Markus; Cortina, Niña Socorro; Erb, Tobias J; Kerfeld, Cheryl A

2017-02-16

Many bacteria encode proteinaceous bacterial microcompartments (BMCs) that encapsulate sequential enzymatic reactions of diverse metabolic pathways. Well-characterized BMCs include carboxysomes for CO 2 -fixation, and propanediol- and ethanolamine-utilizing microcompartments that contain B 12 -dependent enzymes. Genes required to form BMCs are typically organized in gene clusters, which promoted their distribution across phyla by horizontal gene transfer. Recently, BMCs associated with glycyl radical enzymes (GREs) were discovered; these are widespread and comprise at least three functionally distinct types. Previously, we predicted one type of these GRE-associated microcompartments (GRMs) represents a B 12 -independent propanediol-utilizing BMC. Here we functionally and structurally characterize enzymes of the GRM of Rhodopseudomonas palustris BisB18 and demonstrate their concerted function in vitro. The GRM signature enzyme, the GRE, is a dedicated 1,2-propanediol dehydratase with a new type of intramolecular encapsulation peptide. It forms a complex with its activating enzyme and, in conjunction with an aldehyde dehydrogenase, converts 1,2-propanediol to propionyl-CoA. Notably, homologous GRMs are also encoded in pathogenic Escherichia coli strains. Our high-resolution crystal structures of the aldehyde dehydrogenase lead to a revised reaction mechanism. The successful in vitro reconstitution of a part of the GRM metabolism provides insights into the metabolic function and steps in the assembly of this BMC.

In Vitro Characterization and Concerted Function of Three Core Enzymes of a Glycyl Radical Enzyme - Associated Bacterial Microcompartment

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

Zarzycki, Jan; Sutter, Markus; Cortina, Niña Socorro

Many bacteria encode proteinaceous bacterial microcompartments (BMCs) that encapsulate sequential enzymatic reactions of diverse metabolic pathways. Well-characterized BMCs include carboxysomes for CO 2-fixation, and propanediol- and ethanolamine-utilizing microcompartments that contain B 12-dependent enzymes. Genes thus required to form BMCs are typically organized in gene clusters, which promoted their distribution across phyla by horizontal gene transfer. Recently, BMCs associated with glycyl radical enzymes (GREs) were discovered; these are widespread and comprise at least three functionally distinct types. Previously, we predicted one type of these GRE-associated microcompartments (GRMs) represents a B 12-independent propanediol-utilizing BMC. We functionally and structurally characterize enzymes of themore » GRM of Rhodopseudomonas palustris BisB18 and demonstrate their concerted function in vitro. The GRM signature enzyme, the GRE, is a dedicated 1,2-propanediol dehydratase with a new type of intramolecular encapsulation peptide. It forms a complex with its activating enzyme and, in conjunction with an aldehyde dehydrogenase, converts 1,2-propanediol to propionyl-CoA. Notably, homologous GRMs are also encoded in pathogenic Escherichia coli strains. Our high-resolution crystal structures of the aldehyde dehydrogenase lead to a revised reaction mechanism. The successful in vitro reconstitution of a part of the GRM metabolism provides insights into the metabolic function and steps in the assembly of this BMC.« less

In Vitro Characterization and Concerted Function of Three Core Enzymes of a Glycyl Radical Enzyme - Associated Bacterial Microcompartment

DOE PAGES

Zarzycki, Jan; Sutter, Markus; Cortina, Niña Socorro; ...

2017-02-16

Many bacteria encode proteinaceous bacterial microcompartments (BMCs) that encapsulate sequential enzymatic reactions of diverse metabolic pathways. Well-characterized BMCs include carboxysomes for CO 2-fixation, and propanediol- and ethanolamine-utilizing microcompartments that contain B 12-dependent enzymes. Genes thus required to form BMCs are typically organized in gene clusters, which promoted their distribution across phyla by horizontal gene transfer. Recently, BMCs associated with glycyl radical enzymes (GREs) were discovered; these are widespread and comprise at least three functionally distinct types. Previously, we predicted one type of these GRE-associated microcompartments (GRMs) represents a B 12-independent propanediol-utilizing BMC. We functionally and structurally characterize enzymes of themore » GRM of Rhodopseudomonas palustris BisB18 and demonstrate their concerted function in vitro. The GRM signature enzyme, the GRE, is a dedicated 1,2-propanediol dehydratase with a new type of intramolecular encapsulation peptide. It forms a complex with its activating enzyme and, in conjunction with an aldehyde dehydrogenase, converts 1,2-propanediol to propionyl-CoA. Notably, homologous GRMs are also encoded in pathogenic Escherichia coli strains. Our high-resolution crystal structures of the aldehyde dehydrogenase lead to a revised reaction mechanism. The successful in vitro reconstitution of a part of the GRM metabolism provides insights into the metabolic function and steps in the assembly of this BMC.« less

Bioinformatic Characterization of Glycyl Radical Enzyme-Associated Bacterial Microcompartments

PubMed Central

Zarzycki, Jan; Erbilgin, Onur

2015-01-01

Bacterial microcompartments (BMCs) are proteinaceous organelles encapsulating enzymes that catalyze sequential reactions of metabolic pathways. BMCs are phylogenetically widespread; however, only a few BMCs have been experimentally characterized. Among them are the carboxysomes and the propanediol- and ethanolamine-utilizing microcompartments, which play diverse metabolic and ecological roles. The substrate of a BMC is defined by its signature enzyme. In catabolic BMCs, this enzyme typically generates an aldehyde. Recently, it was shown that the most prevalent signature enzymes encoded by BMC loci are glycyl radical enzymes, yet little is known about the function of these BMCs. Here we characterize the glycyl radical enzyme-associated microcompartment (GRM) loci using a combination of bioinformatic analyses and active-site and structural modeling to show that the GRMs comprise five subtypes. We predict distinct functions for the GRMs, including the degradation of choline, propanediol, and fuculose phosphate. This is the first family of BMCs for which identification of the signature enzyme is insufficient for predicting function. The distinct GRM functions are also reflected in differences in shell composition and apparently different assembly pathways. The GRMs are the counterparts of the vitamin B12-dependent propanediol- and ethanolamine-utilizing BMCs, which are frequently associated with virulence. This study provides a comprehensive foundation for experimental investigations of the diverse roles of GRMs. Understanding this plasticity of function within a single BMC family, including characterization of differences in permeability and assembly, can inform approaches to BMC bioengineering and the design of therapeutics. PMID:26407889

Measuring specificity in multi-substrate/product systems as a tool to investigate selectivity in vivo.

PubMed

Kuo, Yin-Ming; Henry, Ryan A; Andrews, Andrew J

2016-01-01

Multiple substrate enzymes present a particular challenge when it comes to understanding their activity in a complex system. Although a single target may be easy to model, it does not always present an accurate representation of what that enzyme will do in the presence of multiple substrates simultaneously. Therefore, there is a need to find better ways to both study these enzymes in complicated systems, as well as accurately describe the interactions through kinetic parameters. This review looks at different methods for studying multiple substrate enzymes, as well as explores options on how to most accurately describe an enzyme's activity within these multi-substrate systems. Identifying and defining this enzymatic activity should help clear the way to using in vitro systems to accurately predicting the behavior of multi-substrate enzymes in vivo. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. Copyright © 2015. Published by Elsevier B.V.

Cloning of the Arabidopsis and Rice Formaldehyde Dehydrogenase Genes: Implications for the Origin of Plant Adh Enzymes

PubMed Central

Dolferus, R.; Osterman, J. C.; Peacock, W. J.; Dennis, E. S.

1997-01-01

This article reports the cloning of the genes encoding the Arabidopsis and rice class III ADH enzymes, members of the alcohol dehydrogenase or medium chain reductase/dehydrogenase superfamily of proteins with glutathione-dependent formaldehyde dehydrogenase activity (GSH-FDH). Both genes contain eight introns in exactly the same positions, and these positions are conserved in plant ethanol-active Adh genes (class P). These data provide further evidence that plant class P genes have evolved from class III genes by gene duplication and acquisition of new substrate specificities. The position of introns and similarities in the nucleic acid and amino acid sequences of the different classes of ADH enzymes in plants and humans suggest that plant and animal class III enzymes diverged before they duplicated to give rise to plant and animal ethanol-active ADH enzymes. Plant class P ADH enzymes have gained substrate specificities and evolved promoters with different expression properties, in keeping with their metabolic function as part of the alcohol fermentation pathway. PMID:9215914

Mechanisms, biology and inhibitors of deubiquitinating enzymes.

PubMed

Love, Kerry Routenberg; Catic, André; Schlieker, Christian; Ploegh, Hidde L

2007-11-01

The addition of ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers to proteins serves to modulate function and is a key step in protein degradation, epigenetic modification and intracellular localization. Deubiquitinating enzymes and Ubl-specific proteases, the proteins responsible for the removal of Ub and Ubls, act as an additional level of control over the ubiquitin-proteasome system. Their conservation and widespread occurrence in eukaryotes, prokaryotes and viruses shows that these proteases constitute an essential class of enzymes. Here, we discuss how chemical tools, including activity-based probes and suicide inhibitors, have enabled (i) discovery of deubiquitinating enzymes, (ii) their functional profiling, crystallographic characterization and mechanistic classification and (iii) development of molecules for therapeutic purposes.

Protein Tyrosine Phosphatases: From Housekeeping Enzymes to Master-Regulators of Signal Transduction

PubMed Central

Tonks, Nicholas K.

2013-01-01

There are many misconceptions surrounding the roles of protein phosphatases in the regulation of signal transduction, perhaps the most damaging of which is the erroneous view that these enzymes exert their effects merely as constitutively active housekeeping enzymes. On the contrary, the phosphatases are critical, specific regulators of signaling in their own right and serve an essential function, in a coordinated manner with the kinases, to determine the response to a physiological stimulus. This review is a personal perspective on the development of our understanding of the protein tyrosine phosphatase (PTP) family of enzymes. I have discussed various aspects of the structure, regulation and function of the PTP family, which I hope will illustrate the fundamental importance of these enzymes to the control of signal transduction. PMID:23176256

Relief of autoinhibition by conformational switch explains enzyme activation by a catalytically dead paralog

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

Volkov, Oleg A.; Kinch, Lisa; Ariagno, Carson

Catalytically inactive enzyme paralogs occur in many genomes. Some regulate their active counterparts but the structural principles of this regulation remain largely unknown. We report X-ray structures ofTrypanosoma brucei S-adenosylmethionine decarboxylase alone and in functional complex with its catalytically dead paralogous partner, prozyme. We show monomericTbAdoMetDC is inactive because of autoinhibition by its N-terminal sequence. Heterodimerization with prozyme displaces this sequence from the active site through a complex mechanism involving acis-to-transproline isomerization, reorganization of a β-sheet, and insertion of the N-terminal α-helix into the heterodimer interface, leading to enzyme activation. We propose that the evolution of this intricate regulatory mechanismmore » was facilitated by the acquisition of the dimerization domain, a single step that can in principle account for the divergence of regulatory schemes in the AdoMetDC enzyme family. These studies elucidate an allosteric mechanism in an enzyme and a plausible scheme by which such complex cooperativity evolved.« less

The interdigitating loop of the enolase superfamily as a specificity binding determinant or 'flying buttress'.

PubMed

Bearne, Stephen L

2017-05-01

Enzymes of the enolase superfamily (ENS) are mechanistically diverse, yet share a common partial reaction (abstraction of the α-proton from a carboxylate substrate). While the catalytic machinery responsible for the deprotonation reaction has been conserved, divergent evolution has led to numerous ENS members that catalyze different overall reactions. This rich functional diversity has made the ENS an excellent model system for developing the approaches necessary to validate enzyme function. However, enzymes of the ENS also share a common bidomain structure ((β/α) 7 β-barrel domain and α+β capping domain) which makes validation of function from structural information challenging. This review presents a comparative survey of the structural data obtained over the past decade for enzymes from all seven subgroups that comprise the ENS. Of the seven ENS subgroups (enolase, mandelate racemase (MR), muconate lactonizing enzyme, β-methylaspartate ammonia lyase, d-glucarate dehydratase, d-mannonate dehydratase (ManD), and galactarate dehydratase 2), only enzymes of the MR and ManD subgroups exhibit an additional feature of structural complexity-an interdigitating loop. This loop emanates from one protomer of a homodimeric pair and penetrates into the adjacent, symmetry-related protomer to either contribute a binding determinant to the active site of the adjacent protomer, or act as a 'flying buttress' to support residues of the active site. The analysis presented in this review suggests that the interdigitating loop is the only gross structural element that permits functional distinction between ENS subgroups at the tertiary level of protein structure. Copyright © 2017 Elsevier B.V. All rights reserved.

Biochemical characterization of rice trehalose-6-phosphate phosphatases supports distinctive functions of these plant enzymes.

PubMed

Shima, Shuhei; Matsui, Hirokazu; Tahara, Satoshi; Imai, Ryozo

2007-03-01

Substantial levels of trehalose accumulate in bacteria, fungi, and invertebrates, where it serves as a storage carbohydrate or as a protectant against environmental stresses. In higher plants, trehalose is detected at fairly low levels; therefore, a regulatory or signaling function has been proposed for this molecule. In many organisms, trehalose-6-phosphate phosphatase is the enzyme governing the final step of trehalose biosynthesis. Here we report that OsTPP1 and OsTPP2 are the two major trehalose-6-phosphate phosphatase genes expressed in vegetative tissues of rice. Similar to results obtained from our previous OsTPP1 study, complementation analysis of a yeast trehalose-6-phosphate phosphatase mutant and activity measurement of the recombinant protein demonstrated that OsTPP2 encodes a functional trehalose-6-phosphate phosphatase enzyme. OsTPP2 expression is transiently induced in response to chilling and other abiotic stresses. Enzymatic characterization of recombinant OsTPP1 and OsTPP2 revealed stringent substrate specificity for trehalose 6-phosphate and about 10 times lower K(m) values for trehalose 6-phosphate as compared with trehalose-6-phosphate phosphatase enzymes from microorganisms. OsTPP1 and OsTPP2 also clearly contrasted with microbial enzymes, in that they are generally unstable, almost completely losing activity when subjected to heat treatment at 50 degrees C for 4 min. These characteristics of rice trehalose-6-phosphate phosphatase enzymes are consistent with very low cellular substrate concentration and tightly regulated gene expression. These data also support a plant-specific function of trehalose biosynthesis in response to environmental stresses.

Interaction of angiotensin-converting enzyme (ACE) with membrane-bound carboxypeptidase M (CPM) - a new function of ACE.

PubMed

Sun, Xiaoou; Wiesner, Burkhard; Lorenz, Dorothea; Papsdorf, Gisela; Pankow, Kristin; Wang, Po; Dietrich, Nils; Siems, Wolf-Eberhard; Maul, Björn

2008-12-01

Angiotensin-converting enzyme (ACE) demonstrates, besides its typical dipeptidyl-carboxypeptidase activity, several unusual functions. Here, we demonstrate with molecular, biochemical, and cellular techniques that the somatic wild-type murine ACE (mACE), stably transfected in Chinese Hamster Ovary (CHO) or Madin-Darby Canine Kidney (MDCK) cells, interacts with endogenous membranal co-localized carboxypeptidase M (CPM). CPM belongs to the group of glycosylphosphatidylinositol (GPI)-anchored proteins. Here we report that ACE, completely independent of its known dipeptidase activities, has GPI-targeted properties. Our results indicate that the spatial proximity between mACE and the endogenous CPM enables an ACE-evoked release of CPM. These results are discussed with respect to the recently proposed GPI-ase activity and function of sperm-bound ACE.

Angiotensin-I Converting Enzyme (ACE) Inhibitory and Anti-Oxidant Activities of Sea Cucumber (Actinopyga lecanora) Hydrolysates

PubMed Central

Ghanbari, Raheleh; Zarei, Mohammad; Ebrahimpour, Afshin; Abdul-Hamid, Azizah; Ismail, Amin; Saari, Nazamid

2015-01-01

In recent years, food protein-derived hydrolysates have received considerable attention because of their numerous health benefits. Amongst the hydrolysates, those with anti-hypertensive and anti-oxidative activities are receiving special attention as both activities can play significant roles in preventing cardiovascular diseases. The present study investigated the angiotensin-I converting enzyme (ACE) inhibitory and anti-oxidative activities of Actinopyga lecanora (A. lecanora) hydrolysates, which had been prepared by alcalase, papain, bromelain, flavourzyme, pepsin, and trypsin under their optimum conditions. The alcalase hydrolysate showed the highest ACE inhibitory activity (69.8%) after 8 h of hydrolysis while the highest anti-oxidative activities measured by 2,2-diphenyl 1-1-picrylhydrazyl radical scavenging (DPPH) (56.00%) and ferrous ion-chelating (FIC) (59.00%) methods were exhibited after 24 h and 8 h of hydrolysis, respectively. The ACE-inhibitory and anti-oxidative activities displayed dose-dependent trends, and increased with increasing protein hydrolysate concentrations. Moreover, strong positive correlations between angiotensin-I converting enzyme (ACE) inhibitory and anti-oxidative activities were also observed. This study indicates that A. lecanora hydrolysate can be exploited as a source of functional food owing to its anti-oxidant as well as anti-hypertension functions. PMID:26690117

Amy63, a novel type of marine bacterial multifunctional enzyme possessing amylase, agarase and carrageenase activities

PubMed Central

Liu, Ge; Wu, Shimei; Jin, Weihua; Sun, Chaomin

2016-01-01

A multifunctional enzyme is one that performs multiple physiological functions, thus benefiting the organism. Characterization of multifunctional enzymes is important for researchers to understand how organisms adapt to different environmental challenges. In the present study, we report the discovery of a novel multifunctional enzyme Amy63 produced by marine bacterium Vibrio alginolyticus 63. Remarkably, Amy63 possesses amylase, agarase and carrageenase activities. Amy63 is a substrate promiscuous α-amylase, with the substrate priority order of starch, carrageenan and agar. Amy63 maintains considerable amylase, carrageenase and agarase activities and stabilities at wide temperature and pH ranges, and optimum activities are detected at temperature of 60 °C and pH of 6.0, respectively. Moreover, the heteroexpression of Amy63 dramatically enhances the ability of E. coli to degrade starch, carrageenan and agar. Motif searching shows three continuous glycosyl hydrolase 70 (GH70) family homologs existed in Amy63 encoding sequence. Combining serial deletions and phylogenetic analysis of Amy63, the GH70 homologs are proposed as the determinants of enzyme promiscuity. Notably, such enzymes exist in all kingdoms of life, thus providing an expanded perspective on studies of multifunctional enzymes. To our knowledge, this is the first report of an amylase having additional agarase and carrageenase activities. PMID:26725302

Ligand-binding specificity and promiscuity of the main lignocellulolytic enzyme families as revealed by active-site architecture analysis.

PubMed

Tian, Li; Liu, Shijia; Wang, Shuai; Wang, Lushan

2016-03-24

Biomass can be converted into sugars by a series of lignocellulolytic enzymes, which belong to the glycoside hydrolase (GH) families summarized in CAZy databases. Here, using a structural bioinformatics method, we analyzed the active site architecture of the main lignocellulolytic enzyme families. The aromatic amino acids Trp/Tyr and polar amino acids Glu/Asp/Asn/Gln/Arg occurred at higher frequencies in the active site architecture than in the whole enzyme structure. And the number of potential subsites was significantly different among different families. In the cellulase and xylanase families, the conserved amino acids in the active site architecture were mostly found at the -2 to +1 subsites, while in β-glucosidase they were mainly concentrated at the -1 subsite. Families with more conserved binding amino acid residues displayed strong selectivity for their ligands, while those with fewer conserved binding amino acid residues often exhibited promiscuity when recognizing ligands. Enzymes with different activities also tended to bind different hydroxyl oxygen atoms on the ligand. These results may help us to better understand the common and unique structural bases of enzyme-ligand recognition from different families and provide a theoretical basis for the functional evolution and rational design of major lignocellulolytic enzymes.

Mechanistic insight into the functional transition of the enzyme guanylate kinase induced by a single mutation

PubMed Central

Zhang, Yuebin; Niu, Huiyan; Li, Yan; Chu, Huiying; Shen, Hujun; Zhang, Dinglin; Li, Guohui

2015-01-01

Dramatic functional changes of enzyme usually require scores of alterations in amino acid sequence. However, in the case of guanylate kinase (GK), the functional novelty is induced by a single (S→P) mutation, leading to the functional transition of the enzyme from a phosphoryl transfer kinase into a phosphorprotein interaction domain. Here, by using molecular dynamic (MD) and metadynamics simulations, we provide a comprehensive description of the conformational transitions of the enzyme after mutating serine to proline. Our results suggest that the serine plays a crucial role in maintaining the closed conformation of wild-type GK and the GMP recognition. On the contrary, the S→P mutant exhibits a stable open conformation and loses the ability of ligand binding, which explains its functional transition from the GK enzyme to the GK domain. Furthermore, the free energy profiles (FEPs) obtained by metadymanics clearly demonstrate that the open-closed conformational transition in WT GK is positive correlated with the process of GMP binding, indicating the GMP-induced closing motion of GK enzyme, which is not observed in the mutant. In addition, the FEPs show that the S→P mutation can also leads to the mis-recognition of GMP, explaining the vanishing of catalytic activity of the mutant. PMID:25672880

Mechanistic insight into the functional transition of the enzyme guanylate kinase induced by a single mutation.

PubMed

Zhang, Yuebin; Niu, Huiyan; Li, Yan; Chu, Huiying; Shen, Hujun; Zhang, Dinglin; Li, Guohui

2015-02-12

Dramatic functional changes of enzyme usually require scores of alterations in amino acid sequence. However, in the case of guanylate kinase (GK), the functional novelty is induced by a single (S→P) mutation, leading to the functional transition of the enzyme from a phosphoryl transfer kinase into a phosphorprotein interaction domain. Here, by using molecular dynamic (MD) and metadynamics simulations, we provide a comprehensive description of the conformational transitions of the enzyme after mutating serine to proline. Our results suggest that the serine plays a crucial role in maintaining the closed conformation of wild-type GK and the GMP recognition. On the contrary, the S→P mutant exhibits a stable open conformation and loses the ability of ligand binding, which explains its functional transition from the GK enzyme to the GK domain. Furthermore, the free energy profiles (FEPs) obtained by metadymanics clearly demonstrate that the open-closed conformational transition in WT GK is positive correlated with the process of GMP binding, indicating the GMP-induced closing motion of GK enzyme, which is not observed in the mutant. In addition, the FEPs show that the S→P mutation can also leads to the mis-recognition of GMP, explaining the vanishing of catalytic activity of the mutant.

From protein engineering to immobilization: promising strategies for the upgrade of industrial enzymes.

PubMed

Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

2013-01-10

Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes.

Refined molecular hinge between allosteric and catalytic domain determines allosteric regulation and stability of fungal chorismate mutase

PubMed Central

Helmstaedt, Kerstin; Heinrich, Gabriele; Lipscomb, William N.; Braus, Gerhard H.

2002-01-01

The yeast chorismate mutase is regulated by tyrosine as feedback inhibitor and tryptophan as crosspathway activator. The monomer consists of a catalytic and a regulatory domain covalently linked by the loop L220s (212–226), which functions as a molecular hinge. Two monomers form the active dimeric enzyme stabilized by hydrophobic interactions in the vicinity of loop L220s. The role of loop L220s and its environment for enzyme regulation, dimerization, and stability was analyzed. Substitution of yeast loop L220s in place of the homologous loop from the corresponding and similarly regulated Aspergillus enzyme (and the reverse substitution) changed tyrosine inhibition to activation. Yeast loop L220s substituted into the Aspergillus enzyme resulted in a tryptophan-inhibitable enzyme. Monomeric yeast chorismate mutases could be generated by substituting two hydrophobic residues in and near the hinge region. The resulting Thr-212→Asp–Phe-28→Asp enzyme was as stable as wild type, but lost allosteric regulation and showed reduced catalytic activity. These results underline the crucial role of this molecular hinge for inhibition, activation, quaternary structure, and stability of yeast chorismate mutase. PMID:11997452

Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes

PubMed Central

Fischbach, Michael A.; Lai, Jonathan R.; Roche, Eric D.; Walsh, Christopher T.; Liu, David R.

2007-01-01

Nonribosomal peptides (NRPs) are produced by NRP synthetase (NRPS) enzymes that function as molecular assembly lines. The modular architecture of NRPSs suggests that a domain responsible for activating a building block could be replaced with a domain from a foreign NRPS to create a chimeric assembly line that produces a new variant of a natural NRP. However, such chimeric NRPS modules are often heavily impaired, impeding efforts to create novel NRP variants by swapping domains from different modules or organisms. Here we show that impaired chimeric NRPSs can be functionally restored by directed evolution. Using rounds of mutagenesis coupled with in vivo screens for NRP production, we rapidly isolated variants of two different chimeric NRPSs with ≈10-fold improvements in enzyme activity and product yield, including one that produces new derivatives of the potent NRP/polyketide antibiotic andrimid. Because functional restoration in these examples required only modest library sizes (103 to 104 clones) and three or fewer rounds of screening, our approach may be widely applicable even for NRPSs from genetically challenging hosts. PMID:17620609

Multiplexed Activity-based Protein Profiling of the Human Pathogen Aspergillus fumigatus Reveals Large Functional Changes upon Exposure to Human Serum*

PubMed Central

Wiedner, Susan D.; Burnum, Kristin E.; Pederson, LeeAnna M.; Anderson, Lindsey N.; Fortuin, Suereta; Chauvigné-Hines, Lacie M.; Shukla, Anil K.; Ansong, Charles; Panisko, Ellen A.; Smith, Richard D.; Wright, Aaron T.

2012-01-01

Environmental adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised host lung. We hypothesized that exposure of the fungal pathogen to human serum would lead to significant alterations to the organism's physiology, including metabolic activity and stress response. Shifts in functional pathway and corresponding enzyme reactivity of A. fumigatus upon exposure to the human host may represent much needed prognostic indicators of fungal infection. To address this, we employed a multiplexed activity-based protein profiling (ABPP) approach coupled to quantitative mass spectrometry-based proteomics to measure broad enzyme reactivity of the fungus cultured with and without human serum. ABPP showed a shift from aerobic respiration to ethanol fermentation and utilization over time in the presence of human serum, which was not observed in serum-free culture. Our approach provides direct insight into this pathogen's ability to survive, adapt, and proliferate. Additionally, our multiplexed ABPP approach captured a broad swath of enzyme reactivity and functional pathways and provides a method for rapid assessment of the A. fumigatus response to external stimuli. PMID:22865858

Multiplexed activity-based protein profiling of the human pathogen Aspergillus fumigatus reveals large functional changes upon exposure to human serum.

PubMed

Wiedner, Susan D; Burnum, Kristin E; Pederson, LeeAnna M; Anderson, Lindsey N; Fortuin, Suereta; Chauvigné-Hines, Lacie M; Shukla, Anil K; Ansong, Charles; Panisko, Ellen A; Smith, Richard D; Wright, Aaron T

2012-09-28

Environmental adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised host lung. We hypothesized that exposure of the fungal pathogen to human serum would lead to significant alterations to the organism's physiology, including metabolic activity and stress response. Shifts in functional pathway and corresponding enzyme reactivity of A. fumigatus upon exposure to the human host may represent much needed prognostic indicators of fungal infection. To address this, we employed a multiplexed activity-based protein profiling (ABPP) approach coupled to quantitative mass spectrometry-based proteomics to measure broad enzyme reactivity of the fungus cultured with and without human serum. ABPP showed a shift from aerobic respiration to ethanol fermentation and utilization over time in the presence of human serum, which was not observed in serum-free culture. Our approach provides direct insight into this pathogen's ability to survive, adapt, and proliferate. Additionally, our multiplexed ABPP approach captured a broad swath of enzyme reactivity and functional pathways and provides a method for rapid assessment of the A. fumigatus response to external stimuli.

An active second dihydrofolate reductase enzyme is not a feature of rat and mouse, but they do have activity in their mitochondria.

PubMed

Hughes, Linda; Carton, Robert; Minguzzi, Stefano; McEntee, Gráinne; Deinum, Eva E; O'Connell, Mary J; Parle-McDermott, Anne

2015-07-08

The identification of a second functional dihydrofolate reductase enzyme in humans, DHFRL1, led us to consider whether this is also a feature of rodents. We demonstrate that dihydrofolate reductase activity is also a feature of the mitochondria in both rat and mouse but this is not due to a second enzyme. While our phylogenetic analysis revealed that RNA-mediated DHFR duplication events did occur across the mammal tree, the duplicates in brown rat and mouse are likely to be processed pseudogenes. Humans have evolved the need for two separate enzymes while laboratory rats and mice have just one. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Regio- and stereodivergent antibiotic oxidative carbocyclizations catalysed by Rieske oxygenase-like enzymes

NASA Astrophysics Data System (ADS)

Sydor, Paulina K.; Barry, Sarah M.; Odulate, Olanipekun M.; Barona-Gomez, Francisco; Haynes, Stuart W.; Corre, Christophe; Song, Lijiang; Challis, Gregory L.

2011-05-01

Oxidative cyclizations, exemplified by the biosynthetic assembly of the penicillin nucleus from a tripeptide precursor, are arguably the most synthetically powerful implementation of C-H activation reactions in nature. Here, we show that Rieske oxygenase-like enzymes mediate regio- and stereodivergent oxidative cyclizations to form 10- and 12-membered carbocyclic rings in the key steps of the biosynthesis of the antibiotics streptorubin B and metacycloprodigiosin, respectively. These reactions represent the first examples of oxidative carbocyclizations catalysed by non-haem iron-dependent oxidases and define a novel type of catalytic activity for Rieske enzymes. A better understanding of how these enzymes achieve such remarkable regio- and stereocontrol in the functionalization of unactivated hydrocarbon chains will greatly facilitate the development of selective man-made C-H activation catalysts.

Role of Disulfide Bridges in the Activity and Stability of a Cold-Active α-Amylase

PubMed Central

Siddiqui, Khawar Sohail; Poljak, Anne; Guilhaus, Michael; Feller, Georges; D'Amico, Salvino; Gerday, Charles; Cavicchioli, Ricardo

2005-01-01

The cold-adapted α-amylase from Pseudoalteromonas haloplanktis unfolds reversibly and cooperatively according to a two-state mechanism at 30°C and unfolds reversibly and sequentially with two transitions at temperatures below 12°C. To examine the role of the four disulfide bridges in activity and conformational stability of the enzyme, the eight cysteine residues were reduced with β-mercaptoethanol or chemically modified using iodoacetamide or iodoacetic acid. Matrix-assisted laser desorption-time of flight mass spectrometry analysis confirmed that all of the cysteines were modified. The iodoacetamide-modified enzyme reversibly folded/unfolded and retained approximately one-third of its activity. Removal of all disulfide bonds resulted in stabilization of the least stable region of the enzyme (including the active site), with a concomitant decrease in activity (increase in activation enthalpy). Disulfide bond removal had a greater impact on enzyme activity than on stability (particularly the active-site region). The functional role of the disulfide bridges appears to be to prevent the active site from developing ionic interactions. Overall, the study demonstrated that none of the four disulfide bonds are important in stabilizing the native structure of enzyme, and instead, they appear to promote a localized destabilization to preserve activity. PMID:16109962

Ectomycorrhizal fungi enhance nitrogen and phosphorus nutrition of Nothofagus dombeyi under drought conditions by regulating assimilative enzyme activities.

PubMed

Alvarez, Maricel; Huygens, Dries; Olivares, Erick; Saavedra, Isabel; Alberdi, Miren; Valenzuela, Eduardo

2009-08-01

Drought stress conditions (DC) reduce plant growth and nutrition, restraining the sustainable reestablishment of Nothofagus dombeyi in temperate south Chilean forest ecosystems. Ectomycorrhizal symbioses have been documented to enhance plant nitrogen (N) and phosphorus (P) uptake under drought, but the regulation of involved assimilative enzymes remains unclear. We studied 1-year-old N. dombeyi (Mirb.) Oerst. plants in association with the ectomycorrhizal fungi Pisolithus tinctorius (Pers.) Coker & Couch. and Descolea antartica Sing. In greenhouse experiments, shoot and root dry weights, mycorrhizal colonization, foliar N and P concentrations, and root enzyme activities [glutamate synthase (glutamine oxoglutarate aminotransferase (GOGAT), EC 1.4.1.13-14), glutamine synthetase (GS, EC 6.3.1.2), glutamate dehydrogenase (GDH, EC 1.4.1.2-4), nitrate reductase (NR, EC 1.6.6.1), and acid phosphomonoesterase (PME, EC 3.1.3.1-2)] were determined as a function of soil-water content. Inoculation of N. dombeyi with P. tinctorius and D. antartica significantly stimulated plant growth and increased plant foliar N and P concentrations, especially under DC. Ectomycorrhizal inoculation increased the activity of all studied enzymes relative to non-mycorrhizal plants under drought. We speculate that GDH is a key enzyme involved in the enhancement of ectomycorrhizal carbon (C) availability by fuelling the tricarboxylic acid (TCA) cycle under conditions of drought-induced carbon deficit. All studied assimilative enzymes of the ectomycorrhizal associations, involved in C, N, and P transfers, are closely interlinked and interdependent. The up-regulation of assimilative enzyme activities by ectomycorrhizal fungal root colonizers acts as a functional mechanism to increase seedling endurance to drought. We insist upon incorporating ectomycorrhizal inoculation in existing Chilean afforestation programs.

The Enzyme Function Initiative†

PubMed Central

Gerlt, John A.; Allen, Karen N.; Almo, Steven C.; Armstrong, Richard N.; Babbitt, Patricia C.; Cronan, John E.; Dunaway-Mariano, Debra; Imker, Heidi J.; Jacobson, Matthew P.; Minor, Wladek; Poulter, C. Dale; Raushel, Frank M.; Sali, Andrej; Shoichet, Brian K.; Sweedler, Jonathan V.

2011-01-01

The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily-specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include: 1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation); 2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia; 3) computational and bioinformatic tools for using the strategy; 4) provision of experimental protocols and/or reagents for enzyme production and characterization; and 5) dissemination of data via the EFI’s website, enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal and pharmaceutical efforts. PMID:21999478

The Enzyme Function Initiative.

PubMed

Gerlt, John A; Allen, Karen N; Almo, Steven C; Armstrong, Richard N; Babbitt, Patricia C; Cronan, John E; Dunaway-Mariano, Debra; Imker, Heidi J; Jacobson, Matthew P; Minor, Wladek; Poulter, C Dale; Raushel, Frank M; Sali, Andrej; Shoichet, Brian K; Sweedler, Jonathan V

2011-11-22

The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic, we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include (1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation), (2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia, (3) computational and bioinformatic tools for using the strategy, (4) provision of experimental protocols and/or reagents for enzyme production and characterization, and (5) dissemination of data via the EFI's Website, http://enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal, and pharmaceutical efforts. © 2011 American Chemical Society

Rational approaches for engineering novel functionalities in carbon-carbon bond forming enzymes

PubMed Central

Baker, Perrin; Seah, Stephen Y. K.

2012-01-01

Enzymes that catalyze carbon-carbon bond formation can be exploited as biocatalyst for synthetic organic chemistry. However, natural enzymes frequently do not possess the required properties or specificities to catalyze industrially useful transformations. This mini-review describes recent work using knowledge-guided site-specific mutagenesis of key active site residues to alter substrate specificity, stereospecificity and reaction specificity of these enzymes. In addition, examples of de novo designed enzymes that catalyze C-C bond reactions not found in nature will be discussed. PMID:24688644

High yield of functional metagenomic library from mangroves constructed in fosmid vector.

PubMed

Gonçalves, A C S; dos Santos, A C F; dos Santos, T F; Pessoa, T B A; Dias, J C T; Rezende, R P

2015-10-02

In the present study, metagenomic technique and fosmid vectors were used to construct a library of clones for exploring the biotechnological potential of mangrove soils by isolation of functional genes encoding hydrolytic enzymes. The library was built with genomic DNA from the soil samples of mangrove sediments and the functional screening of 1824 clones (~64 Mbp) was performed to detect the hydrolytic activity specific for cellulases, amylases (at acidic, neutral and basic pH), lipases/esterases, proteases, and nitrilases. Significant numbers of clones, positive for the tested enzyme activities were obtained. Our results indicate the importance and biotechnological potential of mangrove soils especially when compared to those obtained using other soil metagenomic libraries.

Structural Determinants of an Insect β-N-Acetyl-d-hexosaminidase Specialized as a Chitinolytic Enzyme*

PubMed Central

Liu, Tian; Zhang, Haitao; Liu, Fengyi; Wu, Qingyue; Shen, Xu; Yang, Qing

2011-01-01

β-N-Acetyl-d-hexosaminidase has been postulated to have a specialized function. However, the structural basis of this specialization is not yet established. OfHex1, the enzyme from the Asian corn borer Ostrinia furnacalis (one of the most destructive pests) has previously been reported to function merely in chitin degradation. Here the vital role of OfHex1 during the pupation of O. furnacalis was revealed by RNA interference, and the crystal structures of OfHex1 and OfHex1 complexed with TMG-chitotriomycin were determined at 2.1 Å. The mechanism of selective inhibition by TMG-chitotriomycin was related to the existence of the +1 subsite at the active pocket of OfHex1 and a key residue, Trp490, at this site. Mutation of Trp490 to Ala led to a 2,277-fold decrease in sensitivity toward TMG-chitotriomycin as well as an 18-fold decrease in binding affinity for the substrate (GlcNAc)2. Although the overall topology of the catalytic domain of OfHex1 shows a high similarity with the human and bacterial enzymes, OfHex1 is distinguished from these enzymes by large conformational changes linked to an “open-close” mechanism at the entrance of the active site, which is characterized by the “lid” residue, Trp448. Mutation of Trp448 to Ala or Phe resulted in a more than 1,000-fold loss in enzyme activity, due mainly to the effect on kcat. The current work has increased our understanding of the structure-function relationship of OfHex1, shedding light on the structural basis that accounts for the specialized function of β-N-acetyl-d-hexosaminidase as well as making the development of species-specific pesticides a likely reality. PMID:21106526

Deletion of the Thyroid Hormone-Activating Type 2 Deiodinase Rescues Cone Photoreceptor Degeneration but Not Deafness in Mice Lacking Type 3 Deiodinase.

PubMed

Ng, Lily; Liu, Hong; St Germain, Donald L; Hernandez, Arturo; Forrest, Douglas

2017-06-01

Type 2 deiodinase amplifies and type 3 deiodinase depletes levels of the active form of thyroid hormone, triiodothyronine. Given the opposing activities of these enzymes, we tested the hypothesis that they counteract each other's developmental functions by investigating whether deletion of type 2 deiodinase (encoded by Dio2) modifies sensory phenotypes in type 3 deiodinase-deficient (Dio3-/-) mice. Dio3-/- mice display degeneration of retinal cones, the photoreceptors that mediate daylight and color vision. In Dio2-/- mice, cone function was largely normal but deletion of Dio2 in Dio3-/- mice markedly recovered cone numbers and electroretinogram responses, suggesting counterbalancing roles for both enzymes in cone survival. Both Dio3-/- and Dio2-/- strains exhibit deafness with cochlear abnormalities. In Dio3-/-;Dio2-/- mice, deafness was exacerbated rather than alleviated, suggesting unevenly balanced actions by these enzymes during auditory development. Dio3-/- mice also exhibit an atrophic thyroid gland, low thyroxine, and high triiodothyronine levels, but this phenotype was ameliorated in Dio3-/-;Dio2-/- mice, indicating counterbalancing roles for the enzymes in determining the thyroid hormone status. The results suggest that the composite action of these two enzymes is a critical determinant in visual and auditory development and in setting the systemic thyroid hormone status.

Testing Geometrical Discrimination within an Enzyme Active Site: Constrained Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole

PubMed Central

Sigala, Paul A.; Kraut, Daniel A.; Caaveiro, Jose M. M.; Pybus, Brandon; Ruben, Eliza A.; Ringe, Dagmar; Petsko, Gregory A.; Herschlag, Daniel

2009-01-01

Enzymes are classically proposed to accelerate reactions by binding substrates within active site environments that are structurally preorganized to optimize binding interactions with reaction transition states rather than ground states. This is a remarkably formidable task considering the limited 0.1 – 1 Å scale of most substrate rearrangements. The flexibility of active site functional groups along the coordinate of substrate rearrangement, the distance scale on which enzymes can distinguish structural rearrangement, and the energetic significance of discrimination on that scale remain open questions that are fundamental to a basic physical understanding of enzyme active sites and catalysis. We bring together high resolution X-ray crystallography, 1H and 19F NMR spectroscopy, quantum mechanical calculations, and transition state analog binding measurements to test the distance scale on which non-covalent forces can constrain side chain and ligand relaxation or translation along a specific coordinate and the energetic consequences of such geometric constraints within the active site of bacterial ketosteroid isomerase (KSI). Our results strongly suggest that packing and binding interactions within the KSI active site can constrain local side chain reorientation and prevent hydrogen bond shortening by 0.1 Å or less. Further, this constraint has substantial energetic effects on ligand binding and stabilization of negative charge within the oxyanion hole. These results provide evidence that subtle geometric effects, indistinguishable in most X-ray crystallographic structures, can have significant energetic consequences and highlight the importance of using synergistic experimental approaches to dissect enzyme function. PMID:18808119

Does the Clock Make the Poison? Circadian Variation in Response to Pesticides

PubMed Central

Hooven, Louisa A.; Sherman, Katherine A.; Butcher, Shawn; Giebultowicz, Jadwiga M.

2009-01-01

Background Circadian clocks govern daily physiological and molecular rhythms, and putative rhythms in expression of xenobiotic metabolizing (XM) genes have been described in both insects and mammals. Such rhythms could have important consequences for outcomes of chemical exposures at different times of day. To determine whether reported XM gene expression rhythms result in functional rhythms, we examined daily profiles of enzyme activity and dose responses to the pesticides propoxur, deltamethrin, fipronil, and malathion. Methodology/Principal Findings Published microarray expression data were examined for temporal patterns. Male Drosophila were collected for ethoxycoumarin-O-deethylase (ECOD), esterase, glutathione-S-transferase (GST), and, and uridine 5′-diphosphoglucosyltransferase (UGT) enzyme activity assays, or subjected to dose-response tests at four hour intervals throughout the day in both light/dark and constant light conditions. Peak expression of several XM genes cluster in late afternoon. Significant diurnal variation was observed in ECOD and UGT enzyme activity, however, no significant daily variation was observed in esterase or GST activity. Daily profiles of susceptibility to lethality after acute exposure to propoxur and fipronil showed significantly increased resistance in midday, while susceptibility to deltamethrin and malathion varied little. In constant light, which interferes with clock function, the daily variation in susceptibility to propoxur and in ECOD and UGT enzyme activity was depressed. Conclusions/Significance Expression and activities of specific XM enzymes fluctuate during the day, and for specific insecticides, the concentration resulting in 50% mortality varies significantly during the day. Time of day of chemical exposure should be an important consideration in experimental design, use of pesticides, and human risk assessment. PMID:19649249

Expression and characterization of fifteen Rhizopus oryzae 99-880 polygalacturonase enzymes in Pichia pastoris.

PubMed

Mertens, Jeffrey A; Bowman, Michael J

2011-04-01

Polygalacturonase (PG) enzymes hydrolyze the long polygalacturonic acid chains found in the smooth regions of pectin. Interest in this enzyme class continues due to their ability to macerate tissues of economically important crops and their use in a number of industrial processes. Rhizopus oryzae has a large PG gene family with 15 of 18 genes encoding unique active enzymes. The PG enzymes, 12 endo-PG and 3 exo-galacturonases, were expressed in Pichia pastoris and purified enabling biochemical characterization to gain insight into the maintenance of this large gene family within the Rhizopus genome. The 15 PG enzymes have a pH optima ranging from 4.0 to 5.0. Temperature optima of the 15 PG enzymes vary from 30 to 40 °C. While the pH and temperature optima do little to separate the enzymes, the specific activity of the enzymes is highly variable ranging from over 200 to less than 1 μmol/min/mg. A general pattern related to the groupings found in the phylogentic tree was visible with the group containing the exo-PG enzymes demonstrating the lowest specific activity. Finally, the progress curves of the PG enzymes, contained within the phylogenetic group that includes the exo-PG enzymes, acting on trigalacturonic acid lend additional support to the idea that the ancestral form of PG in Rhizopus is endolytic and exolytic function evolved later.

Full activation of RNaseL in animal cells requires binding of 2-5A within ankyrin repeats 6 to 9 of this interferon-inducible enzyme.

PubMed

Díaz-Guerra, M; Rivas, C; Esteban, M

1999-02-01

To define protein domains important for activation of the interferon (IFN)-induced enzyme 2-5A-dependent RNaseL, we have generated vaccinia virus (VV) recombinants able to express in cultured cells truncated forms of this protein and compared their biologic activities with those producing the wild-type enzyme, with and without coexpression of 2-5A synthetase. Our results show that full activation of RNaseL requires binding of 2-5A oligonucleotides within amino acid positions 212-339, corresponding to ankyrin repeats 6 to 9. The protein kinase and ribonuclease domains of RNaseL, amino acids 340-741, are sufficient for a constitutively active enzyme that is unresponsive to excess 2-5A. These results demonstrate in vivo the importance of the ankyrin domains in the biologic function of RNaseL. We suggest that ankyrin repeats act as key modulators of RNaseL activity.

Gut-based antioxidant enzymes in a polyphagous and a graminivorous grasshopper.

PubMed

Barbehenn, Raymond V

2002-07-01

Graminivorous species of grasshoppers develop lethal lesions in their midgut epithelia when they ingest tannic acid, whereas polyphagous grasshoppers are unaffected by ingested tannins. This study tests the hypothesis that polyphagous species are defended by higher activities of antioxidant enzymes (constitutive or inducible) in their guts than are graminivorous species. Comparisons were made between four antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APOX), and glutathione transferase peroxidase (GSTPX). Enzyme activities were measured in the gut lumens and midgut tissues of Melanoplus sanguinipes (polyphagous) and Aulocara ellioti (graminivorous). The results of this study do not support the hypothesis that M. sanguinipes is better defended by antioxidant enzymes than is A. ellioti, nor are these enzymes more inducible in M. sanguinipes than in A. ellioti when insects consume food containing 15% dry weight tannic acid. Instead, tannic acid consumption reduced SOD, APOX, and GSTPX activities in both species. This study reports the first evidence that SOD is secreted into the midgut lumen in insects, with activities two- to fourfold higher than those found in midgut tissues. The spatial distribution of GSTPX and APOX activities observed in both species suggests that ingested plant antioxidant enzymes may function as acquired defenses in grasshoppers. In addition, the results of this study permit the first comparison between the antioxidant enzyme defenses of Orthoptera and Lepidoptera. Most notably, grasshoppers have higher SOD activities than caterpillars, but completely lack APOX in their midgut tissues.

A new methodology for the determination of enzyme activity based on carbon nanotubes and glucose oxidase.

PubMed

Yeşiller, Gülden; Sezgintürk, Mustafa Kemal

2015-11-10

In this research, a novel enzyme activity analysis methodology is introduced as a new perspective for this area. The activity of elastase enzyme, which is a digestive enzyme mostly of found in the digestive system of vertebrates, was determined by an electrochemical device composed of carbon nanotubes and a second enzyme, glucose oxidase, which was used as a signal generator enzyme. In this novel methodology, a complex bioactive layer was constructed by using carbon nanotubes, glucose oxidase and a supporting protein, gelatin on a solid, conductive substrate. The activity of elastase was determined by monitoring the hydrolysis rate of elastase enzyme in the bioactive layer. As a result of this hydrolysis of elastase, glucose oxidase was dissociated from the bioactive layer, and following this the electrochemical signal due to glucose oxidase was decreased. The progressive elastase-catalyzed digestion of the bioactive layer containing glucose oxidase decreased the layer's enzymatic efficiency, resulting in a decrease of the glucose oxidation current as a function of the enzyme activity. The ratio of the decrease was correlated to elastase activity level. In this study, optimization experiments of bioactive components and characterization of the resulting new electrochemical device were carried out. A linear calibration range from 0.0303U/mL to 0.0729U/mL of elastase was reported. Real sample analyses were also carried out by the new electrochemical device. Copyright © 2015 Elsevier B.V. All rights reserved.

Who is who in litter decomposition? Metaproteomics reveals major microbial players and their biogeochemical functions

PubMed Central

Schneider, Thomas; Keiblinger, Katharina M; Schmid, Emanuel; Sterflinger-Gleixner, Katja; Ellersdorfer, Günther; Roschitzki, Bernd; Richter, Andreas; Eberl, Leo; Zechmeister-Boltenstern, Sophie; Riedel, Kathrin

2012-01-01

Leaf-litter decomposition is a central process in carbon cycling; however, our knowledge about the microbial regulation of this process is still scarce. Metaproteomics allows us to link the abundance and activity of enzymes during nutrient cycling to their phylogenetic origin based on proteins, the ‘active building blocks' in the system. Moreover, we employed metaproteomics to investigate the influence of environmental factors and nutrients on the decomposer structure and function during beech litter decomposition. Litter was collected at forest sites in Austria with different litter nutrient content. Proteins were analyzed by 1-D-SDS-PAGE followed by liquid-chromatography and tandem mass-spectrometry. Mass spectra were assigned to phylogenetic and functional groups by a newly developed bioinformatics workflow, assignments being validated by complementary approaches. We provide evidence that the litter nutrient content and the stoichiometry of C:N:P affect the decomposer community structure and activity. Fungi were found to be the main producers of extracellular hydrolytic enzymes, with no bacterial hydrolases being detected by our metaproteomics approach. Detailed investigation of microbial succession suggests that it is influenced by litter nutrient content. Microbial activity was stimulated at higher litter nutrient contents via a higher abundance and activity of extracellular enzymes. PMID:22402400

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

Bianchetti, Christopher M.; Takasuka, Taichi E.; Deutsch, Sam

The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium. Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define themore » active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ~30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Furthermore, application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties.« less

Catalytic activity of various pepsin reduced Au nanostructures towards reduction of nitroarenes and resazurin

NASA Astrophysics Data System (ADS)

Sharma, Bhagwati; Mandani, Sonam; Sarma, Tridib K.

2015-01-01

Pepsin, a digestive protease enzyme, could function as a reducing as well as stabilizing agent for the synthesis of Au nanostructures of various size and shape under different reaction conditions. The simple tuning of the pH of the reaction medium led to the formation of spherical Au nanoparticles, anisotropic Au nanostructures such as triangles, hexagons, etc., as well as ultra small fluorescent Au nanoclusters. The activity of the enzyme was significantly inhibited after its participation in the formation of Au nanoparticles due to conformational changes in the native structure of the enzyme which was studied by fluorescence, circular dichroism (CD), and infra red spectroscopy. However, the Au nanoparticle-enzyme composites served as excellent catalyst for the reduction of p-nitrophenol and resazurin, with the catalytic activity varying with size and shape of the nanoparticles. The presence of pepsin as the surface stabilizer played a crucial role in the activity of the Au nanoparticles as reduction catalysts, as the approach of the reacting molecules to the nanoparticle surface was actively controlled by the stabilizing enzyme.

Effect of mitoguazone on polyamine oxidase activity in rat liver.

PubMed

Ferioli, Maria Elena; Berselli, Debora; Caimi, Samuela

2004-12-01

Mitoguazone is a known inhibitor of polyamine biosynthesis through competitive inhibition of S-adenosylmethionine decarboxylase. A recent renewed interest in mitoguazone as an antineoplastic agent prompted us to investigate the effect of the drug on polyamine catabolism in rat liver, since the organ plays an important role in detoxification mechanisms. Thus, the purpose of this work was to evaluate the effect of in vivo mitoguazone administration on polyamine catabolic enzymes. In particular, our interest was directed to the changes in polyamine oxidase activity, since this enzyme has been recently confirmed to exert important functions that until now were underestimated. Mitoguazone administration induced hepatic polyamine oxidase activity starting at 4 h after administration, and the enzyme returned to basal levels 96 h after treatment. The changes in enzyme activity were accompanied by changes in putrescine concentrations, which increased starting at 4 h until 72 h after treatment. We also evaluated the activity of the newly identified spermine oxidase, which was not significantly changed by mitoguazone treatment. Therefore, we hypothesized that the enzyme involved in mitoguazone response of the liver is the polyamine oxidase, which acts on acetylated polyamines as substrate.

Identification of essential active-site residues in the cyanogenic beta-glucosidase (linamarase) from cassava (Manihot esculenta Crantz) by site-directed mutagenesis.

PubMed Central

Keresztessy, Z; Brown, K; Dunn, M A; Hughes, M A

2001-01-01

The coding sequence of the mature cyanogenic beta-glucosidase (beta-glucoside glucohydrolase, EC 3.2.1.21; linamarase) was cloned into the vector pYX243 modified to contain the SUC2 yeast secretion signal sequence and expressed in Saccharomyces cerevisiae. The recombinant enzyme is active, glycosylated and showed similar stability to the plant protein. Michaelis constants for hydrolysis of the natural substrate, linamarin (K(m)=1.06 mM) and the synthetic p-nitrophenyl beta-D-glucopyranoside (PNP-Glc; K(m)=0.36 mM), as well as apparent pK(a) values of the free enzyme and the enzyme-substrate complexes (pK(E)(1)=4.4-4.8, pK(E)(2)=6.7-7.2, pK(ES)(1)=3.9-4.4, pK(ES)(2)=8.3) were very similar to those of the plant enzyme. Site-directed mutagenesis was carried out to study the function of active-site residues based on a homology model generated for the enzyme using the MODELLER program. Changing Glu-413 to Gly destroyed enzyme activity, consistent with it being the catalytic nucleophile. The Gln-339Glu mutation also abolished activity, confirming a function in positioning the catalytic diad. The Ala-201Val mutation shifted the pK(a) of the acid/base catalyst Glu-198 from 7.22 to 7.44, reflecting a change in its hydrophobic environment. A Phe-269Asn change increased K(m) for linamarin hydrolysis 16-fold (16.1 mM) and that for PNP-Glc only 2.5-fold (0.84 mM), demonstrating that Phe-269 contributes to the cyanogenic specificity of the cassava beta-glucosidase. PMID:11139381

Bacterial Community Composition and Extracellular Enzyme Activity in Temperate Streambed Sediment during Drying and Rewetting

PubMed Central

Pohlon, Elisabeth; Ochoa Fandino, Adriana; Marxsen, Jürgen

2013-01-01

Droughts are among the most important disturbance events for stream ecosystems; they not only affect stream hydrology but also the stream biota. Although desiccation of streams is common in Mediterranean regions, phases of dryness in headwaters have been observed more often and for longer periods in extended temperate regions, including Central Europe, reflecting global climate change and enhanced water withdrawal. The effects of desiccation and rewetting on the bacterial community composition and extracellular enzyme activity, a key process in the carbon flow of streams and rivers, were investigated in a typical Central European stream, the Breitenbach (Hesse, Germany). Wet streambed sediment is an important habitat in streams. It was sampled and exposed in the laboratory to different drying scenarios (fast, intermediate, slow) for 13 weeks, followed by rewetting of the sediment from the fast drying scenario via a sediment core perfusion technique for 2 weeks. Bacterial community structure was analyzed using CARD-FISH and TGGE, and extracellular enzyme activity was assessed using fluorogenic model substrates. During desiccation the bacterial community composition shifted toward composition in soil, exhibiting increasing proportions of Actinobacteria and Alphaproteobacteria and decreasing proportions of Bacteroidetes and Betaproteobacteria. Simultaneously the activities of extracellular enzymes decreased, most pronounced with aminopeptidases and less pronounced with enzymes involved in the degradation of polymeric carbohydrates. After rewetting, the general ecosystem functioning, with respect to extracellular enzyme activity, recovered after 10 to 14 days. However, the bacterial community composition had not yet achieved its original composition as in unaffected sediments within this time. Thus, whether the bacterial community eventually recovers completely after these events remains unknown. Perhaps this community undergoes permanent changes, especially after harsh desiccation, followed by loss of the specialized functions of specific groups of bacteria. PMID:24386188

Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes.

PubMed

Wei, Hui; Wang, Erkang

2013-07-21

Over the past few decades, researchers have established artificial enzymes as highly stable and low-cost alternatives to natural enzymes in a wide range of applications. A variety of materials including cyclodextrins, metal complexes, porphyrins, polymers, dendrimers and biomolecules have been extensively explored to mimic the structures and functions of naturally occurring enzymes. Recently, some nanomaterials have been found to exhibit unexpected enzyme-like activities, and great advances have been made in this area due to the tremendous progress in nano-research and the unique characteristics of nanomaterials. To highlight the progress in the field of nanomaterial-based artificial enzymes (nanozymes), this review discusses various nanomaterials that have been explored to mimic different kinds of enzymes. We cover their kinetics, mechanisms and applications in numerous fields, from biosensing and immunoassays, to stem cell growth and pollutant removal. We also summarize several approaches to tune the activities of nanozymes. Finally, we make comparisons between nanozymes and other catalytic materials (other artificial enzymes, natural enzymes, organic catalysts and nanomaterial-based catalysts) and address the current challenges and future directions (302 references).

Functional and composition differences between mitochondrial complex II in Arabidopsis and rice are correlated with the complex genetic history of the enzyme.

PubMed

Huang, Shaobai; Taylor, Nicolas L; Narsai, Reena; Eubel, Holger; Whelan, James; Millar, A Harvey

2010-02-01

Complex II plays a central role in mitochondrial metabolism as a component of both the electron transport chain and the tricarboxylic acid cycle. However, the composition and function of the plant enzyme has been elusive and differs from the well-characterised enzymes in mammals and bacteria. Herewith, we demonstrate that mitochondrial Complex II from Arabidopsis and rice differ significantly in several aspects: (1) Stability-Rice complex II in contrast to Arabidopsis is not stable when resolved by native electrophoresis and activity staining. (2) Composition-Arabidopsis complex II contains 8 subunits, only 7 of which have homologs in the rice genome. SDH 1 and 2 subunits display high levels of amino acid identity between two species, while the remainder of the subunits are not well conserved at a sequence level, indicating significant divergence. (3) Gene expression-the pairs of orthologous SDH1 and SDH2 subunits were universally expressed in both Arabidopsis and rice. The very divergent genes for SDH3 and SDH4 were co-expressed in both species, consistent with their functional co-ordination to form the membrane anchor. The plant-specific SDH5, 6 and 7 subunits with unknown functions appeared to be differentially expressed in both species. (4) Biochemical regulation -succinate-dependent O(2) consumption and SDH activity of isolated Arabidopsis mitochondria were substantially stimulated by ATP, but a much more minor effect of ATP was observed for the rice enzyme. The ATP activation of succinate-dependent reduction of DCPIP in frozen-thawed and digitonin-solubilised mitochondrial samples, and with or without the uncoupler CCCP, indicate that the differential ATP effect on SDH is not via the protonmotive force but likely due to an allosteric effect on the plant SDH enzyme itself, in contrast to the enzyme in other organisms.

Tempol Supplementation Restores Diaphragm Force and Metabolic Enzyme Activities in mdx Mice

PubMed Central

Burns, David P.; Ali, Izza; Rieux, Clement; Healy, James; Jasionek, Greg; O’Halloran, Ken D.

2017-01-01

Duchenne muscular dystrophy (DMD) is characterized by striated muscle weakness, cardiomyopathy, and respiratory failure. Since oxidative stress is recognized as a secondary pathology in DMD, the efficacy of antioxidant intervention, using the superoxide scavenger tempol, was examined on functional and biochemical status of dystrophin-deficient diaphragm muscle. Diaphragm muscle function was assessed, ex vivo, in adult male wild-type and dystrophin-deficient mdx mice, with and without a 14-day antioxidant intervention. The enzymatic activities of muscle citrate synthase, phosphofructokinase, and lactate dehydrogenase were assessed using spectrophotometric assays. Dystrophic diaphragm displayed mechanical dysfunction and altered biochemical status. Chronic tempol supplementation in the drinking water increased diaphragm functional capacity and citrate synthase and lactate dehydrogenase enzymatic activities, restoring all values to wild-type levels. Chronic supplementation with tempol recovers force-generating capacity and metabolic enzyme activity in mdx diaphragm. These findings may have relevance in the search for therapeutic strategies in neuromuscular disease. PMID:29210997

Lipid-induced NOX2 activation inhibits autophagic flux by impairing lysosomal enzyme activity[S

PubMed Central

Jaishy, Bharat; Zhang, Quanjiang; Chung, Heaseung S.; Riehle, Christian; Soto, Jamie; Jenkins, Stephen; Abel, Patrick; Cowart, L. Ashley; Van Eyk, Jennifer E.; Abel, E. Dale

2015-01-01

Autophagy is a catabolic process involved in maintaining energy and organelle homeostasis. The relationship between obesity and the regulation of autophagy is cell type specific. Despite adverse consequences of obesity on cardiac structure and function, the contribution of altered cardiac autophagy in response to fatty acid overload is incompletely understood. Here, we report the suppression of autophagosome clearance and the activation of NADPH oxidase (Nox)2 in both high fat-fed murine hearts and palmitate-treated H9C2 cardiomyocytes (CMs). Defective autophagosome clearance is secondary to superoxide-dependent impairment of lysosomal acidification and enzyme activity in palmitate-treated CMs. Inhibition of Nox2 prevented superoxide overproduction, restored lysosome acidification and enzyme activity, and reduced autophagosome accumulation in palmitate-treated CMs. Palmitate-induced Nox2 activation was dependent on the activation of classical protein kinase Cs (PKCs), specifically PKCβII. These findings reveal a novel mechanism linking lipotoxicity with a PKCβ-Nox2-mediated impairment in pH-dependent lysosomal enzyme activity that diminishes autophagic turnover in CMs. PMID:25529920

Enhanced hepatic and kidney cytochrome p-450 activities in nandrolone decanoate treated albino mice.

PubMed

Acharjee, B K; Mahanta, R

2009-04-01

Anabolic androgenic steroids are the xenobiotic substrates that are metabolized in the body by the protective enzyme systems. Mixed function oxygenase enzymes include a group of enzymes which play an essential role in the metabolism of a broad range of xenobiotics including endogenous and exogenous substrates. Cytochrome P-450, a member of mixed function oxygenase enzymes, plays an important role in oxidative metabolism of drugs and xenobiotics entering human body. Various anabolic steroids are found either to increase or decrease the activity of cytochrome P-450. However, effect of nandrolone decanoate, most commonly abused anabolic steroid, on cytochrome P-450 activity is still fragmentary. In the present study, albino mice were administered intramuscular 2.5 mg of nandrolone decanoate injection at 15 days interval. Cytochrome P-450 activity is determined by following the method of Omura and Sato (1964) in liver and kidney tissues of both normal and experimental groups upto 90 days. Investigation shows a significant (p <0.01) increase of cytochrome P-450 (nmol/mg) activity in liver tissue as compared to that of kidney tissues. A tissue specific and dose specific increase of cytochrome P-450 activity is observed. Mean cytochrome P-450 is found highest in liver tissue on 45(th) day whereas the activity in kidney tissue is noticed on 90(th) day of treatment. From the above observation, nandrolone decanoate can be suggested as a potent inducer of cytochrome P-450 activity like other anabolic steroids.

De novo active sites for resurrected Precambrian enzymes

NASA Astrophysics Data System (ADS)

Risso, Valeria A.; Martinez-Rodriguez, Sergio; Candel, Adela M.; Krüger, Dennis M.; Pantoja-Uceda, David; Ortega-Muñoz, Mariano; Santoyo-Gonzalez, Francisco; Gaucher, Eric A.; Kamerlin, Shina C. L.; Bruix, Marta; Gavira, Jose A.; Sanchez-Ruiz, Jose M.

2017-07-01

Protein engineering studies often suggest the emergence of completely new enzyme functionalities to be highly improbable. However, enzymes likely catalysed many different reactions already in the last universal common ancestor. Mechanisms for the emergence of completely new active sites must therefore either plausibly exist or at least have existed at the primordial protein stage. Here, we use resurrected Precambrian proteins as scaffolds for protein engineering and demonstrate that a new active site can be generated through a single hydrophobic-to-ionizable amino acid replacement that generates a partially buried group with perturbed physico-chemical properties. We provide experimental and computational evidence that conformational flexibility can assist the emergence and subsequent evolution of new active sites by improving substrate and transition-state binding, through the sampling of many potentially productive conformations. Our results suggest a mechanism for the emergence of primordial enzymes and highlight the potential of ancestral reconstruction as a tool for protein engineering.

Regional variation in muscle metabolic enzymes in individual American shad (Alosa sapidissima)

USGS Publications Warehouse

Leonard, J.B.K.

1999-01-01

Evaluation of the activity of metabolic enzymes is often used to asses metabolic capacity at the tissue level, but the amount of regional variability within a tissue in an individual fish of a given species is frequently unknown. The activities of four enzymes (citrate synthase (CS), phosphofructokinase, lactate dehydrogenase (LDH), and ??-hydroxyacyl coenzyme A dehydrogenase (HOAD) were assayed in red and white muscle at 10 sites along the body of adult American shad (Alosa sapidissima). Red and white muscle HOAD and white muscle CS and LDH varied significantly, generally increasing posteriorly. Maximal variation occurs in red muscle HOAD (~450%) and white muscle LDH (~60%) activity. Differences between the sexes also vary with sampling location. This study suggests that the variability in enzyme activity may be linked to functional differences in the muscle at different locations, and also provides guidelines for sample collection in this species.

AMP-activated protein kinase: Role in metabolism and therapeutic implications.

PubMed

Schimmack, Greg; Defronzo, Ralph A; Musi, Nicolas

2006-11-01

AMP-activated protein kinase (AMPK) is an enzyme that works as a fuel gauge which becomes activated in situations of energy consumption. AMPK functions to restore cellular ATP levels by modifying diverse metabolic and cellular pathways. In the skeletal muscle, AMPK is activated during exercise and is involved in contraction-stimulated glucose transport and fatty acid oxidation. In the heart, AMPK activity increases during ischaemia and functions to sustain ATP, cardiac function and myocardial viability. In the liver, AMPK inhibits the production of glucose, cholesterol and triglycerides and stimulates fatty acid oxidation. Recent studies have shown that AMPK is involved in the mechanism of action of metformin and thiazolidinediones, and the adipocytokines leptin and adiponectin. These data, along with evidence that pharmacological activation of AMPK in vivo improves blood glucose homeostasis, cholesterol concentrations and blood pressure in insulin-resistant rodents, make this enzyme an attractive pharmacological target for the treatment of type 2 diabetes, ischaemic heart disease and other metabolic diseases.

Gastrointestinal Functionality of Aquatic Animal (Oreochromis niloticus) Carcass in Water Allows Estimating Time of Death.

PubMed

Hahor, Waraporn; Thongprajukaew, Karun; Yoonram, Krueawan; Rodjaroen, Somrak

2016-11-01

Postmortem changes have been previously studied in some terrestrial animal models, but no prior information is available on aquatic species. Gastrointestinal functionality was investigated in terms of indices, protein concentration, digestive enzyme activity, and scavenging activity, in an aquatic animal model, Nile tilapia, to assess the postmortem changes. Dead fish were floated indoors, and samples were collected within 48 h after death. Stomasomatic index decreased with postmortem time and correlated positively with protein, pepsin-specific activity, and stomach scavenging activity. Also intestosomatic index decreased significantly and correlated positively with protein, specific activity of trypsin, chymotrypsin, amylase, lipase, and intestinal scavenging activity. In their postmortem changes, the digestive enzymes exhibited earlier lipid degradation than carbohydrate or protein. The intestine changed more rapidly than the stomach. The findings suggest that the postmortem changes of gastrointestinal functionality can serve as primary data for the estimation of time of death of an aquatic animal. © 2016 American Academy of Forensic Sciences.

Gold and silver nanoparticles for biomolecule immobilization and enzymatic catalysis

PubMed Central

2012-01-01

In this work, a simple method for alcohol synthesis with high enantiomeric purity was proposed. For this, colloidal gold and silver surface modifications with 3-mercaptopropanoic acid and cysteamine were used to generate carboxyl and amine functionalized gold and silver nanoparticles of 15 and 45 nm, respectively. Alcohol dehydrogenase from Thermoanaerobium brockii (TbADH) and its cofactor (NADPH) were physical and covalent (through direct adsorption and using cross-linker) immobilized on nanoparticles' surface. In contrast to the physical and covalent immobilizations that led to a loss of 90% of the initial enzyme activity and 98% immobilization, the use of a cross-linker in immobilization process promoted a loss to 30% of the initial enzyme activity and >92% immobilization. The yield of NADPH immobilization was about 80%. The best results in terms of activity were obtained with Ag-citr nanoparticle functionalized with carboxyl groups (Ag-COOH), Au-COOH(CTAB), and Au-citr functionalized with amine groups and stabilized with CTAB (Au-NH2(CTAB)) nanoparticles treated with 0.7% and 1.0% glutaraldehyde. Enzyme conformation upon immobilization was studied using fluorescence and circular dichroism spectroscopies. Shift in ellipticity at 222 nm with about 4 to 7 nm and significant decreasing in fluorescence emission for all bioconjugates were observed by binding of TbADH to silver/gold nanoparticles. Emission redshifting of 5 nm only for Ag-COOH-TbADH bioconjugate demonstrated change in the microenvironment of TbADH. Enzyme immobilization on glutaraldehyde-treated Au-NH2(CTAB) nanoparticles promotes an additional stabilization preserving about 50% of enzyme activity after 15 days storage. Nanoparticles attached-TbADH-NADPH systems were used for enantioselective (ee > 99%) synthesis of (S)-7-hydroxy-2-tetralol. PMID:22655978

Gold and silver nanoparticles for biomolecule immobilization and enzymatic catalysis.

PubMed

Petkova, Galina A; Záruba, Capital Ka Cyrillicamil; Zvátora, Pavel; Král, Vladimír

2012-06-01

In this work, a simple method for alcohol synthesis with high enantiomeric purity was proposed. For this, colloidal gold and silver surface modifications with 3-mercaptopropanoic acid and cysteamine were used to generate carboxyl and amine functionalized gold and silver nanoparticles of 15 and 45 nm, respectively. Alcohol dehydrogenase from Thermoanaerobium brockii (TbADH) and its cofactor (NADPH) were physical and covalent (through direct adsorption and using cross-linker) immobilized on nanoparticles' surface. In contrast to the physical and covalent immobilizations that led to a loss of 90% of the initial enzyme activity and 98% immobilization, the use of a cross-linker in immobilization process promoted a loss to 30% of the initial enzyme activity and >92% immobilization. The yield of NADPH immobilization was about 80%. The best results in terms of activity were obtained with Ag-citr nanoparticle functionalized with carboxyl groups (Ag-COOH), Au-COOH(CTAB), and Au-citr functionalized with amine groups and stabilized with CTAB (Au-NH2(CTAB)) nanoparticles treated with 0.7% and 1.0% glutaraldehyde. Enzyme conformation upon immobilization was studied using fluorescence and circular dichroism spectroscopies. Shift in ellipticity at 222 nm with about 4 to 7 nm and significant decreasing in fluorescence emission for all bioconjugates were observed by binding of TbADH to silver/gold nanoparticles. Emission redshifting of 5 nm only for Ag-COOH-TbADH bioconjugate demonstrated change in the microenvironment of TbADH. Enzyme immobilization on glutaraldehyde-treated Au-NH2(CTAB) nanoparticles promotes an additional stabilization preserving about 50% of enzyme activity after 15 days storage. Nanoparticles attached-TbADH-NADPH systems were used for enantioselective (ee > 99%) synthesis of (S)-7-hydroxy-2-tetralol.

Function, structure, and stability of enzymes confined in agarose gels.

PubMed

Kunkel, Jeffrey; Asuri, Prashanth

2014-01-01

Research over the past few decades has attempted to answer how proteins behave in molecularly confined or crowded environments when compared to dilute buffer solutions. This information is vital to understanding in vivo protein behavior, as the average spacing between macromolecules in the cell cytosol is much smaller than the size of the macromolecules themselves. In our study, we attempt to address this question using three structurally and functionally different model enzymes encapsulated in agarose gels of different porosities. Our studies reveal that under standard buffer conditions, the initial reaction rates of the agarose-encapsulated enzymes are lower than that of the solution phase enzymes. However, the encapsulated enzymes retain a higher percentage of their activity in the presence of denaturants. Moreover, the concentration of agarose used for encapsulation had a significant effect on the enzyme functional stability; enzymes encapsulated in higher percentages of agarose were more stable than the enzymes encapsulated in lower percentages of agarose. Similar results were observed through structural measurements of enzyme denaturation using an 8-anilinonaphthalene-1-sulfonic acid fluorescence assay. Our work demonstrates the utility of hydrogels to study protein behavior in highly confined environments similar to those present in vivo; furthermore, the enhanced stability of gel-encapsulated enzymes may find use in the delivery of therapeutic proteins, as well as the design of novel strategies for biohybrid medical devices.

Long-Term Persistence of Bi-functionality Contributes to the Robustness of Microbial Life through Exaptation

PubMed Central

Sterner, Reinhard; Merkl, Rainer

2016-01-01

Modern enzymes are highly optimized biocatalysts that process their substrates with extreme efficiency. Many enzymes catalyze more than one reaction; however, the persistence of such ambiguities, their consequences and evolutionary causes are largely unknown. As a paradigmatic case, we study the history of bi-functionality for a time span of approximately two billion years for the sugar isomerase HisA from histidine biosynthesis. To look back in time, we computationally reconstructed and experimentally characterized three HisA predecessors. We show that these ancient enzymes catalyze not only the HisA reaction but also the isomerization of a similar substrate, which is commonly processed by the isomerase TrpF in tryptophan biosynthesis. Moreover, we found that three modern-day HisA enzymes from Proteobacteria and Thermotogae also possess low TrpF activity. We conclude that this bi-functionality was conserved for at least two billion years, most likely without any evolutionary pressure. Although not actively selected for, this trait can become advantageous in the case of a gene loss. Such exaptation is exemplified by the Actinobacteria that have lost the trpF gene but possess the bi-functional HisA homolog PriA, which adopts the roles of both HisA and TrpF. Our findings demonstrate that bi-functionality can perpetuate in the absence of selection for very long time-spans. PMID:26824644

Metabolic enzymes dysregulation in heart failure: the prospective therapy.

PubMed

Parihar, Priyanka; Parihar, Mordhwaj Singh

2017-01-01

The heart failure accounts for the highest mortality rate all over the world. The development of preventive therapeutic approaches is still in their infancy. Owing to the extremely high energy demand of the heart, the bioenergetics pathways need to respond efficiently based on substrate availability. The metabolic regulation of such heart bioenergetics is mediated by various rate limiting enzymes involved in energy metabolism. Although all the pertinent mechanisms are not clearly understood, the progressive decline in the activity of metabolic enzymes leading to diminished ATP production is known to cause progression of the heart failure. Therefore, metabolic therapy that can maintain the appropriate activities of metabolic enzymes can be a promising approach for the prevention and treatment of the heart failure. The flavonoids that constitute various human dietary ingredients also effectively offer a variety of health benefits. The flavonoids target a variety of metabolic enzymes and facilitate effective management of the equilibrium between production and utilization of energy in the heart. This review discusses the broad impact of metabolic enzymes in the heart functions and explains how the dysregulated enzyme activity causes the heart failure. In addition, the prospects of targeting dysregulated metabolic enzymes by developing flavonoid-based metabolic approaches are discussed.

Ionizable side chains at catalytic active sites of enzymes.

PubMed

Jimenez-Morales, David; Liang, Jie; Eisenberg, Bob

2012-05-01

Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1,072 Å(3). The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes.

Ionizable Side Chains at Catalytic Active Sites of Enzymes

PubMed Central

Jimenez-Morales, David; Liang, Jie

2012-01-01

Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1072 Å3. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes. PMID:22484856

Studies on Marek's Disease Virus Encoded Ribonucleotide Reductase

USDA-ARS?s Scientific Manuscript database

Ribonucleotide reductase (RR) is an essential enzyme for the conversion of ribonucleotides to deoxyribonucleotides in prokaryotic and eukaryotic cells. The enzyme consists of two subunits namely RR1 and RR2, both of which associate to form an active holoenzyme. Herpesviruses express a functional R...

High-Resolution X-Ray Structures of Two Functionally Distinct Members of the Cyclic Amide Hydrolase Family of Toblerone Fold Enzymes

PubMed Central

Peat, Thomas S.; Balotra, Sahil; Wilding, Matthew; Hartley, Carol J.; Newman, Janet

2017-01-01

ABSTRACT The Toblerone fold was discovered recently when the first structure of the cyclic amide hydrolase, AtzD (a cyanuric acid hydrolase), was elucidated. We surveyed the cyclic amide hydrolase family, finding a strong correlation between phylogenetic distribution and specificity for either cyanuric acid or barbituric acid. One of six classes (IV) could not be tested due to a lack of expression of the proteins from it, and another class (V) had neither cyanuric acid nor barbituric acid hydrolase activity. High-resolution X-ray structures were obtained for a class VI barbituric acid hydrolase (1.7 Å) from a Rhodococcus species and a class V cyclic amide hydrolase (2.4 Å) from a Frankia species for which we were unable to identify a substrate. Both structures were homologous with the tetrameric Toblerone fold enzyme AtzD, demonstrating a high degree of structural conservation within the cyclic amide hydrolase family. The barbituric acid hydrolase structure did not contain zinc, in contrast with early reports of zinc-dependent activity for this enzyme. Instead, each barbituric acid hydrolase monomer contained either Na+ or Mg2+, analogous to the structural metal found in cyanuric acid hydrolase. The Frankia cyclic amide hydrolase contained no metal but instead formed unusual, reversible, intermolecular vicinal disulfide bonds that contributed to the thermal stability of the protein. The active sites were largely conserved between the three enzymes, differing at six positions, which likely determine substrate specificity. IMPORTANCE The Toblerone fold enzymes catalyze an unusual ring-opening hydrolysis with cyclic amide substrates. A survey of these enzymes shows that there is a good correlation between physiological function and phylogenetic distribution within this family of enzymes and provide insights into the evolutionary relationships between the cyanuric acid and barbituric acid hydrolases. This family of enzymes is structurally and mechanistically distinct from other enzyme families; however, to date the structure of just two, physiologically identical, enzymes from this family has been described. We present two new structures: a barbituric acid hydrolase and an enzyme of unknown function. These structures confirm that members of the CyAH family have the unusual Toblerone fold, albeit with some significant differences. PMID:28235873

Biofunctionalization of carbon nanostructures through enzyme immobilization in colloidal silica

NASA Astrophysics Data System (ADS)

Goulet, Evan M.

Multi-walled carbon nanotubes (MWNT) and carbon nanopipettes (CNP) provide interesting high aspect ratio scaffolds on which to base functionally gradient materials. In this dissertation, we present a general method for the production of an enzymatically active composite material based on MWNTs. Polyethyleneimine (PEI) was applied to purified MWNTs, generating a positive electrostatic potential on the MWNTs. This positive potential was used to apply negatively charged colloidal silica particle in the presence of a high concentration of enzyme. The silica coating continued to grow via localized condensation of silica particles driven by the buffered saline conditions, immobilizing the enzyme within the coating. The mesoporous nanostructure was characterized via transmission electron microscopy. Optical spectroscopy experiments on the material employed as an active suspension showed that the immobilized enzymes horseradish peroxidase (HRP) and tyrosinase (TV) retained their activity upon incorporation into the material. Using HRP as a model enzyme, it was determined that the MWNT-HRP-Silica material showed similar pH and temperature dependencies in activity to those of free HRP in solution. An examination of the Michaelis-Menten kinetics showed that the material had a slightly higher value of KM than did free HRP. The MWNT-HRP-Silica material was also employed as an active filter membrane, which allowed us to explore the reusable nature of the material. We were able to show the denaturation of the filter due to the loss of Ca2+ cations at low pH and then restore the activity by soaking the filter membrane in 1 mM CaCl2. The MWNT-HRP-Silica material was used to modify a carbon microelectrode and produce a functioning electrochemical sensor for H2O2 . Utilizing cyclic voltammetry, the sensor was shown to have a linear response in limiting current versus concentration of H2O2 of 4.26 pA/microM. We also determined a lower detection limit of 0.67 microM H2O2. CNPs were investigated as functional microelectrodes. Colloidal silica was applied to the CNP with HRP, but it was difficult to prove functionality. One irregularly coated CNP showed a clear response to H2O2, but we were not able to reproduce the response in other samples. This work indicated the CNPs have promise as functional microelectrodes.

Large-Scale Analysis Exploring Evolution of Catalytic Machineries and Mechanisms in Enzyme Superfamilies.

PubMed

Furnham, Nicholas; Dawson, Natalie L; Rahman, Syed A; Thornton, Janet M; Orengo, Christine A

2016-01-29

Enzymes, as biological catalysts, form the basis of all forms of life. How these proteins have evolved their functions remains a fundamental question in biology. Over 100 years of detailed biochemistry studies, combined with the large volumes of sequence and protein structural data now available, means that we are able to perform large-scale analyses to address this question. Using a range of computational tools and resources, we have compiled information on all experimentally annotated changes in enzyme function within 379 structurally defined protein domain superfamilies, linking the changes observed in functions during evolution to changes in reaction chemistry. Many superfamilies show changes in function at some level, although one function often dominates one superfamily. We use quantitative measures of changes in reaction chemistry to reveal the various types of chemical changes occurring during evolution and to exemplify these by detailed examples. Additionally, we use structural information of the enzymes active site to examine how different superfamilies have changed their catalytic machinery during evolution. Some superfamilies have changed the reactions they perform without changing catalytic machinery. In others, large changes of enzyme function, in terms of both overall chemistry and substrate specificity, have been brought about by significant changes in catalytic machinery. Interestingly, in some superfamilies, relatives perform similar functions but with different catalytic machineries. This analysis highlights characteristics of functional evolution across a wide range of superfamilies, providing insights that will be useful in predicting the function of uncharacterised sequences and the design of new synthetic enzymes. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Swit_4259, an acetoacetate decarboxylase-like enzyme from Sphingomonas wittichii RW1

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

Mydy, Lisa S.; Mashhadi, Zahra; Knight, T. William

The Gram-negative bacteriumSphingomonas wittichiiRW1 is notable for its ability to metabolize a variety of aromatic hydrocarbons. Not surprisingly, theS. wittichiigenome contains a number of putative aromatic hydrocarbon-degrading gene clusters. One of these includes an enzyme of unknown function, Swit_4259, which belongs to the acetoacetate decarboxylase-like superfamily (ADCSF). Here, it is reported that Swit_4259 is a small (28.8 kDa) tetrameric ADCSF enzyme that, unlike the prototypical members of the superfamily, does not have acetoacetate decarboxylase activity. Structural characterization shows that the tertiary structure of Swit_4259 is nearly identical to that of the true decarboxylases, but there are important differences in themore » fine structure of the Swit_4259 active site that lead to a divergence in function. In addition, it is shown that while it is a poor substrate, Swit_4259 can catalyze the hydration of 2-oxo-hex-3-enedioate to yield 2-oxo-4-hydroxyhexanedioate. It is also demonstrated that Swit_4259 has pyruvate aldolase-dehydratase activity, a feature that is common to all of the family V ADCSF enzymes studied to date. The enzymatic activity, together with the genomic context, suggests that Swit_4259 may be a hydratase with a role in the metabolism of an as-yet-unknown hydrocarbon. These data have implications for engineering bioremediation pathways to degrade specific pollutants, as well as structure–function relationships within the ADCSF in general.« less

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

Genomics Review of Holocellulose Deconstruction by Aspergilli

PubMed Central

Segato, Fernando; Damásio, André R. L.; de Lucas, Rosymar C.; Squina, Fabio M.

2014-01-01

SUMMARY Biomass is constructed of dense recalcitrant polymeric materials: proteins, lignin, and holocellulose, a fraction constituting fibrous cellulose wrapped in hemicellulose-pectin. Bacteria and fungi are abundant in soil and forest floors, actively recycling biomass mainly by extracting sugars from holocellulose degradation. Here we review the genome-wide contents of seven Aspergillus species and unravel hundreds of gene models encoding holocellulose-degrading enzymes. Numerous apparent gene duplications followed functional evolution, grouping similar genes into smaller coherent functional families according to specialized structural features, domain organization, biochemical activity, and genus genome distribution. Aspergilli contain about 37 cellulase gene models, clustered in two mechanistic categories: 27 hydrolyze and 10 oxidize glycosidic bonds. Within the oxidative enzymes, we found two cellobiose dehydrogenases that produce oxygen radicals utilized by eight lytic polysaccharide monooxygenases that oxidize glycosidic linkages, breaking crystalline cellulose chains and making them accessible to hydrolytic enzymes. Among the hydrolases, six cellobiohydrolases with a tunnel-like structural fold embrace single crystalline cellulose chains and cooperate at nonreducing or reducing end termini, splitting off cellobiose. Five endoglucanases group into four structural families and interact randomly and internally with cellulose through an open cleft catalytic domain, and finally, seven extracellular β-glucosidases cleave cellobiose and related oligomers into glucose. Aspergilli contain, on average, 30 hemicellulase and 7 accessory gene models, distributed among 9 distinct functional categories: the backbone-attacking enzymes xylanase, mannosidase, arabinase, and xyloglucanase, the short-side-chain-removing enzymes xylan α-1,2-glucuronidase, arabinofuranosidase, and xylosidase, and the accessory enzymes acetyl xylan and feruloyl esterases. PMID:25428936

The structure of Plasmodium falciparum serine hydroxymethyltransferase reveals a novel redox switch that regulates its activities

PubMed Central

Chitnumsub, Penchit; Ittarat, Wanwipa; Jaruwat, Aritsara; Noytanom, Krittikar; Amornwatcharapong, Watcharee; Pornthanakasem, Wichai; Chaiyen, Pimchai; Yuthavong, Yongyuth; Leartsakulpanich, Ubolsree

2014-01-01

Plasmodium falciparum serine hydroxymethyltransferase (PfSHMT), an enzyme in the dTMP synthesis cycle, is an antimalarial target because inhibition of its expression or function has been shown to be lethal to the parasite. As the wild-type enzyme could not be crystallized, protein engineering of residues on the surface was carried out. The surface-engineered mutant PfSHMT-F292E was successfully crystallized and its structure was determined at 3 Å resolution. The PfSHMT-F292E structure is a good representation of PfSHMT as this variant revealed biochemical properties similar to those of the wild type. Although the overall structure of PfSHMT is similar to those of other SHMTs, unique features including the presence of two loops and a distinctive cysteine pair formed by Cys125 and Cys364 in the tetrahydrofolate (THF) substrate binding pocket were identified. These structural characteristics have never been reported in other SHMTs. Biochemical characterization and mutation analysis of these two residues confirm that they act as a disulfide/sulfhydryl switch to regulate the THF-dependent catalytic function of the enzyme. This redox switch is not present in the human enzyme, in which the cysteine pair is absent. The data reported here can be further exploited as a new strategy to specifically disrupt the activity of the parasite enzyme without interfering with the function of the human enzyme. PMID:24914963

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

PubMed Central

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

2008-01-01

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

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

PubMed

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

2006-05-09

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

Biochemical and Structural Characterization of WlbA from Bordetella pertussis and Chromobacterium violaceum: Enzymes Required for the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid

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

Thoden, James B.; Holden, Hazel M.

2011-12-22

The unusual sugar 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, or ManNAc3NAcA, has been observed in the lipopolysaccharides of both pathogenic and nonpathogenic Gram-negative bacteria. It is added to the lipopolysaccharides of these organisms by glycosyltransferases that use as substrates UDP-ManNAc3NAcA. Five enzymes are ultimately required for the biosynthesis of UDP-ManNAc3NAcA starting from UDP-N-acetylglucosamine. The second enzyme in the pathway, encoded by the wlba gene and referred to as WlbA, catalyzes the NAD-dependent oxidation of the C-3' hydroxyl group of the UDP-linked sugar. Here we describe a combined structural and functional investigation of the WlbA enzymes from Bordetella pertussis and Chromobacterium violaceum. For this investigation,more » ternary structures were determined in the presence of NAD(H) and substrate to 2.13 and 1.5 {angstrom} resolution, respectively. Both of the enzymes display octameric quaternary structures with their active sites positioned far apart. The octamers can be envisioned as tetramers of dimers. Kinetic studies demonstrate that the reaction mechanisms for these enzymes are sequential and that they do not require {alpha}-ketoglutarate for activity. These results are in sharp contrast to those recently reported for the WlbA enzymes from Pseudomonas aeruginosa and Thermus thermophilus, which function via ping-pong mechanisms that involve {alpha}-ketoglutarate. Taken together, the results reported here demonstrate that there are two distinct families of WlbA enzymes, which differ with respect to amino acid sequences, quaternary structures, active site architectures, and kinetic mechanisms.« less

Biochemical and Structural Characterization of WlbA from Bordetella pertussis and Chromobacterium violaceum: Enzymes Required for the Biosynthesis of 2,3-Diacetamido-2,3-Dideoxy-d-Mannuronic Acid¶

PubMed Central

Thoden, James B.; Holden, Hazel M.

2011-01-01

The unusual sugar 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, or ManNAc3NAcA1, has been observed in the lipopolysaccharides of both pathogenic and nonpathogenic Gram-negative bacteria. It is added to the lipopolysaccharides of these organisms by glycosyltransferases that use as substrates, UDP-ManNAc3NAcA. Five enzymes are ultimately required for the biosynthesis of UDP-ManNAc3NAcA starting from UDP-N-acetylglucosamine. The second enzyme in the pathway, encoded by the wlba gene and referred to as WlbA, catalyzes the NAD-dependent oxidation of the C-3' hydroxyl group of the UDP-linked sugar. Here we describe a combined structural and functional investigation of the WlbA enzymes from Bordetella pertussis and Chromobacterium violaceum. For this investigation, ternary structures were determined in the presence of NAD(H) and substrate to 2.13 Å and 1.5 Å resolution, respectively. Both of the enzymes display octameric quaternary structures with their active sites positioned far apart. The octamers can be envisioned as tetramers of dimers. Kinetic studies demonstrate that the reaction mechanisms for these enzymes are sequential and that they do not require α-ketoglutarate for activity. These results are in sharp contrast to those recently reported for the WlbA enzymes from Pseudomonas aeruginosa and Thermus thermophilus, which function via ping-pong mechanisms that involve α-ketoglutarate. Taken together the results reported here demonstrate that there are two distinct families of WlbA enzymes, which differ with respect to amino acid sequences, quaternary structures, active site architectures, and kinetic mechanisms. PMID:21241053

Neofunctionalization of a duplicate hatching enzyme gene during the evolution of teleost fishes.

PubMed

Sano, Kaori; Kawaguchi, Mari; Watanabe, Satoshi; Yasumasu, Shigeki

2014-10-19

Duplication and subsequent neofunctionalization of the teleostean hatching enzyme gene occurred in the common ancestor of Euteleostei and Otocephala, producing two genes belonging to different phylogenetic clades (clade I and II). In euteleosts, the clade I enzyme inherited the activity of the ancestral enzyme of swelling the egg envelope by cleavage of the N-terminal region of egg envelope proteins. The clade II enzyme gained two specific cleavage sites, N-ZPd and mid-ZPd but lost the ancestral activity. Thus, euteleostean clade II enzymes assumed a new function; solubilization of the egg envelope by the cooperative action with clade I enzyme. However, in Otocephala, the clade II gene was lost during evolution. Consequently, in a late group of Otocephala, only the clade I enzyme is present to swell the egg envelope. We evaluated the egg envelope digestion properties of clade I and II enzymes in Gonorynchiformes, an early diverging group of Otocephala, using milkfish, and compared their digestion with those of other fishes. Finally, we propose a hypothesis of the neofunctionalization process. The milkfish clade II enzyme cleaved N-ZPd but not mid-ZPd, and did not cause solubilization of the egg envelope. We conclude that neofunctionalization is incomplete in the otocephalan clade II enzymes. Comparison of clade I and clade II enzyme characteristics implies that the specificity of the clade II enzymes gradually changed during evolution after the duplication event, and that a change in substrate was required for the addition of the mid-ZPd site and loss of activity at the N-terminal region. We infer the process of neofunctionalization of the clade II enzyme after duplication of the gene. The ancestral clade II gene gained N-ZPd cleavage activity in the common ancestral lineage of the Euteleostei and Otocephala. Subsequently, acquisition of cleavage activity at the mid-ZPd site and loss of cleavage activity in the N-terminal region occurred during the evolution of Euteleostei, but not of Otocephala. The clade II enzyme provides an example of the development of a neofunctional gene for which the substrate, the egg envelope protein, has adapted to a gradual change in the specificity of the corresponding enzyme.

Molecular characterization of novel pyridoxal-5'-phosphate-dependent enzymes from the human microbiome.

PubMed

Fleischman, Nicholas M; Das, Debanu; Kumar, Abhinav; Xu, Qingping; Chiu, Hsiu-Ju; Jaroszewski, Lukasz; Knuth, Mark W; Klock, Heath E; Miller, Mitchell D; Elsliger, Marc-André; Godzik, Adam; Lesley, Scott A; Deacon, Ashley M; Wilson, Ian A; Toney, Michael D

2014-08-01

Pyridoxal-5'-phosphate or PLP, the active form of vitamin B6, is a highly versatile cofactor that participates in a large number of mechanistically diverse enzymatic reactions in basic metabolism. PLP-dependent enzymes account for ∼1.5% of most prokaryotic genomes and are estimated to be involved in ∼4% of all catalytic reactions, making this an important class of enzymes. Here, we structurally and functionally characterize three novel PLP-dependent enzymes from bacteria in the human microbiome: two are from Eubacterium rectale, a dominant, nonpathogenic, fecal, Gram-positive bacteria, and the third is from Porphyromonas gingivalis, which plays a major role in human periodontal disease. All adopt the Type I PLP-dependent enzyme fold and structure-guided biochemical analysis enabled functional assignments as tryptophan, aromatic, and probable phosphoserine aminotransferases. © 2014 The Protein Society.

Forizymes - functionalised artificial forisomes as a platform for the production and immobilisation of single enzymes and multi-enzyme complexes.

PubMed

Visser, Franziska; Müller, Boje; Rose, Judith; Prüfer, Dirk; Noll, Gundula A

2016-08-09

The immobilisation of enzymes plays an important role in many applications, including biosensors that require enzyme activity, stability and recyclability in order to function efficiently. Here we show that forisomes (plant-derived mechanoproteins) can be functionalised with enzymes by translational fusion, leading to the assembly of structures designated as forizymes. When forizymes are expressed in the yeast Saccharomyces cerevisiae, the enzymes are immobilised by the self-assembly of forisome subunits to form well-structured protein bodies. We used glucose-6-phosphate dehydrogenase (G6PDH) and hexokinase 2 (HXK2) as model enzymes for the one-step production and purification of catalytically active forizymes. These structures retain the typical stimulus-response reaction of the forisome and the enzyme remains active even after multiple assay cycles, which we demonstrated using G6PDH forizymes as an example. We also achieved the co-incorporation of both HXK2 and G6PDH in a single forizyme, facilitating a two-step reaction cascade that was 30% faster than the coupled reaction using the corresponding enzymes on different forizymes or in solution. Our novel forizyme immobilisation technique therefore not only combines the sensory properties of forisome proteins with the catalytic properties of enzymes but also allows the development of multi-enzyme complexes for incorporation into technical devices.

Differentiated roles for MreB-actin isologues and autolytic enzymes in Bacillus subtilis morphogenesis.

PubMed

Domínguez-Cuevas, Patricia; Porcelli, Ida; Daniel, Richard A; Errington, Jeff

2013-09-01

Cell morphogenesis in most bacteria is governed by spatiotemporal growth regulation of the peptidoglycan cell wall layer. Much is known about peptidoglycan synthesis but regulation of its turnover by hydrolytic enzymes is much less well understood. Bacillus subtilis has a multitude of such enzymes. Two of the best characterized are CwlO and LytE: cells lacking both enzymes have a lethal block in cell elongation. Here we show that activity of CwlO is regulated by an ABC transporter, FtsEX, which is required for cell elongation, unlike cell division as in Escherichia coli. Actin-like MreB proteins are thought to play a key role in orchestrating cell wall morphogenesis. B. subtilis has three MreB isologues with partially differentiated functions. We now show that the three MreB isologues have differential roles in regulation of the CwlO and LytE systems and that autolysins control different aspects of cell morphogenesis. The results add major autolytic activities to the growing list of functions controlled by MreB isologues in bacteria and provide new insights into the different specialized functions of essential cell wall autolysins. © 2013 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

OXP1/YKL215c encodes an ATP-dependent 5-oxoprolinase in Saccharomyces cerevisiae: functional characterization, domain structure and identification of actin-like ATP-binding motifs in eukaryotic 5-oxoprolinases.

PubMed

Kumar, Akhilesh; Bachhawat, Anand Kumar

2010-06-01

OXP1/YKL215c, an uncharacterized ORF of Saccharomyces cerevisiae, encodes a functional ATP-dependent 5-oxoprolinase of 1286 amino acids. The yeast 5-oxoprolinase activity was demonstrated in vivo by utilization of 5-oxoproline as a source of glutamate and OTC, a 5-oxoproline sulfur analogue, as a source of sulfur in cells overexpressing OXP1. In vitro characterization by expression and purification of the recombinant protein in S. cerevisiae revealed that the enzyme exists and functions as a dimer, and has a K(m) of 159 microM and a V(max) of 3.5 nmol h(-1) microg(-1) protein. The enzyme was found to be functionally separable in two distinct domains. An 'actin-like ATPase motif' could be identified in 5-oxprolinases, and mutation of key residues within this motif led to complete loss in ATPase and 5-oxoprolinase activity of the enzyme. The results are discussed in the light of the previously postulated truncated gamma-glutamyl cycle of yeasts.

Functional gene profiling through metaRNAseq approach reveals diet-dependent variation in rumen microbiota of buffalo (Bubalus bubalis).

PubMed

Hinsu, Ankit T; Parmar, Nidhi R; Nathani, Neelam M; Pandit, Ramesh J; Patel, Anand B; Patel, Amrutlal K; Joshi, Chaitanya G

2017-04-01

Recent advances in next generation sequencing technology have enabled analysis of complex microbial community from genome to transcriptome level. In the present study, metatranscriptomic approach was applied to elucidate functionally active bacteria and their biological processes in rumen of buffalo (Bubalus bubalis) adapted to different dietary treatments. Buffaloes were adapted to a diet containing 50:50, 75:25 and 100:0 forage to concentrate ratio, each for 6 weeks, before ruminal content sample collection. Metatranscriptomes from rumen fiber adherent and fiber-free active bacteria were sequenced using Ion Torrent PGM platform followed by annotation using MG-RAST server and CAZYmes (Carbohydrate active enzymes) analysis toolkit. In all the samples Bacteroidetes was the most abundant phylum followed by Firmicutes. Functional analysis using KEGG Orthology database revealed Metabolism as the most abundant category at level 1 within which Carbohydrate metabolism was dominating. Diet treatments also exerted significant differences in proportion of enzymes involved in metabolic pathways for VFA production. Carbohydrate Active Enzyme(CAZy) analysis revealed the abundance of genes encoding glycoside hydrolases with the highest representation of GH13 CAZy family in all the samples. The findings provide an overview of the activities occurring in the rumen as well as active bacterial population and the changes occurring through different dietary treatments. Copyright © 2017 Elsevier Ltd. All rights reserved.

Enzymatic processing of pigmented and non pigmented rice bran on changes in oryzanol, polyphenols and antioxidant activity.

PubMed

Prabhu, Ashish A; Jayadeep, A

2015-10-01

Bran from different rice varieties is a treasure of nutrients and nutraceuticals, and its use is limited due to the poor sensory and functional properties. Application of enzymes can alter the functional and phytochemical properties. So the effect of endo-xylanase, cellulase and their combination on microstructural, nutraceutical and antioxidant properties of pigmented (Jyothi) and non-pigmented (IR64) rice bran were investigated. Scanning electron micrograph revealed micro structural changes in fibre structures on processing. All the enzymatic processing methods resulted in an increase in the content of oryzanol, soluble, bound and total polyphenols, flavonoid and tannin. It also showed an increase in the bioactivity with respect to free radical scavenging activity and total antioxidant activity. However, extent of the increase in bio-actives varied with the type of bran and enzyme application method. Endo-xylanase showed higher percentage difference compared to controls of Jyothi and IR64 bran extracts respectively in the content of the bound (10 & 19 %) and total (20 & 14 %) polyphenols. Combination of both the enzymes resulted in higher percentage increase of bioactive components and properties. It resulted in greater percentage difference compared to controls of Jyothi and IR64 extracts respectively in the content of soluble (58 & 17 %) and total (21 & 14 %) polyphenols, flavonoids (12 & 38 %), γ-oryzanol (10 & 12 %), free radical scavenging activity (64 & 30 %) and total antioxidant activity (82 & 136 %). It may be concluded that enzymatic bio-processing of bran with cellulose and hemicellulose degrading enzymes can improve its nutraceutical properties, and it may be used for development of functional foods.

Nuclear localization of metabolic enzymes in immunity and metastasis.

PubMed

He, Yuchen; Gao, Menghui; Cao, Yiqu; Tang, Haosheng; Liu, Shuang; Tao, Yongguang

2017-12-01

Metabolism is essential to all living organisms that provide cells with energy, regulators, building blocks, enzyme cofactors and signaling molecules, and is in tune with nutritional conditions and the function of cells to make the appropriate developmental decisions or maintain homeostasis. As a fundamental biological process, metabolism state affects the production of multiple metabolites and the activation of various enzymes that participate in regulating gene expression, cell apoptosis, cancer progression and immunoreactions. Previous studies generally focus on the function played by the metabolic enzymes in the cytoplasm and mitochondrion. In this review, we conclude the role of them in the nucleus and their implications for cancer progression, immunity and metastasis. Copyright © 2017 Elsevier B.V. All rights reserved.

Active site and laminarin binding in glycoside hydrolase family 55

DOE PAGES

Bianchetti, Christopher M.; Takasuka, Taichi E.; Deutsch, Sam; ...

2015-03-09

The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium. Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define themore » active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ~30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Furthermore, application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties.« less

Advancing understanding of microbial bioenergy conversion processes by activity-based protein profiling

DOE PAGES

Liu, Yun; Fredrickson, James K.; Sadler, Natalie C.; ...

2015-09-25

Here, the development of renewable biofuels is a global priority, but success will require novel technologies that greatly improve our understanding of microbial systems biology. An approach with great promise in enabling functional characterization of microbes is activity-based protein profiling (ABPP), which employs chemical probes to directly measure enzyme function in discrete enzyme classes in vivo and/or in vitro, thereby facilitating the rapid discovery of new biocatalysts and enabling much improved biofuel production platforms. We review general design strategies in ABPP, and highlight recent advances that are or could be pivotal to biofuels processes including applications of ABPP to cellulosicmore » bioethanol, biodiesel, and phototrophic production of hydrocarbons. We also examine the key challenges and opportunities of ABPP in renewable biofuels research. The integration of ABPP with molecular and systems biology approaches will shed new insight on the catalytic and regulatory mechanisms of functional enzymes and their synergistic effects in the field of biofuels production.« less

Bioinformatics approaches for structural and functional analysis of proteins in secondary metabolism in Withania somnifera.

PubMed

Sanchita; Singh, Swati; Sharma, Ashok

2014-11-01

Withania somnifera (Ashwagandha) is an affluent storehouse of large number of pharmacologically active secondary metabolites known as withanolides. These secondary metabolites are produced by withanolide biosynthetic pathway. Very less information is available on structural and functional aspects of enzymes involved in withanolides biosynthetic pathways of Withiana somnifera. We therefore performed a bioinformatics analysis to look at functional and structural properties of these important enzymes. The pathway enzymes taken for this study were 3-Hydroxy-3-methylglutaryl coenzyme A reductase, 1-Deoxy-D-xylulose-5-phosphate synthase, 1-Deoxy-D-xylulose-5-phosphate reductase, farnesyl pyrophosphate synthase, squalene synthase, squalene epoxidase, and cycloartenol synthase. The prediction of secondary structure was performed for basic structural information. Three-dimensional structures for these enzymes were predicted. The physico-chemical properties such as pI, AI, GRAVY and instability index were also studied. The current information will provide a platform to know the structural attributes responsible for the function of these protein until experimental structures become available.

Non-metabolic functions of glycolytic enzymes in tumorigenesis.

PubMed

Yu, X; Li, S

2017-05-11

Cancer cells reprogram their metabolism to meet the requirement for survival and rapid growth. One hallmark of cancer metabolism is elevated aerobic glycolysis and reduced oxidative phosphorylation. Emerging evidence showed that most glycolytic enzymes are deregulated in cancer cells and play important roles in tumorigenesis. Recent studies revealed that all essential glycolytic enzymes can be translocated into nucleus where they participate in tumor progression independent of their canonical metabolic roles. These noncanonical functions include anti-apoptosis, regulation of epigenetic modifications, modulation of transcription factors and co-factors, extracellular cytokine, protein kinase activity and mTORC1 signaling pathway, suggesting that these multifaceted glycolytic enzymes not only function in canonical metabolism but also directly link metabolism to epigenetic and transcription programs implicated in tumorigenesis. These findings underscore our understanding about how tumor cells adapt to nutrient and fuel availability in the environment and most importantly, provide insights into development of cancer therapy.

Mechanistic Models of Light-limited and Light-saturated Rates of Photosynthesis

DTIC Science & Technology

1997-09-30

concentrations of the rate-limiting enzyme ribulose-1,5-bisphosphate carboxylase ( Rubisco ) that catalyzes carbon fixation. This work is supported by...saturated photosynthesis as a function of the activity of the key photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase ( Rubisco ) for a Report...concentration and its specific rate. The Rubisco activities were determined at the same temperature as the photosynthetic measurements, and hence did not

Molecular dynamics simulations and statistical coupling analysis of GPI12 in L. major: functional co-evolution and conservedness reveals potential drug-target sites.

PubMed

Singh, Shailza; Mandlik, Vineetha; Shinde, Sonali

2015-03-01

GPI12 represents an important enzyme in the GPI biosynthetic pathway of several parasites like 'Leishmania'. GPI activity is generally regulated through either the hindrance in GPI complex assembly formation or the modulation of the lipophosphoglycan (LPG) flux to either reduce or enhance the pathogenicity in an organism. Of the various GPI molecules known, GPI12 is an important enzyme in the GPI biosynthetic pathway which can be exploited as a target due to the substrate specificity difference in parasites and humans. In the present study, the functional importance of the co-evolving residues of the GPI12 protein of Leishmania has been highlighted using the GPI proteins belonging to the GlcNAC-deacetylase family. Exploring the active site of the GPI12 protein and designing inhibitors against the functional residues provide ways and means to change the efficiency of deacetylation activity of the enzyme. The activity of de-N-acetylase is low in the absence of metal ions like zinc. Hence we designed eight small molecules in order to modulate the activity of GPI12. Compound 8 was found to be an appropriate choice to target the agonist (GPI12) active site thereby targeting the residues which were essential in the Zn binding and chelation activity. Inhibition of these sites offered a strong constraint to block the protein activity and in turn GPI biosynthesis.

The effect of deep eutectic solvents on catalytic function and structure of bovine liver catalase.

PubMed

Harifi-Mood, Ali Reza; Ghobadi, Roohollah; Divsalar, Adeleh

2017-02-01

Aqueous solutions of reline and glyceline, the most common deep eutectic solvents, were used as a medium for Catalase reaction. By some spectroscopic methods such as UV-vis, fluorescence and circular dichroism (CD) function and structure of Catalase were investigated in aqueous solutions of reline and glyceline. These studies showed that the binding affinity of the substrate to the enzyme increased in the presence of 100mM glyceline solution, which contrasts with reline solution that probably relates to instructive changes in secondary structure of protein. Meanwhile, enzyme remained nearly 70% and 80% active in this concentration of glyceline and reline solutions respectively. In the high concentration of DES solutions, enzyme became mainly inactive but surprisingly stayed in nearly 40% active in choline chloride solution, which is the common ion species in reline and glyceline solvents. It is proposed that the chaotropic nature of choline cation might stop the reducing trend of activity in concentrated choline chloride solutions but this instructive effect is lost in aqueous deep eutectic solvents. In this regard, the presence of various concentrations of deep eutectic solvents in the aqueous media of human cells would be an activity adjuster for this important enzyme in its different operation conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

A Novel Multifunctional β-N-Acetylhexosaminidase Revealed through Metagenomics of an Oil-Spilled Mangrove

PubMed Central

Soares, Fábio Lino; Marcon, Joelma; Khakhum, Nittaya; Cerdeira, Louise Teixeira; Domingos, Daniela Ferreira; Taketani, Rodrigo Gouvea; de Oliveira, Valéria Maia; Lima, André Oliveira de Souza

2017-01-01

The use of culture-independent approaches, such as metagenomics, provides complementary access to environmental microbial diversity. Mangrove environments represent a highly complex system with plenty of opportunities for finding singular functions. In this study we performed a functional screening of fosmid libraries obtained from an oil contaminated mangrove site, with the purpose of identifying clones expressing hydrolytic activities. A novel gene coding for a β-N-acetylhexosaminidase with 355 amino acids and 43KDa was retrieved and characterized. The translated sequence showed only 38% similarity to a β-N-acetylhexosaminidase gene in the genome of Veillonella sp. CAG:933, suggesting that it might constitute a novel enzyme. The enzyme was expressed, purified, and characterized for its enzymatic activity on carboxymethyl cellulose, p-Nitrophenyl-2acetamide-2deoxy-β-d-glucopyranoside, p-Nitrophenyl-2acetamide-2deoxy-β-d-galactopyranoside, and 4-Nitrophenyl β-d-glucopyranoside, presenting β-N-acetylglucosaminidase, β-glucosidase, and β-1,4-endoglucanase activities. The enzyme showed optimum activity at 30 °C and pH 5.5. The characterization of the putative novel β-N-acetylglucosaminidase enzyme reflects similarities to characteristics of the environment explored, which differs from milder conditions environments. This work exemplifies the application of cultivation-independent molecular techniques to the mangrove microbiome for obtaining a novel biotechnological product. PMID:28952541

[The experimental research of inner ear metabolism and electrical physiology of autoimmune sensorineural hearing loss].

PubMed

Tan, C; Cao, Y; Hu, P

1998-09-01

Inquire into the mechanism of inner ear pathological physiology in autoimmune sensorineural hearing loss (ASHL). With the auditory electric-physiological techniques and enzyme-histochemical method, the change of inner ear hearing function and enzyme activity were observed. These animals, which threshold of auditory nerve compound active potential (CAP) and cochlear microphonic potential(CM) heightening evidently, showed that the amplitude of endolymphatic potential(EP) (include-EP) bring down in various degrees, which was related to the change of the active of Na(+)-K(+)-ATPase and SDH in vascularis stria and endolymphatic sac. The abnormality of enzymes metabolism in inner ear tissues, which following autoimmune inflammation damage, is the pathological foundation of hearing dysfunction.

Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation.

PubMed

Droux, M; Miginiac-Maslow, M; Jacquot, J P; Gadal, P; Crawford, N A; Kosower, N S; Buchanan, B B

1987-07-01

The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with [14C]iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.

Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

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

Droux, M.; Miginiac-Maslow, M.; Jacquot, J.P.

The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with (/sup 14/C)iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn,more » reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.« less

Functionalized Anodic Aluminum Oxide Membrane–Electrode System for Enzyme Immobilization

PubMed Central

2015-01-01

A nanoporous membrane system with directed flow carrying reagents to sequentially attached enzymes to mimic nature’s enzyme complex system was demonstrated. Genetically modified glycosylation enzyme, OleD Loki variant, was immobilized onto nanometer-scale electrodes at the pore entrances/exits of anodic aluminum oxide membranes through His6-tag affinity binding. The enzyme activity was assessed in two reactions—a one-step “reverse” sugar nucleotide formation reaction (UDP-Glc) and a two-step sequential sugar nucleotide formation and sugar nucleotide-based glycosylation reaction. For the one-step reaction, enzyme specific activity of 6–20 min–1 on membrane supports was seen to be comparable to solution enzyme specific activity of 10 min–1. UDP-Glc production efficiencies as high as 98% were observed at a flow rate of 0.5 mL/min, at which the substrate residence time over the electrode length down pore entrances was matched to the enzyme activity rate. This flow geometry also prevented an unwanted secondary product hydrolysis reaction, as observed in the test homogeneous solution. Enzyme utilization increased by a factor of 280 compared to test homogeneous conditions due to the continuous flow of fresh substrate over the enzyme. To mimic enzyme complex systems, a two-step sequential reaction using OleD Loki enzyme was performed at membrane pore entrances then exits. After UDP-Glc formation at the entrance electrode, aglycon 4-methylumbelliferone was supplied at the exit face of the reactor, affording overall 80% glycosylation efficiency. The membrane platform showed the ability to be regenerated with purified enzyme as well as directly from expression crude, thus demonstrating a single-step immobilization and purification process. PMID:25025628

Insights into Substrate Specificity and Metal Activation of Mammalian Tetrahedral Aspartyl Aminopeptidase*

PubMed Central

Chen, Yuanyuan; Farquhar, Erik R.; Chance, Mark R.; Palczewski, Krzysztof; Kiser, Philip D.

2012-01-01

Aminopeptidases are key enzymes involved in the regulation of signaling peptide activity. Here, we present a detailed biochemical and structural analysis of an evolutionary highly conserved aspartyl aminopeptidase called DNPEP. We show that this peptidase can cleave multiple physiologically relevant substrates, including angiotensins, and thus may play a key role in regulating neuron function. Using a combination of x-ray crystallography, x-ray absorption spectroscopy, and single particle electron microscopy analysis, we provide the first detailed structural analysis of DNPEP. We show that this enzyme possesses a binuclear zinc-active site in which one of the zinc ions is readily exchangeable with other divalent cations such as manganese, which strongly stimulates the enzymatic activity of the protein. The plasticity of this metal-binding site suggests a mechanism for regulation of DNPEP activity. We also demonstrate that DNPEP assembles into a functionally relevant tetrahedral complex that restricts access of peptide substrates to the active site. These structural data allow rationalization of the enzyme's preference for short peptide substrates with N-terminal acidic residues. This study provides a structural basis for understanding the physiology and bioinorganic chemistry of DNPEP and other M18 family aminopeptidases. PMID:22356908

Enzyme-Responsive Nanomaterials for Controlled Drug Delivery

PubMed Central

Hu, Quanyin; Katti, Prateek S.; Gu, Zhen

2015-01-01

Enzymes underpin physiological function and exhibit dysregulation in many disease-associated microenvironments and aberrant cell processes. Exploiting altered enzyme activity and expression for diagnostics, drug targeting, and drug release is tremendously promising. When combined with booming research in nanobiotechnology, enzyme-responsive nanomaterials for controlled drug release have achieved significant development and been studied as an important class of drug delivery devices in nanomedicine. In this review, we describe enzymes such as proteases, phospholipase and oxidoreductases that serve as delivery triggers. Subsequently, we explore recently developed enzyme-responsive nanomaterials with versatile applications for extracellular and intracellular drug delivery. We conclude by discussing future opportunities and challenges in this area. PMID:25251024

Enzyme-responsive nanomaterials for controlled drug delivery

NASA Astrophysics Data System (ADS)

Hu, Quanyin; Katti, Prateek S.; Gu, Zhen

2014-10-01

Enzymes underpin physiological function and exhibit dysregulation in many disease-associated microenvironments and aberrant cell processes. Exploiting altered enzyme activity and expression for diagnostics, drug targeting, and drug release is tremendously promising. When combined with booming research in nanobiotechnology, enzyme-responsive nanomaterials used for controlled drug release have achieved significant development and have been studied as an important class of drug delivery strategies in nanomedicine. In this review, we describe enzymes such as proteases, phospholipases and oxidoreductases that serve as delivery triggers. Subsequently, we explore recently developed enzyme-responsive nanomaterials with versatile applications for extracellular and intracellular drug delivery. We conclude by discussing future opportunities and challenges in this area.

Exploiting bacterial drug resistance: a single construct for the diagnosis and treatment of drug resistant infections

NASA Astrophysics Data System (ADS)

Sallum, Ulysses W.; Zheng, Xiang; Verma, Sarika; Hasan, Tayyaba

2009-06-01

β-lactamase enzyme-activated photosensitizer (β-LEAP). We aim to exploit drug resistance mechanisms to selectively release photosensitizers (PSs) for a specific photodynamic antimicrobial effect and reduced host tissue damage. Consequently, the fluorescence emission intensity of the PSs increases and allows for the detection of enzyme activity. In this work we sought to evaluate β-LEAP for use as a sensitive molecular probe. We have reported the enzyme specific antibacterial action of β-LEAP. Here we report the use of β-LEAP for the rapid functional definition of a β-lactamase.

The functional consequences of mis-sense mutations affecting an intra-molecular salt bridge in arylsulphatase A.

PubMed Central

Schestag, Frank; Yaghootfam, Afshin; Habetha, Matthias; Poeppel, Peter; Dietz, Frank; Klein, Roger A; Zlotogora, Joel; Gieselmann, Volkmar

2002-01-01

Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulphatase A. We describe the functional consequences of three mis-sense mutations in the arylsulphatase A gene (Asp-335-Val, Arg-370-Trp and Arg-370-Gln), affecting an apparent intramolecular Asp-335 to Arg-370 salt bridge, and interpret the effects and clinical consequences on the basis of the three-dimensional structure of arylsulphatase A. Asp-335-Val and Arg-370-Trp substitutions each cause a complete loss of enzyme activity and are associated with the most severe form of the human disease, whereas the Arg-370-Gln-substituted enzyme retains some residual activity, being found in a patient suffering from the milder juvenile form of the disease. Detailed analysis reveals that formation of the apparent salt bridge depends critically on the presence of aspartic acid and arginine residues at positions 335 and 370, respectively. Substitution by various other amino acids, including glutamic acid and lysine, affects enzyme function severely. Biosynthesis and immunoprecipitation studies indicate that the Asp-335-Val substitution affects folding of arylsulphatase A more severely than either the Arg-370-Trp or Arg-370-Gln substitutions. In vitro mutagenesis data show that clinical severity correlates with the space occupied by residue 370. The combination with structural data suggests that the bulky tryptophan residue broadens the cleft held together by the apparent salt bridge, whereas the smaller glutamine residue still allows the cleft to close, yielding a less severely affected enzyme. The position of residue 370 in the three-dimensional structure of the enzyme provides a plausible explanation for the differing severities in loss of enzyme function caused by the mutations and thus the clinical phenotype. PMID:12086582

Two Alternative Pathways for the Synthesis of the Rare Compatible Solute Mannosylglucosylglycerate in Petrotoga mobilis▿

PubMed Central

Fernandes, Chantal; Mendes, Vitor; Costa, Joana; Empadinhas, Nuno; Jorge, Carla; Lamosa, Pedro; Santos, Helena; da Costa, Milton S.

2010-01-01

The compatible solute mannosylglucosylglycerate (MGG), recently identified in Petrotoga miotherma, also accumulates in Petrotoga mobilis in response to hyperosmotic conditions and supraoptimal growth temperatures. Two functionally connected genes encoding a glucosyl-3-phosphoglycerate synthase (GpgS) and an unknown glycosyltransferase (gene Pmob_1143), which we functionally characterized as a mannosylglucosyl-3-phosphoglycerate synthase and designated MggA, were identified in the genome of Ptg. mobilis. This enzyme used the product of GpgS, glucosyl-3-phosphoglycerate (GPG), as well as GDP-mannose to produce mannosylglucosyl-3-phosphoglycerate (MGPG), the phosphorylated precursor of MGG. The MGPG dephosphorylation was determined in cell extracts, and the native enzyme was partially purified and characterized. Surprisingly, a gene encoding a putative glucosylglycerate synthase (Ggs) was also identified in the genome of Ptg. mobilis, and an active Ggs capable of producing glucosylglycerate (GG) from ADP-glucose and d-glycerate was detected in cell extracts and the recombinant enzyme was characterized, as well. Since GG has never been identified in this organism nor was it a substrate for the MggA, we anticipated the existence of a nonphosphorylating pathway for MGG synthesis. We putatively identified the corresponding gene, whose product had some sequence homology with MggA, but it was not possible to recombinantly express a functional enzyme from Ptg. mobilis, which we named mannosylglucosylglycerate synthase (MggS). In turn, a homologous gene from Thermotoga maritima was successfully expressed, and the synthesis of MGG was confirmed from GDP-mannose and GG. Based on the measurements of the relevant enzyme activities in cell extracts and on the functional characterization of the key enzymes, we propose two alternative pathways for the synthesis of the rare compatible solute MGG in Ptg. mobilis. PMID:20061481

Chemical modification of L-asparaginase from Cladosporium sp. for improved activity and thermal stability.

PubMed

Mohan Kumar, N S; Kishore, Vijay; Manonmani, H K

2014-01-01

L-Asparaginase (ASNase), an antileukemia enzyme, is facing problems with antigenicity in the blood. Modification of L-asparaginase from Cladosporium sp. was tried to obtain improved stability and improved functionality. In our experiment, modification of the enzyme was tried with bovine serum albumin, ovalbumin by crosslinking using glutaraldehyde, N-bromosuccinimide, and mono-methoxy polyethylene glycol. Modified enzymes were studied for activity, temperature stability, rate constants (kd), and protection to proteolytic digestion. Modification with ovalbumin resulted in improved enzyme activity that was 10-fold higher compared to native enzyme, while modification with bovine serum albumin through glutaraldehyde cross-linking resulted in high stability of L-asparaginase that was 8.5- and 7.62-fold more compared to native enzyme at 28°C and 37°C by the end of 24 hr. These effects were dependent on the quantity of conjugate formed. Modification also markedly prolonged L-asparaginase half-life and serum stability. N-Bromosuccinimide-modified ASNase presented greater stability with prolonged in vitro half-life of 144 hr to proteolytic digestion relative to unmodified enzyme (93 h). The present work could be seen as producing a modified L-asparaginase with improved activity and stability and can be a potential source for developing therapeutic agents for cancer treatment.

Enzyme inhibitory metabolites from endophytic Penicillium citrinum isolated from Boswellia sacra.

PubMed

Ali, Sajid; Khan, Abdul Latif; Ali, Liaqat; Rizvi, Tania Shamim; Khan, Sumera Afzal; Hussain, Javid; Hamayun, Muhammad; Al-Harrasi, Ahmed

2017-07-01

Fungal endophytes establish an important niche within the host plant through the secretion of chemical constituents. Isolation of bioactive metabolites could be a vital source for inhibiting the function of enzymes such as α-glucosidase and urease. The present study aimed to elucidate the potential of endophytes associated with Boswellia sacra through bioassay-guided isolation and identification of secondary metabolites with enzyme inhibitory ability. Endophytic fungal strains viz. Penicillium citrinum, P. spinulosum, Fusarium oxysporum, Alternaria alternata and Aspergillus caespitosus were identified through genomic DNA extraction, PCR amplification, sequencing and phylogenetic analysis. The enzymes inhibition analysis of the ethyl acetate extract from pure cultures suggested that P. citrinum possess significantly higher enzyme inhibitory activities compared to other strains. The active strain was subjected to chromatographic isolation and nuclear magnetic resonance methods to identify bioactive compounds. The bioactive extracts resulted in the isolation of 11-oxoursonic acid benzyl ester (1), n-nonane (2), 3-decene-1-ol (3), 2-Hydroxyphenyl acetic acid (4), and Glochidacuminosides A (5). Among pure compound, 11-oxoursonic acid benzyl ester (1) showed significantly higher enzyme inhibition activity compared to other metabolites. Our results suggest that the endophytic microorganism associated with the arid-land tree can offer a rich source of biologically active chemical constituents that could help discover lead drugs for enzyme inhibition.

Demonstration of 3 alpha(17 beta)-hydroxysteroid dehydrogenase distinct from 3 alpha-hydroxysteroid dehydrogenase in hamster liver.

PubMed Central

Ohmura, M; Hara, A; Nakagawa, M; Sawada, H

1990-01-01

NAD(+)-linked and NADP(+)-linked 3 alpha-hydroxysteroid dehydrogenases were purified to homogeneity from hamster liver cytosol. The two monomeric enzymes, although having similar molecular masses of 38,000, differed from each other in pI values, activation energy and heat stability. The two proteins also gave different fragmentation patterns by gel electrophoresis after digestion with protease. The NADP(+)-linked enzyme catalysed the oxidoreduction of various 3 alpha-hydroxysteroids, whereas the NAD(+)-linked enzyme oxidized the 3 alpha-hydroxy group of pregnanes and some bile acids, and the 17 beta-hydroxy group of testosterone and androstanes. The thermal stabilities of the 3 alpha- and 17 beta-hydroxysteroid dehydrogenase activities of the NAD(+)-linked enzyme were identical, and the two enzyme activities were inhibited by mixing 17 beta- and 3 alpha-hydroxysteroid substrates, respectively. Medroxyprogesterone acetate, hexoestrol and 3 beta-hydroxysteroids competitively inhibited 3 alpha- and 17 beta-hydroxysteroid dehydrogenase activities of the enzyme. These results show that hamster liver contains a 3 alpha(17 beta)-hydroxysteroid dehydrogenase structurally and functionally distinct from 3 alpha-hydroxysteroid dehydrogenase. Images Fig. 1. Fig. 2. PMID:2317205

Crystal structure of a cold-active protease (Pro21717) from the psychrophilic bacterium, Pseudoalteromonas arctica PAMC 21717, at 1.4 Å resolution: Structural adaptations to cold and functional analysis of a laundry detergent enzyme.

PubMed

Park, Ha Ju; Lee, Chang Woo; Kim, Dockyu; Do, Hackwon; Han, Se Jong; Kim, Jung Eun; Koo, Bon-Hun; Lee, Jun Hyuck; Yim, Joung Han

2018-01-01

Enzymes isolated from organisms found in cold habitats generally exhibit higher catalytic activity at low temperatures than their mesophilic homologs and are therefore known as cold-active enzymes. Cold-active proteases are very useful in a variety of biotechnological applications, particularly as active ingredients in laundry and dishwashing detergents, where they provide strong protein-degrading activity in cold water. We identified a cold-active protease (Pro21717) from a psychrophilic bacterium, Pseudoalteromonas arctica PAMC 21717, and determined the crystal structure of its catalytic domain (CD) at a resolution of 1.4 Å. The Pro21717-CD structure shows a conserved subtilisin-like fold with a typical catalytic triad (Asp185, His244, and Ser425) and contains four calcium ions and three disulfide bonds. Interestingly, we observed an unexpected electron density at the substrate-binding site from a co-purified peptide. Although the sequence of this peptide is unknown, analysis of the peptide-complexed structure nonetheless provides some indication of the substrate recognition and binding mode of Pro21717. Moreover, various parameters, including a wide substrate pocket size, an abundant active-site loop content, and a flexible structure provide potential explanations for the cold-adapted properties of Pro21717. In conclusion, this is first structural characterization of a cold-adapted subtilisin-like protease, and these findings provide a structural and functional basis for industrial applications of Pro21717 as a cold-active laundry or dishwashing detergent enzyme.

Crystal structure of a cold-active protease (Pro21717) from the psychrophilic bacterium, Pseudoalteromonas arctica PAMC 21717, at 1.4 Å resolution: Structural adaptations to cold and functional analysis of a laundry detergent enzyme

PubMed Central

Do, Hackwon; Han, Se Jong; Kim, Jung Eun; Koo, Bon-Hun; Yim, Joung Han

2018-01-01

Enzymes isolated from organisms found in cold habitats generally exhibit higher catalytic activity at low temperatures than their mesophilic homologs and are therefore known as cold-active enzymes. Cold-active proteases are very useful in a variety of biotechnological applications, particularly as active ingredients in laundry and dishwashing detergents, where they provide strong protein-degrading activity in cold water. We identified a cold-active protease (Pro21717) from a psychrophilic bacterium, Pseudoalteromonas arctica PAMC 21717, and determined the crystal structure of its catalytic domain (CD) at a resolution of 1.4 Å. The Pro21717-CD structure shows a conserved subtilisin-like fold with a typical catalytic triad (Asp185, His244, and Ser425) and contains four calcium ions and three disulfide bonds. Interestingly, we observed an unexpected electron density at the substrate-binding site from a co-purified peptide. Although the sequence of this peptide is unknown, analysis of the peptide-complexed structure nonetheless provides some indication of the substrate recognition and binding mode of Pro21717. Moreover, various parameters, including a wide substrate pocket size, an abundant active-site loop content, and a flexible structure provide potential explanations for the cold-adapted properties of Pro21717. In conclusion, this is first structural characterization of a cold-adapted subtilisin-like protease, and these findings provide a structural and functional basis for industrial applications of Pro21717 as a cold-active laundry or dishwashing detergent enzyme. PMID:29466378

Modified TMV Particles as Beneficial Scaffolds to Present Sensor Enzymes

PubMed Central

Koch, Claudia; Wabbel, Katrin; Eber, Fabian J.; Krolla-Sidenstein, Peter; Azucena, Carlos; Gliemann, Hartmut; Eiben, Sabine; Geiger, Fania; Wege, Christina

2015-01-01

Tobacco mosaic virus (TMV) is a robust nanotubular nucleoprotein scaffold increasingly employed for the high density presentation of functional molecules such as peptides, fluorescent dyes, and antibodies. We report on its use as advantageous carrier for sensor enzymes. A TMV mutant with a cysteine residue exposed on every coat protein (CP) subunit (TMVCys) enabled the coupling of bifunctional maleimide-polyethylene glycol (PEG)-biotin linkers (TMVCys/Bio). Its surface was equipped with two streptavidin [SA]-conjugated enzymes: glucose oxidase ([SA]-GOx) and horseradish peroxidase ([SA]-HRP). At least 50% of the CPs were decorated with a linker molecule, and all thereof with active enzymes. Upon use as adapter scaffolds in conventional “high-binding” microtiter plates, TMV sticks allowed the immobilization of up to 45-fold higher catalytic activities than control samples with the same input of enzymes. Moreover, they increased storage stability and reusability in relation to enzymes applied directly to microtiter plate wells. The functionalized TMV adsorbed to solid supports showed a homogeneous distribution of the conjugated enzymes and structural integrity of the nanorods upon transmission electron and atomic force microscopy. The high surface-increase and steric accessibility of the viral scaffolds in combination with the biochemical environment provided by the plant viral coat may explain the beneficial effects. TMV can, thus, serve as a favorable multivalent nanoscale platform for the ordered presentation of bioactive proteins. PMID:26734040

Exploring the potential impact of an expanded genetic code on protein function

DOE PAGES

Xiao, Han; Nasertorabi, Fariborz; Choi, Sei -hyun; ...

2015-05-18

With few exceptions, all living organisms encode the same 20 canonical amino acids; however, it remains an open question whether organisms with additional amino acids beyond the common 20 might have an evolutionary advantage. In this paper, we begin to test that notion by making a large library of mutant enzymes in which 10 structurally distinct noncanonical amino acids were substituted at single sites randomly throughout TEM-1 β-lactamase. A screen for growth on the β-lactam antibiotic cephalexin afforded a unique p-acrylamido-phenylalanine (AcrF) mutation at Val-216 that leads to an increase in catalytic efficiency by increasing k cat, but not significantlymore » affecting K M. To understand the structural basis for this enhanced activity, we solved the X-ray crystal structures of the ligand-free mutant enzyme and of the deacylation-defective wild-type and mutant cephalexin acyl-enzyme intermediates. These structures show that the Val-216–AcrF mutation leads to conformational changes in key active site residues—both in the free enzyme and upon formation of the acyl-enzyme intermediate—that lower the free energy of activation of the substrate transacylation reaction. Finally, the functional changes induced by this mutation could not be reproduced by substitution of any of the 20 canonical amino acids for Val-216, indicating that an expanded genetic code may offer novel solutions to proteins as they evolve new activities.« less

A small-molecule switch for Golgi sulfotransferases.

PubMed

de Graffenried, Christopher L; Laughlin, Scott T; Kohler, Jennifer J; Bertozzi, Carolyn R

2004-11-30

The study of glycan function is a major frontier in biology that could benefit from small molecules capable of perturbing carbohydrate structures on cells. The widespread role of sulfotransferases in modulating glycan function makes them prime targets for small-molecule modulators. Here, we report a system for conditional activation of Golgi-resident sulfotransferases using a chemical inducer of dimerization. Our approach capitalizes on two features shared by these enzymes: their requirement of Golgi localization for activity on cellular substrates and the modularity of their catalytic and localization domains. Fusion of these domains to the proteins FRB and FKBP enabled their induced assembly by the natural product rapamycin. We applied this strategy to the GlcNAc-6-sulfotransferases GlcNAc6ST-1 and GlcNAc6ST-2, which collaborate in the sulfation of L-selectin ligands. Both the activity and specificity of the inducible enzymes were indistinguishable from their WT counterparts. We further generated rapamycin-inducible chimeric enzymes comprising the localization domain of a sulfotransferase and the catalytic domain of a glycosyltransferase, demonstrating the generality of the system among other Golgi enzymes. The approach provides a means for studying sulfate-dependent processes in cellular systems and, potentially, in vivo.

Tryptophan biosynthetic enzymes of Staphylococcus aureus.

PubMed

Proctor, A R; Kloos, W E

1973-04-01

Tryptophan biosynthetic enzymes were assayed in various tryptophan mutants of Staphylococcus aureus strain 655 and the wild-type parent. All mutants, except trpB mutants, lacked only the activity corresponding to the particular biosynthetic block, as suggested previously by analysis of accumulated intermediates and auxonography. Tryptophan synthetase A was not detected in extracts of either trpA or trpB mutants but appeared normal in other mutants. Mutants in certain other classes exhibited partial loss of another particular tryptophan enzyme activity. Tryptophan synthetase B activity was not detected in cell extract preparations but was detected in whole cells. The original map order proposed for the S. aureus tryptophan gene cluster was clarified by the definition of trpD (phosphoribosyl transferase(-)) and trpF (phosphoribosyl anthranilate isomerase(-)) mutants. These mutants were previously unresolved and designated as trp(DF) mutants (anthranilate accumulators). Phosphoribosyl anthranilate isomerase and indole-3-glycerol phosphate synthetase enzymes were separable by molecular sieve chromatography, suggesting that these functions are coded by separate loci. Molecular sieve chromatography failed to reveal aggregates involving anthranilate synthetase, phosphoribosyl transferase, phosphoribosyl anthranilate isomerase, and indole-3-glycerol phosphate synthetase, and this procedure provided an estimate of the molecular weights of these enzymes. Tryptophan was shown to repress synthesis of all six tryptophan biosynthetic enzymes, and derepression of all six activities was incident upon tryptophan starvation. Tryptophan inhibited the activity of anthranilate synthetase, the first enzyme of the pathway.

Synthesis and evaluation of novel triazoles and mannich bases functionalized 1,4-dihydropyridine as angiotensin converting enzyme (ACE) inhibitors.

PubMed

Kumbhare, Ravindra M; Kosurkar, Umesh B; Bagul, Pankaj K; Kanwal, Abhinav; Appalanaidu, K; Dadmal, Tulshiram L; Banerjee, Sanjay Kumar

2014-11-01

A series of novel diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate embedded triazole and mannich bases were synthesized, and evaluated for their angiotensin converting enzyme (ACE) inhibitory activity. Screening of above synthesized compounds for ACE inhibition showed that triazoles functionalized compounds have better ACE inhibitory activity compared to that of mannich bases analogues. Among all triazoles we found 6 h, 6 i and 6 j to have good ACE inhibition activity with IC50 values 0.713 μM, 0.409 μM and 0.653 μM, respectively. Among mannich bases series compounds, only 7c resulted as most active ACE inhibitor with IC50 value of 0.928 μM. Copyright © 2014. Published by Elsevier Ltd.

A designed bifunctional laccase/β-1,3-1,4-glucanase enzyme shows synergistic sugar release from milled sugarcane bagasse.

PubMed

Furtado, G P; Ribeiro, L F; Lourenzoni, M R; Ward, R J

2013-01-01

A bifunctional enzyme has been created by fusing two Bacillus subtilis enzymes: the β-1,3-1,4-glucanase (BglS, EC 3.2.1.73) that hydrolyzes plant cell wall β-glucans and the copper-dependent oxidase laccase (CotA, EC 1.10.3.2) that catalyzes the oxidation of aromatic compounds with simultaneous reduction of oxygen to water. The chimeric laccase/β-1,3-1,4-glucanase was created by insertion fusion of the bglS and cotA genes, and expressed in Escherichia coli. The affinity-purified recombinant chimeric enzyme showed both laccase and glucanase activities, with a maximum laccase activity at pH 4.5 and 75°C that showed a V(max) 30% higher than observed for the parental laccase. The maximum glucanase activity in the chimeric enzyme was at pH 6.0 and 50°C, with a slight reduction in V(max) by ∼10% compared with the parental glucanase. A decreased K(M) resulted in an overall increase in the K(cat)/K(M) value for the glucanase activity of the chimeric enzyme. The hydrolytic activity of the chimera was 20% higher against natural milled sugarcane bagasse as compared with equimolar mixtures of the separate parental enzymes. Molecular dynamics simulations indicated the approximation of the two catalytic domains in the chimeric enzyme, and the formation of an inter-domain interface may underlie the improved catalytic function.

Interactions of β-Lactamases with Sanfetrinem (GV 104326) Compared to Those with Imipenem and with Oral β-Lactams

PubMed Central

Babini, Gioia S.; Yuan, Meifang; Livermore, David M.

1998-01-01

Sanfetrinem is a trinem β-lactam which can be administered orally as a hexatil ester. We examined whether its β-lactamase interactions resembled those of the available carbapenems, i.e., stable to AmpC and extended-spectrum β-lactamases but labile to class B and functional group 2f enzymes. The comparator drugs were imipenem, oral cephalosporins, and amoxicillin. MICs were determined for β-lactamase expression variants, and hydrolysis was examined directly with representative enzymes. Sanfetrinem was a weak inducer of AmpC β-lactamases below the MIC and had slight lability, with a kcat of 0.00033 s−1 for the Enterobacter cloacae enzyme. Its MICs for AmpC-derepressed E. cloacae and Citrobacter freundii were 4 to 8 μg/ml, compared with MICs of 0.12 to 2 μg/ml for AmpC-inducible and -basal strains; MICs for AmpC-derepressed Serratia marcescens and Morganella morganii were not raised. Cefixime and cefpodoxime were more labile than sanfetrinem to the E. cloacae AmpC enzyme, and AmpC-derepressed mutants showed much greater resistance; imipenem was more stable and retained full activity against derepressed mutants. Like imipenem, sanfetrinem was stable to TEM-1 and TEM-10 enzymes and retained full activity against isolates and transconjugants with various extended-spectrum TEM and SHV enzymes, whereas these organisms were resistant to cefixime and cefpodoxime. Sanfetrinem, like imipenem and cefixime but unlike cefpodoxime, also retained activity against Proteus vulgaris and Klebsiella oxytoca strains that hyperproduced potent chromosomal class A β-lactamases. Functional group 2f enzymes, including Sme-1, NMC-A, and an unnamed enzyme from Acinetobacter spp., increased the sanfetrinem MICs by up to 64-fold. These enzymes also compromised the activities of imipenem and amoxicillin but not those of the cephalosporins. The hydrolysis of sanfetrinem was examined with a purified Sme-1 enzyme, and biphasic kinetics were found. Finally, zinc β-lactamases, including IMP-1 and the L1 enzyme of Stenotrophomonas maltophilia, conferred resistance to sanfetrinem and all other β-lactams tested, and hydrolysis was confirmed with the IMP-1 enzyme. We conclude that sanfetrinem has β-lactamase interactions similar to those of the available carbapenems except that it is a weaker inducer of AmpC types, with some tendency to select derepressed mutants, unlike imipenem and meropenem. PMID:9593145

From Protein Engineering to Immobilization: Promising Strategies for the Upgrade of Industrial Enzymes

PubMed Central

Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

2013-01-01

Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes. PMID:23306150

A bacterial hydrogen-dependent CO2 reductase forms filamentous structures.

PubMed

Schuchmann, Kai; Vonck, Janet; Müller, Volker

2016-04-01

Interconversion of CO2 and formic acid is an important reaction in bacteria. A novel enzyme complex that directly utilizes molecular hydrogen as electron donor for the reversible reduction of CO2 has recently been identified in the Wood-Ljungdahl pathway of an acetogenic bacterium. This pathway is utilized for carbon fixation as well as energy conservation. Here we describe the further characterization of the quaternary structure of this enzyme complex and the unexpected behavior of this enzyme in polymerizing into filamentous structures. Polymerization of metabolic enzymes into similar structures has been observed only in rare cases but the increasing number of examples point towards a more general characteristic of enzyme functioning. Polymerization of the purified enzyme into ordered filaments of more than 0.1 μm in length was only dependent on the presence of divalent cations. Polymerization was a reversible process and connected to the enzymatic activity of the oxygen-sensitive enzyme with the filamentous form being the most active state. © 2016 Federation of European Biochemical Societies.

Rapid bursts and slow declines: on the possible evolutionary trajectories of enzymes.

PubMed

Newton, Matilda S; Arcus, Vickery L; Patrick, Wayne M

2015-06-06

The evolution of enzymes is often viewed as following a smooth and steady trajectory, from barely functional primordial catalysts to the highly active and specific enzymes that we observe today. In this review, we summarize experimental data that suggest a different reality. Modern examples, such as the emergence of enzymes that hydrolyse human-made pesticides, demonstrate that evolution can be extraordinarily rapid. Experiments to infer and resurrect ancient sequences suggest that some of the first organisms present on the Earth are likely to have possessed highly active enzymes. Reconciling these observations, we argue that rapid bursts of strong selection for increased catalytic efficiency are interspersed with much longer periods in which the catalytic power of an enzyme erodes, through neutral drift and selection for other properties such as cellular energy efficiency or regulation. Thus, many enzymes may have already passed their catalytic peaks. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

Functional characterization of a thermostable endoglucanase belonging to glycoside hydrolase family 45 from Fomitopsis palustris.

PubMed

Cha, Ju-Hee; Yoon, Jeong-Jun; Cha, Chang-Jun

2018-05-22

A gene encoding an endoglucanase belonging to subfamily C of glycoside hydrolase family 45 (GH45) was identified in the brown rot fungus Fomitopsis palustris and functionally expressed in Pichia pastoris. The recombinant protein displayed hydrolytic activities toward various substrates such as carboxymethyl cellulose, phosphoric acid swollen cellulose, glucomannan, lichenan, and β-glucan. In particular, the enzyme had a unique catalytic efficiency on β-1,4-glucans rather than mixed β-1,3/1,4-glucans as compared to other GH45 endoglucanases. The fungal enzyme was relatively thermostable, retaining more than 91.4% activity at 80 °C for 1 h. Site-directed mutagenesis studies revealed that the mutants N95D and D117N had significantly reduced enzymatic activities, indicating that both residues are essential for the catalytic reaction. Our study expands knowledge and understanding of the catalytic mechanism of GH45 subfamily C enzymes and also suggests that this thermostable endoglucanase from F. palustris has great potential in industrial applications.

New insights into the early steps of oil body mobilization during pollen germination

PubMed Central

Castro, Antonio Jesús

2013-01-01

In some plants, pollen grains accumulate storage lipids that serve as energy supply during germination. Here, three enzymes involved in early steps of oil body mobilization in the male gametophyte were functionally characterized for the first time. The effect of extracellular sugars on pollen performance and oil body dynamics was also analysed. Olive pollen oil bodies showed phospholipase A, lipase, and lipoxygenase activities on their surface. Enzyme activity levels increased during germination with a maximum after 3h. Removal of extracellular sugars from the germination medium did not affect pollen performance but increased enzyme activity rates and sped up oil body mobilization. Inhibitors seriously hampered pollen germination and pollen tube growth, leading to a characteristic accumulation of oil bodies in the germinative aperture. It can be concluded that storage lipids are sufficient for proper olive pollen germination. A lipase and a lipoxygenase are likely involved in oil body mobilization. Extracellular sugars may modulate their function, while a phospholipase A may promote their access to the storage lipids. PMID:23132905

Increased collagenase and dipeptidyl peptidase I activity in leucocytes from healthy elderly people

PubMed Central

Llorente, L; Richaud-Patin, Y; Díaz-Borjón, A; Jakez-Ocampo, J; Alvarado-De La Barrera, C

1999-01-01

The incidence of infectious diseases increases with ageing. The enzymatic activity of leucocytes may have a relevant role in the morbidity and mortality due to infections in the elderly. In this study we have compared the activity of enzymes involved in the inflammatory response in leucocytes from young and elderly women. A total of 35 healthy females was studied, 20 volunteers aged 78–98 years (mean 89.1 years) and 15 young controls aged 19–34 years (mean 26 years). All of them were in good clinical condition, without any acute or chronic disease. Intracellular enzyme activity was analysed by flow cytometry in leucocytes from young and elderly women. The enzyme substrates employed were for oxidative burst, l-aminopeptidase, collagenase, cathepsin B, C, D and, G and dipeptidyl peptidase I. The intracellular enzyme activity assessed by flow cytometry in leucocytes from young and elderly women was similar, as far as oxidative burst, l-aminopeptidase, cathepsin B, C, D and G are concerned. An increased collagenase activity was detected in granulocytes from elders. The mean fluorescence channels for this enzyme corresponded to 86 ± 23 and 60 ± 15 in cells from elders and controls, respectively (P = 0.01224). An increased dipeptidyl peptidase I activity was detected in lymphocytes from elderly women. The corresponding values for this enzyme in elders and the young were 65.9 ± 43.3 and 17.3 ± 5, respectively (P = 0.0036). The proper functional activity of intracellular enzymes involved in inflammatory responses is likely to be determinant for successful ageing. PMID:10361229

Rubisco Activases: AAA+ Chaperones Adapted to Enzyme Repair.

PubMed

Bhat, Javaid Y; Thieulin-Pardo, Gabriel; Hartl, F Ulrich; Hayer-Hartl, Manajit

2017-01-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the key enzyme of the Calvin-Benson-Bassham cycle of photosynthesis, requires conformational repair by Rubisco activase for efficient function. Rubisco mediates the fixation of atmospheric CO 2 by catalyzing the carboxylation of the five-carbon sugar ribulose-1,5-bisphosphate (RuBP). It is a remarkably inefficient enzyme, and efforts to increase crop yields by bioengineering Rubisco remain unsuccessful. This is due in part to the complex cellular machinery required for Rubisco biogenesis and metabolic maintenance. To function, Rubisco must undergo an activation process that involves carboxylation of an active site lysine by a non-substrate CO 2 molecule and binding of a Mg 2+ ion. Premature binding of the substrate RuBP results in an inactive enzyme. Moreover, Rubisco can also be inhibited by a range of sugar phosphates, some of which are "misfire" products of its multistep catalytic reaction. The release of the inhibitory sugar molecule is mediated by the AAA+ protein Rubisco activase (Rca), which couples hydrolysis of ATP to the structural remodeling of Rubisco. Rca enzymes are found in the vast majority of photosynthetic organisms, from bacteria to higher plants. They share a canonical AAA+ domain architecture and form six-membered ring complexes but are diverse in sequence and mechanism, suggesting their convergent evolution. In this review, we discuss recent advances in understanding the structure and function of this important group of client-specific AAA+ proteins.

Spatial localization of the first and last enzymes effectively connects active metabolic pathways in bacteria.

PubMed

Meyer, Pablo; Cecchi, Guillermo; Stolovitzky, Gustavo

2014-12-14

Although much is understood about the enzymatic cascades that underlie cellular biosynthesis, comparatively little is known about the rules that determine their cellular organization. We performed a detailed analysis of the localization of E.coli GFP-tagged enzymes for cells growing exponentially. We found that out of 857 globular enzymes, at least 219 have a discrete punctuate localization in the cytoplasm and catalyze the first or the last reaction in 60% of biosynthetic pathways. A graph-theoretic analysis of E.coli's metabolic network shows that localized enzymes, in contrast to non-localized ones, form a tree-like hierarchical structure, have a higher within-group connectivity, and are traversed by a higher number of feed-forward and feedback loops than their non-localized counterparts. A Gene Ontology analysis of these enzymes reveals an enrichment of terms related to essential metabolic functions in growing cells. Given that these findings suggest a distinct metabolic role for localization, we studied the dynamics of cellular localization of the cell wall synthesizing enzymes in B. subtilis and found that enzymes localize during exponential growth but not during stationary growth. We conclude that active biochemical pathways inside the cytoplasm are organized spatially following a rule where their first or their last enzymes localize to effectively connect the different active pathways and thus could reflect the activity state of the cell's metabolic network.

Evolutionary transitions in enzyme activity of ant fungus gardens.

PubMed

De Fine Licht, Henrik H; Schiøtt, Morten; Mueller, Ulrich G; Boomsma, Jacobus J

2010-07-01

Fungus-growing (attine) ants and their fungal symbionts passed through several evolutionary transitions during their 50 million year old evolutionary history. The basal attine lineages often shifted between two main cultivar clades, whereas the derived higher-attine lineages maintained an association with a monophyletic clade of specialized symbionts. In conjunction with the transition to specialized symbionts, the ants advanced in colony size and social complexity. Here we provide a comparative study of the functional specialization in extracellular enzyme activities in fungus gardens across the attine phylogeny. We show that, relative to sister clades, gardens of higher-attine ants have enhanced activity of protein-digesting enzymes, whereas gardens of leaf-cutting ants also have increased activity of starch-digesting enzymes. However, the enzyme activities of lower-attine fungus gardens are targeted primarily toward partial degradation of plant cell walls, reflecting a plesiomorphic state of nondomesticated fungi. The enzyme profiles of the higher-attine and leaf-cutting gardens appear particularly suited to digest fresh plant materials and to access nutrients from live cells without major breakdown of cell walls. The adaptive significance of the lower-attine symbiont shifts remains unclear. One of these shifts was obligate, but digestive advantages remained ambiguous, whereas the other remained facultative despite providing greater digestive efficiency.

Structure-activity relationships on the study of β-galactosidase folding/unfolding due to interactions with immobilization additives: Triton X-100 and ethanol.

PubMed

Soto, Dayana; Escobar, Sindy; Guzmán, Fanny; Cárdenas, Constanza; Bernal, Claudia; Mesa, Monica

2017-03-01

Improving the enzyme stability is a challenge for allowing their practical application. The surfactants are stabilizing agents, however, there are still questions about their influence on enzyme properties. The structure-activity/stability relationship for β-galactosidase from Bacillus circulans is studied here by Circular Dichroism and activity measurements, as a function of temperature and pH. The tendency of preserving the β-sheet and α-helix structures at temperatures below 65°C and different pH is the result of the balance between the large- and short-range effects, respecting to the active site. This information is fundamental for explaining the structural changes of this enzyme in the presence of Triton X-100 surfactant and ethanol. The enzyme thermal stabilization in the presence of this surfactant responds to the rearrangement of the secondary structure for having optimal activity/stability. The effect of ethanol is more related with changes in the dielectric properties of the aqueous solution than with protein structural transformations. These results contribute to understand the effects of surfactant-enzyme interactions on the enzyme behavior, from the structural point of view and to rationalize the surfactant-based stabilizing strategies for β-galactosidades. Copyright © 2016 Elsevier B.V. All rights reserved.

Functional studies of novel CYP21A2 mutations detected in Norwegian patients with congenital adrenal hyperplasia

PubMed Central

Brønstad, Ingeborg; Breivik, Lars; Methlie, Paal; Wolff, Anette S B; Bratland, Eirik; Nermoen, Ingrid; Løvås, Kristian; Husebye, Eystein S

2014-01-01

In about 95% of cases, congenital adrenal hyperplasia (CAH) is caused by mutations in CYP21A2 gene encoding steroid 21-hydroxylase (21OH). Recently, we have reported four novel CYP21A2 variants in the Norwegian population of patients with CAH, of which p.L388R and p.E140K were associated with salt wasting (SW), p.P45L with simple virilising (SV) and p.V211M+p.V281L with SV to non-classical (NC) phenotypes. We aimed to characterise the novel variants functionally utilising a newly designed in vitro assay of 21OH enzyme activity and structural simulations and compare the results with clinical phenotypes. CYP21A2 mutations and variants were expressed in vitro. Enzyme activity was assayed by assessing the conversion of 17-hydroxyprogesterone to 11-deoxycortisol by liquid chromatography tandem mass spectroscopy. PyMOL 1.3 was used for structural simulations, and PolyPhen2 and PROVEAN for predicting the severity of the mutants. The CYP21A2 mutants, p.L388R and p.E140K, exhibited 1.1 and 11.3% of wt 21OH enzyme activity, respectively, in vitro. We could not detect any functional deficiency of the p.P45L variant in vitro; although prediction tools suggest p.P45L to be pathogenic. p.V211M displayed enzyme activity equivalent to the wt in vitro, which was supported by in silico analyses. We found good correlations between phenotype and the in vitro enzyme activities of the SW mutants, but not for the SV p.P45L variant. p.V211M might have a synergistic effect together with p.V281L, explaining a phenotype between SV and NC CAH. PMID:24671123

Computational protein design and protein-ligand interaction studies for the improvement of organophosphorus degrading potential of Deinococcus radiodurans.

PubMed

Manoharan, Prabu; Sridhar, J

2018-05-01

The organophosphorus hydrolase enzyme is involved in the catalyzing reaction that involve hydrolysis of organophosphate toxic compounds. An enzyme from Deinococcus radiodurans reported as homologous to phosphotriesterase and show activity against organophosphate. In the past activity of this enzyme is low and efforts made to improve the activity by experimental mutation study. However only very few organophosphates tested against very few catalytic site mutations. In order to improve the catalytic power of the organophosphorus hydrolase enzyme, we carried out systematic functional hotspot based protein engineering strategy. The mutants tested against 46 know organophosphate compounds using molecular docking study. Finally, we carried out an extensive molecular docking study to predict the binding of 46 organophosphate compounds to wild-type protein and mutant organophosphorus hydrolase enzyme. At the end we are able to improve the degrading potential of organophosphorus hydrolase enzyme against organophosphate toxic compounds. This preliminary study and the outcome would be useful guide for the experimental scientist involved in the bioremediation of toxic organophosphate compounds. Copyright © 2018 Elsevier Inc. All rights reserved.

Relationship between physiological measurements (SFG -scope for growth-) and the functionality of the digestive gland in Mytilus galloprovincialis.

PubMed

Albentosa, Marina; Sánchez-Hernández, Miriam; Campillo, Juan Antonio; Moyano, Francisco Javier

2012-11-01

The present study was aimed to establish the relationship between the functionality of the digestive gland and physiological rates including SFG (scope for growth) in wild mussels, Mytilus galloprovincilis. The experimental set-up consisted in the evaluation of changes in the morphology of the gland, as well as in the activity of some key digestive enzymes (amylase, laminarinase, cellulase and protease) within a broad range of SFG obtained through manipulation of food ration. The higher SFG values were correlated to an increase in both the size of the digestive gland and the activities of enzymes when expressed in relation to individual. In contrast, no clear relations were observed when the activity of enzymes was expressed in relation to soluble protein, with the exception to amylase. The higher protease activities measured in mussels showing lower SFG may reflect an initial stage of catabolic processes intended to compensate the energy deficit produced by food restriction. The potential use of parameters measured in digestive glands in studies of marine pollution was discussed. Copyright © 2012 Elsevier Inc. All rights reserved.

Hydrolytic and ligninolytic enzyme activities in the Pb contaminated soil inoculated with litter-decomposing fungi.

PubMed

Kähkönen, Mika A; Lankinen, Pauliina; Hatakka, Annele

2008-06-01

The impact of Pb contamination was tested to five hydrolytic (beta-glucosidase, beta-xylosidase, beta-cellobiosidase, alpha-glucosidase and sulphatase) and two ligninolytic (manganese peroxidase, MnP and laccase) enzyme activities in the humus layer in the forest soil. The ability of eight selected litter-degrading fungi to grow and produce extracellular enzymes in the heavily Pb (40 g Pb of kg ww soil(-1)) contaminated and non-contaminated soil in the non-sterile conditions was also studied. The Pb content in the test soil was close to that of the shooting range at Hälvälä (37 g Pb of kg ww soil(-1)) in Southern Finland. The fungi were Agaricus bisporus, Agrocybe praecox, Gymnopus peronatus, Gymnopilus sapineus, Mycena galericulata, Gymnopilus luteofolius, Stropharia aeruginosa and Stropharia rugosoannulata. The Pb contamination (40 g Pb of kg ww soil(-1)) was deleterious to all five studied hydrolytic enzyme activities after five weeks of incubation. All five hydrolytic enzyme activities were significantly higher in the soil than in the extract of the soil indicating that a considerable part of enzymes were particle bound in the soils. Hydrolytic enzyme activities were higher in the non-contaminated soil than in the Pb contaminated soil. Fungal inocula increased the hydrolytic enzyme activities beta-cellobiosidase and beta-glucosidase in non-contaminated soils. All five hydrolytic enzyme activities were similar with fungi and without fungi in the Pb contaminated soil. This was in line that Pb contamination (40 g Pb of kg ww soil(-1)) depressed the growth of all fungi compared to those grown without Pb in the soil. Laccase and MnP activities were low in both Pb contaminated and non-contaminated soil cultures. MnP activities were higher in soil cultures containing Pb than without Pb. Our results showed that Pb in the shooting ranges decreased fungal growth and microbial functioning in the soil.

Threonine-Insensitive Homoserine Dehydrogenase From Soybean: Genomic Organization, Kinetic Mechanism, and In vivo Activity

USDA-ARS?s Scientific Manuscript database

Aspartate kinase (AK) and homoserine dehydrogenase (HSD) functions as key regulatory enzymes at branch points in the aspartate amino acid pathway and are feedback inhibited by threonine. In plants, the biochemical properties of AK and bifunctional AK-HSD enzymes have been characterized, but the mol...

Combinatorial enzyme technology: Conversion of pectin to oligo species and its effect on microbial growth

USDA-ARS?s Scientific Manuscript database

Plant cell wall polysaccharides, which consist of polymeric backbones with various types of substitution, were studied using the concept of combinatorial enzyme technology for conversion of agricultural fibers to functional products. Using citrus pectin as the starting substrate, an active oligo spe...

[Activity of NAD.H-generating enzymes and cytochrome content in mitochondria from rat liver and myocardium under artificial hypobiosis].

PubMed

Mel'nychuk, S D; Khyzhniak, S V; Morozova, V S; Voĭtsits'kyĭ, V M

2013-01-01

The modification particularities of the structural and functional state of the inner mitochondrial membrane of the rat liver and myocardium were observed in conditions of artificial hypobiosis, which was created using hypoxic and hypercapnic gas medium with a body temperature reduction. Under the artificial hypobiosis the activity of NAD.H-generating enzymes of the Krebs cycle of the liver mitochondria decreases. The established changes of the enzymes activity and cytochromes content of the inner mitochondrial membrane indicate the decrease of the oxidative activity of a respiratory chain, that can be limited on a terminal (cytochrome c oxidase) site and leads to the decrease (by 49% at an average) of the H+-ATPase activity of the liver mitochondria. Under the artificial hypobiosis the detected increase of the succinate-KoQ-oxidoreductase activity (by 65% at average) causes the maintaining of the functional activity of a mitochondrial respiratory chain, considering the high (relative to control) cytochrome c oxidase and H+-ATPase activities of the mitochondria of the rats' myocardium. The structural changes of the inner mitochondrial membrane of the liver and myocardium in experimental conditions are accompanied by the increase of hydrophobicity of tryptophan residues microenvironment and the intramolecular modifications of protein molecules.

Metabolic Enzymes Enjoying New Partnerships as RNA-Binding Proteins.

PubMed

Castello, Alfredo; Hentze, Matthias W; Preiss, Thomas

2015-12-01

In the past century, few areas of biology advanced as much as our understanding of the pathways of intermediary metabolism. Initially considered unimportant in terms of gene regulation, crucial cellular fate changes, cell differentiation, or malignant transformation are now known to involve 'metabolic remodeling' with profound changes in the expression of many metabolic enzyme genes. This review focuses on the recent identification of RNA-binding activity of numerous metabolic enzymes. We discuss possible roles of this unexpected second activity in feedback gene regulation ('moonlighting') and/or in the control of enzymatic function. We also consider how metabolism-driven post-translational modifications could regulate enzyme-RNA interactions. Thus, RNA emerges as a new partner of metabolic enzymes with far-reaching possible consequences to be unraveled in the future. Copyright © 2015 Elsevier Ltd. All rights reserved.

Induction of Phase 2 Antioxidant Enzymes by Broccoli Sulforaphane: Perspectives in Maintaining the Antioxidant Activity of Vitamins A, C, and E

PubMed Central

Boddupalli, Sekhar; Mein, Jonathan R.; Lakkanna, Shantala; James, Don R.

2012-01-01

Consumption of fruits and vegetables is recognized as an important part of a healthy diet. Increased consumption of cruciferous vegetables in particular has been associated with a decreased risk of several degenerative and chronic diseases, including cardiovascular disease and certain cancers. Members of the cruciferous vegetable family, which includes broccoli, Brussels sprouts, cauliflower, and cabbage, accumulate significant concentrations of glucosinolates, which are metabolized in vivo to biologically active isothiocyanates (ITCs). The ITC sulforaphane, which is derived from glucoraphanin, has garnered particular interest as an indirect antioxidant due to its extraordinary ability to induce expression of several enzymes via the KEAP1/Nrf2/ARE pathway. Nrf2/ARE gene products are typically characterized as Phase II detoxification enzymes and/or antioxidant (AO) enzymes. Over the last decade, human clinical studies have begun to provide in vivo evidence of both Phase II and AO enzyme induction by SF. Many AO enzymes are redox cycling enzymes that maintain redox homeostasis and activity of free radical scavengers such as vitamins A, C, and E. In this review, we present the existing evidence for induction of PII and AO enzymes by SF, the interactions of SF-induced AO enzymes and proposed maintenance of the essential vitamins A, C, and E, and, finally, the current view of genotypic effects on ITC metabolism and AO enzyme induction and function. PMID:22303412

Sensor potency of the moonlighting enzyme-decorated cytoskeleton: the cytoskeleton as a metabolic sensor

PubMed Central

2013-01-01

Background There is extensive evidence for the interaction of metabolic enzymes with the eukaryotic cytoskeleton. The significance of these interactions is far from clear. Presentation of the hypothesis In the cytoskeletal integrative sensor hypothesis presented here, the cytoskeleton senses and integrates the general metabolic activity of the cell. This activity depends on the binding to the cytoskeleton of enzymes and, depending on the nature of the enzyme, this binding may occur if the enzyme is either active or inactive but not both. This enzyme-binding is further proposed to stabilize microtubules and microfilaments and to alter rates of GTP and ATP hydrolysis and their levels. Testing the hypothesis Evidence consistent with the cytoskeletal integrative sensor hypothesis is presented in the case of glycolysis. Several testable predictions are made. There should be a relationship between post-translational modifications of tubulin and of actin and their interaction with metabolic enzymes. Different conditions of cytoskeletal dynamics and enzyme-cytoskeleton binding should reveal significant differences in local and perhaps global levels and ratios of ATP and GTP. The different functions of moonlighting enzymes should depend on cytoskeletal binding. Implications of the hypothesis The physical and chemical effects arising from metabolic sensing by the cytoskeleton would have major consequences on cell shape, dynamics and cell cycle progression. The hypothesis provides a framework that helps the significance of the enzyme-decorated cytoskeleton be determined. PMID:23398642

Exploring the specific features of interfacial enzymology based on lipase studies.

PubMed

Aloulou, Ahmed; Rodriguez, Jorge A; Fernandez, Sylvie; van Oosterhout, Dirk; Puccinelli, Delphine; Carrière, Frédéric

2006-09-01

Many enzymes are active at interfaces in the living world (such as in the signaling processes at the surface of cell membranes, digestion of dietary lipids, starch and cellulose degradation, etc.), but fundamental enzymology remains largely focused on the interactions between enzymes and soluble substrates. The biochemical and kinetic characterization of lipolytic enzymes has opened up new paths of research in the field of interfacial enzymology. Lipases are water-soluble enzymes hydrolyzing insoluble triglyceride substrates, and studies on these enzymes have led to the development of specific interfacial kinetic models. Structure-function studies on lipases have thrown light on the interfacial recognition sites present in the molecular structure of these enzymes, the conformational changes occurring in the presence of lipids and amphiphiles, and the stability of the enzymes present at interfaces. The pH-dependent activity, substrate specificity and inhibition of these enzymes can all result from both "classical" interactions between a substrate or inhibitor and the active site, as well as from the adsorption of the enzymes at the surface of aggregated substrate particles such as oil drops, lipid bilayers or monomolecular lipid films. The adsorption step can provide an alternative target for improving substrate specificity and developing specific enzyme inhibitors. Several data obtained with gastric lipase, classical pancreatic lipase, pancreatic lipase-related protein 2 and phosphatidylserine-specific phospholipase A1 were chosen here to illustrate these specific features of interfacial enzymology.

Enzyme-Mediated Individual Nanoparticle Release Assay

PubMed Central

Glass, James R.; Dickerson, Janet C.; Schultz, David A.

2007-01-01

Numerous methods have been developed to measure the presence of macromolecular species in a sample, however methods that detect functional activity, or modulators of that activity are more limited. To address this limitation, an approach was developed that utilizes the optical detection of nanoparticles as a measure of enzyme activity. Nanoparticles are increasingly being used as biological labels in static binding assays; here we describe their use in a release assay format where the enzyme-mediated liberation of individual nanoparticles from a surface is measured. A double stranded fragment of DNA is used as the initial tether to bind the nanoparticles to a solid surface. The nanoparticle spatial distribution and number are determined using dark-field optical microscopy and digital image capture. Site specific cleavage of the DNA tether results in nanoparticle release. The methodology and validation of this approach for measuring enzyme-mediated, individual DNA cleavage events, rapidly, with high specificity, and in real-time is described. This approach was used to detect and discriminate between non-methylated and methylated DNA, and demonstrates a novel platform for high-throughput screening of modulators of enzyme activity. PMID:16620746

Variation in soil enzyme activity as a function of vegetation amount, type, and spatial structure in fire-prone Mediterranean shrublands.

PubMed

Mayor, Ángeles G; Goirán, Silvana B; Vallejo, V Ramón; Bautista, Susana

2016-12-15

Fire-prone Mediterranean shrublands may be seriously threatened by land degradation due to progressive opening of the vegetation cover driven by increasing drought and fire recurrence. However, information about the consequences of this opening process for critical ecosystem functions is scant. In this work, we studied the influence of vegetation amount, type, and spatial pattern in the variation of extracellular soil enzyme activity (acid phosphatase, β-glucosidase, and urease) in fire-prone shrublands in eastern Spain. Soil was sampled in vegetation-patch and open-interpatch microsites in 15 shrubland sites affected by large wildfires in 1991. On average, the activities of the three enzymes were 1.5 (β-glucosidase and urease) to 1.7 (acid phosphatase) times higher in soils under vegetation patches than in adjacent interpatches. In addition, phosphatase activity for both microsites significantly decreased with the fragmentation of the vegetation. This result was attributed to a lower influence of roots -the main source of acid phosphatase- in the bigger interpatches of the sites with lower patch cover, and to feedbacks between vegetation pattern, redistribution of resources, and soil quality during post-fire vegetation dynamics. Phosphatase activity was also 1.2 times higher in patches of resprouter plants than in patches of non-resprouters, probably due to the faster post-fire recovery and older age of resprouter patches in these fire-prone ecosystems. The influence on the studied enzymes of topographic and climatic factors acting at the landscape scale was insignificant. According to our results, variations in the cover, pattern, and composition of vegetation patches may have profound impacts on soil enzyme activity and associated nutrient cycling processes in fire-prone Mediterranean shrublands, particularly in those related to phosphorus. Copyright © 2016 Elsevier B.V. All rights reserved.

Cost-efficient entrapment of β-glucosidase in nanoscale latex and silicone polymeric thin films for use as stable biocatalysts.

PubMed

Javed, Muhammad Rizwan; Buthe, Andreas; Rashid, Muhammad Hamid; Wang, Ping

2016-01-01

β-Glucosidase is an ubiquitous enzyme which has enormous biotechnological applications. Its deficiency in natural enzyme preparations is often overcome by exogenous supplementation, which further increases the enzyme utilization cost. Enzyme immobilization offers a potential solution through enzyme recycling and easy recovery. In the present work Aspergillus niger β-glucosidase is immobilized within nanoscale polymeric materials (polyurethane, latex and silicone), through entrapment, and subsequently coated onto a fibrous support. Highest apparent activity (90 U g(-1) polymer) was observed with latex, while highest entrapment efficiency (93%) was observed for the silicone matrix. Immobilization resulted in the thermo-stabilization of the β-glucosidase with an increase in optimum temperature and activation energy for cellobiose hydrolysis. Supplementation to cellulases also resulted in an increased cellulose hydrolysis, while retaining more than 70% functional stability. Hence, the current study describes novel preparations of immobilized β-glucosidase as highly stable and active catalysts for industrial food- and bio-processing applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme

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

Knott, Gavin J.; East-Seletsky, Alexandra; Cofsky, Joshua C.

CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR–Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gatingmore » of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.« less

Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme

DOE PAGES

Knott, Gavin J.; East-Seletsky, Alexandra; Cofsky, Joshua C.; ...

2017-09-11

CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR–Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gatingmore » of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.« less

Dissecting the functional significance of non-catalytic carbohydrate binding modules in the deconstruction of plant cell walls

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

Hahn, Michael G.

The project seeks to investigate the mechanism by which CBMs potentiate the activity of glycoside hydrolases against complete plant cell walls. The project is based on the hypothesis that the wide range of CBMs present in bacterial enzymes maximize the potential target substrates by directing the cognate enzymes not only to different regions of a specific plant cell wall, but also increases the range of plant cell walls that can be degraded. In addition to maximizing substrate access, it was also proposed that CBMs can target specific subsets of hydrolases with complementary activities to the same region of the plantmore » cell wall, thereby maximizing the synergistic interactions between these enzymes. This synergy is based on the premise that the hydrolysis of a specific polysaccharide will increase the access of closely associated polymers to enzyme attack. In addition, it is unclear whether the catalytic module and appended CBM of modular enzymes have evolved unique complementary activities.« less

Enzyme leaps fuel antichemotaxis

PubMed Central

Jee, Ah-Young; Dutta, Sandipan; Cho, Yoon-Kyoung

2018-01-01

There is mounting evidence that enzyme diffusivity is enhanced when the enzyme is catalytically active. Here, using superresolution microscopy [stimulated emission-depletion fluorescence correlation spectroscopy (STED-FCS)], we show that active enzymes migrate spontaneously in the direction of lower substrate concentration (“antichemotaxis”) by a process analogous to the run-and-tumble foraging strategy of swimming microorganisms and our theory quantifies the mechanism. The two enzymes studied, urease and acetylcholinesterase, display two families of transit times through subdiffraction-sized focus spots, a diffusive mode and a ballistic mode, and the latter transit time is close to the inverse rate of catalytic turnover. This biochemical information-processing algorithm may be useful to design synthetic self-propelled swimmers and nanoparticles relevant to active materials. Executed by molecules lacking the decision-making circuitry of microorganisms, antichemotaxis by this run-and-tumble process offers the biological function to homogenize product concentration, which could be significant in situations when the reactant concentration varies from spot to spot. PMID:29255047

Maturation of the [Ni-4Fe-4S] active site of carbon monoxide dehydrogenases.

PubMed

Merrouch, Mériem; Benvenuti, Martino; Lorenzi, Marco; Léger, Christophe; Fourmond, Vincent; Dementin, Sébastien

2018-02-14

Nickel-containing enzymes are diverse in terms of function and active site structure. In many cases, the biosynthesis of the active site depends on accessory proteins which transport and insert the Ni ion. We review and discuss the literature related to the maturation of carbon monoxide dehydrogenases (CODH) which bear a nickel-containing active site consisting of a [Ni-4Fe-4S] center called the C-cluster. The maturation of this center has been much less studied than that of other nickel-containing enzymes such as urease and NiFe hydrogenase. Several proteins present in certain CODH operons, including the nickel-binding proteins CooT and CooJ, still have unclear functions. We question the conception that the maturation of all CODH depends on the accessory protein CooC described as essential for nickel insertion into the active site. The available literature reveals biological variations in CODH active site biosynthesis.

NOpiates: Novel Dual Action Neuronal Nitric Oxide Synthase Inhibitors with μ-Opioid Agonist Activity

PubMed Central

2012-01-01

A novel series of benzimidazole designed multiple ligands (DMLs) with activity at the neuronal nitric oxide synthase (nNOS) enzyme and the μ-opioid receptor was developed. Targeting of the structurally dissimilar heme-containing enzyme and the μ-opioid GPCR was predicated on the modulatory role of nitric oxide on μ-opioid receptor function. Structure–activity relationship studies yielded lead compound 24 with excellent nNOS inhibitory activity (IC50 = 0.44 μM), selectivity over both endothelial nitric oxide synthase (10-fold) and inducible nitric oxide synthase (125-fold), and potent μ-opioid binding affinity, Ki = 5.4 nM. The functional activity as measured in the cyclic adenosine monosphospate secondary messenger assay resulted in full agonist activity (EC50 = 0.34 μM). This work represents a novel approach in the development of new analgesics for the treatment of pain. PMID:24900459

NOpiates: Novel Dual Action Neuronal Nitric Oxide Synthase Inhibitors with μ-Opioid Agonist Activity.

PubMed

Renton, Paul; Green, Brenda; Maddaford, Shawn; Rakhit, Suman; Andrews, John S

2012-03-08

A novel series of benzimidazole designed multiple ligands (DMLs) with activity at the neuronal nitric oxide synthase (nNOS) enzyme and the μ-opioid receptor was developed. Targeting of the structurally dissimilar heme-containing enzyme and the μ-opioid GPCR was predicated on the modulatory role of nitric oxide on μ-opioid receptor function. Structure-activity relationship studies yielded lead compound 24 with excellent nNOS inhibitory activity (IC50 = 0.44 μM), selectivity over both endothelial nitric oxide synthase (10-fold) and inducible nitric oxide synthase (125-fold), and potent μ-opioid binding affinity, K i = 5.4 nM. The functional activity as measured in the cyclic adenosine monosphospate secondary messenger assay resulted in full agonist activity (EC50 = 0.34 μM). This work represents a novel approach in the development of new analgesics for the treatment of pain.

Influence of exocrine and endocrine pancreatic function on intestinal brush border enaymatic activities.

PubMed Central

Caspary, W F; Winckler, K; Lankisch, P G; Creutzfeldt, W

1975-01-01

Digestive enzymatic activities (disaccharidases, alkaline phosphatase, peptide hydrolases) have been determined in the mucosa of 14 patients with chronic pancreatitis. All had an abnormal secretin-pancreozymin test. Four patients had insulin-dependent diabetes mellitus, four a pathological glucose tolerance test. Nine patients had steatorrhoea. Maltase, sucrase, and alkaline phosphatase activity was significantly elevated in patients with exocrine pancreatic insufficiency, whereas those of lactase, trehalase, and peptide hydrolase were normal. Patients with steatorrhoea had higher maltase and sucrase activity than those without steatorrhoea, whereas decreased glucose tolerance had no effect on brush border enzymatic activity. It is suggested thatdecreased exocrine rather than decreased endocrine pancreatic function is responsible for the increase in intestinal disaccharidase and alkaline phosphatase activity, possible by the influence of pacreatic enzymes on the turnover of brush border enzymes from the luminal side of the mucosal membranes or by direct hormonal stimulation though cholecystokinin. PMID:1092602

Promiscuity in the Enzymatic Catalysis of Phosphate and Sulfate Transfer

PubMed Central

2016-01-01

The enzymes that facilitate phosphate and sulfate hydrolysis are among the most proficient natural catalysts known to date. Interestingly, a large number of these enzymes are promiscuous catalysts that exhibit both phosphatase and sulfatase activities in the same active site and, on top of that, have also been demonstrated to efficiently catalyze the hydrolysis of other additional substrates with varying degrees of efficiency. Understanding the factors that underlie such multifunctionality is crucial both for understanding functional evolution in enzyme superfamilies and for the development of artificial enzymes. In this Current Topic, we have primarily focused on the structural and mechanistic basis for catalytic promiscuity among enzymes that facilitate both phosphoryl and sulfuryl transfer in the same active site, while comparing this to how catalytic promiscuity manifests in other promiscuous phosphatases. We have also drawn on the large number of experimental and computational studies of selected model systems in the literature to explore the different features driving the catalytic promiscuity of such enzymes. Finally, on the basis of this comparative analysis, we probe the plausible origins and determinants of catalytic promiscuity in enzymes that catalyze phosphoryl and sulfuryl transfer. PMID:27187273

Activity assessment of microbial fibrinolytic enzymes.

PubMed

Kotb, Essam

2013-08-01

Conversion of fibrinogen to fibrin inside blood vessels results in thrombosis, leading to myocardial infarction and other cardiovascular diseases. In general, there are four therapy options: surgical operation, intake of antiplatelets, anticoagulants, or fibrinolytic enzymes. Microbial fibrinolytic enzymes have attracted much more attention than typical thrombolytic agents because of the expensive prices and the side effects of the latter. The fibrinolytic enzymes were successively discovered from different microorganisms, the most important among which is the genus Bacillus. Microbial fibrinolytic enzymes, especially those from food-grade microorganisms, have the potential to be developed as functional food additives and drugs to prevent or cure thrombosis and other related diseases. There are several assay methods for these enzymes; this may due to the insolubility of substrate, fibrin. Existing assay methods can be divided into three major groups. The first group consists of assay of fibrinolytic activity with natural proteins as substrates, e.g., fibrin plate methods. The second and third groups of assays are suitable for kinetic studies and are based on the determination of hydrolysis of synthetic peptide esters. This review will deal primarily with the microorganisms that have been reported in literature to produce fibrinolytic enzymes and the first review discussing the methods used to assay the fibrinolytic activity.

Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

PubMed Central

Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

2012-01-01

Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor. PMID:23203056

Dual enzyme activities assay by quantitative electrospray ionization quadrupole-time-of-flight mass spectrometry.

PubMed

Cai, Tingting; Zhang, Li; Wang, Haoyang; Zhang, Jing; Wang, Rong; Zhang, Yurong; Guo, Yinlong

2012-01-01

A practical and rapid method based on electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-ToF MS) was developed for detecting activities of both acetylcholinesterase IAChEI and glutathione S-transferase (GST). The simultaneous study of these two enzyme activities is significant for studying human bio-functions, especially for those who take in toxic compounds and have a risk of disease. Here, the enzyme activities were represented by the conversion of enzymatic substrates and determined by quantitatively analyzing enzymatic substrates. Different internal standards were used to quantify each enzymatic substrate and the good linearity of calibration curves demonstrated the feasibility of the internal standards. The Michaelis-Menten constants (Km) of both GST and AChE were measured by this method and were consistent with values previously reported. Furthermore, we applied this approach to detect GST and AChE activities of whole bloods from four deceased and healthy people. The variation in enzyme activity was in accord with information from gas chromatography mass spectrometry [GC/MS). The screening of AChE and GST provided reliable results and strong forensic evidence. This method offers an alternative choice for detecting enzyme activities and is anticipated to have wide applications in pharmaceutical research and prevention in toxic compounds.

Pressure adaptation is linked to thermal adaptation in salt-saturated marine habitats.

PubMed

Alcaide, María; Stogios, Peter J; Lafraya, Álvaro; Tchigvintsev, Anatoli; Flick, Robert; Bargiela, Rafael; Chernikova, Tatyana N; Reva, Oleg N; Hai, Tran; Leggewie, Christian C; Katzke, Nadine; La Cono, Violetta; Matesanz, Ruth; Jebbar, Mohamed; Jaeger, Karl-Erich; Yakimov, Michail M; Yakunin, Alexander F; Golyshin, Peter N; Golyshina, Olga V; Savchenko, Alexei; Ferrer, Manuel

2015-02-01

The present study provides a deeper view of protein functionality as a function of temperature, salt and pressure in deep-sea habitats. A set of eight different enzymes from five distinct deep-sea (3040-4908 m depth), moderately warm (14.0-16.5°C) biotopes, characterized by a wide range of salinities (39-348 practical salinity units), were investigated for this purpose. An enzyme from a 'superficial' marine hydrothermal habitat (65°C) was isolated and characterized for comparative purposes. We report here the first experimental evidence suggesting that in salt-saturated deep-sea habitats, the adaptation to high pressure is linked to high thermal resistance (P value = 0.0036). Salinity might therefore increase the temperature window for enzyme activity, and possibly microbial growth, in deep-sea habitats. As an example, Lake Medee, the largest hypersaline deep-sea anoxic lake of the Eastern Mediterranean Sea, where the water temperature is never higher than 16°C, was shown to contain halopiezophilic-like enzymes that are most active at 70°C and with denaturing temperatures of 71.4°C. The determination of the crystal structures of five proteins revealed unknown molecular mechanisms involved in protein adaptation to poly-extremes as well as distinct active site architectures and substrate preferences relative to other structurally characterized enzymes. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Activity-based metagenomic screening and biochemical characterization of bovine ruminal protozoan glycoside hydrolases.

PubMed

Findley, Seth D; Mormile, Melanie R; Sommer-Hurley, Andrea; Zhang, Xue-Cheng; Tipton, Peter; Arnett, Krista; Porter, James H; Kerley, Monty; Stacey, Gary

2011-11-01

The rumen, the foregut of herbivorous ruminant animals such as cattle, functions as a bioreactor to process complex plant material. Among the numerous and diverse microbes involved in ruminal digestion are the ruminal protozoans, which are single-celled, ciliated eukaryotic organisms. An activity-based screen was executed to identify genes encoding fibrolytic enzymes present in the metatranscriptome of a bovine ruminal protozoan-enriched cDNA expression library. Of the four novel genes identified, two were characterized in biochemical assays. Our results provide evidence for the effective use of functional metagenomics to retrieve novel enzymes from microbial populations that cannot be maintained in axenic cultures.

Effects of Soil Organic Matter Properties and Microbial Community Composition on Enzyme Activities in Cryoturbated Arctic Soils

PubMed Central

Schnecker, Jörg; Wild, Birgit; Hofhansl, Florian; Eloy Alves, Ricardo J.; Bárta, Jiří; Čapek, Petr; Fuchslueger, Lucia; Gentsch, Norman; Gittel, Antje; Guggenberger, Georg; Hofer, Angelika; Kienzl, Sandra; Knoltsch, Anna; Lashchinskiy, Nikolay; Mikutta, Robert; Šantrůčková, Hana; Shibistova, Olga; Takriti, Mounir; Urich, Tim; Weltin, Georg; Richter, Andreas

2014-01-01

Enzyme-mediated decomposition of soil organic matter (SOM) is controlled, amongst other factors, by organic matter properties and by the microbial decomposer community present. Since microbial community composition and SOM properties are often interrelated and both change with soil depth, the drivers of enzymatic decomposition are hard to dissect. We investigated soils from three regions in the Siberian Arctic, where carbon rich topsoil material has been incorporated into the subsoil (cryoturbation). We took advantage of this subduction to test if SOM properties shape microbial community composition, and to identify controls of both on enzyme activities. We found that microbial community composition (estimated by phospholipid fatty acid analysis), was similar in cryoturbated material and in surrounding subsoil, although carbon and nitrogen contents were similar in cryoturbated material and topsoils. This suggests that the microbial community in cryoturbated material was not well adapted to SOM properties. We also measured three potential enzyme activities (cellobiohydrolase, leucine-amino-peptidase and phenoloxidase) and used structural equation models (SEMs) to identify direct and indirect drivers of the three enzyme activities. The models included microbial community composition, carbon and nitrogen contents, clay content, water content, and pH. Models for regular horizons, excluding cryoturbated material, showed that all enzyme activities were mainly controlled by carbon or nitrogen. Microbial community composition had no effect. In contrast, models for cryoturbated material showed that enzyme activities were also related to microbial community composition. The additional control of microbial community composition could have restrained enzyme activities and furthermore decomposition in general. The functional decoupling of SOM properties and microbial community composition might thus be one of the reasons for low decomposition rates and the persistence of 400 Gt carbon stored in cryoturbated material. PMID:24705618

The influence of two imidazolium-based ionic liquids on the structure and activity of glucose oxidase: Experimental and theoretical studies.

PubMed

Janati-Fard, Fatemeh; Housaindokht, Mohammad Reza; Monhemi, Hassan; Esmaeili, Abbas Ali; Nakhaei Pour, Ali

2018-07-15

The search for ionic liquids (ILs) with biochemical and biomedical applications has recently gained great attention. IL containing solvents can change the structure, stability and function of proteins. The study of protein conformation in ILs is important to understand enzymatic activity. In this work, conformational stability and activity of the enzyme in two imidazolium-based ILs (1-butyl 3-methyl-imidozolium and 1-hexyl 3-methyl-imidozoliumbromides) were investigated. We treated glucose oxidase as dimer-active enzyme in different IL concentration and seen that GOx activity was inhibited in the presence of ILs. Our experimental data showed that inhibition of activity and reduction of enzyme tertiary structure are more for hexyl than butyl derivative. These experimental results are in agreement with foregoing observations. To find a possible mechanism, a series of molecular dynamics simulation of the enzyme were performed at different IL concentration. The structure parameters obtained from MD simulation showed that conformational changes at the active site and FAD-binding site support the hypothesis of enzyme inhibition at the presence of ILs. Root mean square deviation and fluctuation calculations indicated that the enzyme has stable conformation at higher IL concentration, in agreement with experimental observation. But hexyl derivative has a much stronger stabilization effect on the protein structure. In summary, the present study could improve our understanding of the molecular mechanism about the ionic liquid effects on the structure and activity of proteins. Copyright © 2018 Elsevier B.V. All rights reserved.

Carbohydrate-active enzymes in Trichoderma harzianum: a bioinformatic analysis bioprospecting for key enzymes for the biofuels industry.

PubMed

Ferreira Filho, Jaire Alves; Horta, Maria Augusta Crivelente; Beloti, Lilian Luzia; Dos Santos, Clelton Aparecido; de Souza, Anete Pereira

2017-10-12

Trichoderma harzianum is used in biotechnology applications due to its ability to produce powerful enzymes for the conversion of lignocellulosic substrates into soluble sugars. Active enzymes involved in carbohydrate metabolism are defined as carbohydrate-active enzymes (CAZymes), and the most abundant family in the CAZy database is the glycoside hydrolases. The enzymes of this family play a fundamental role in the decomposition of plant biomass. In this study, the CAZymes of T. harzianum were identified and classified using bioinformatic approaches after which the expression profiles of all annotated CAZymes were assessed via RNA-Seq, and a phylogenetic analysis was performed. A total of 430 CAZymes (3.7% of the total proteins for this organism) were annotated in T. harzianum, including 259 glycoside hydrolases (GHs), 101 glycosyl transferases (GTs), 6 polysaccharide lyases (PLs), 22 carbohydrate esterases (CEs), 42 auxiliary activities (AAs) and 46 carbohydrate-binding modules (CBMs). Among the identified T. harzianum CAZymes, 47% were predicted to harbor a signal peptide sequence and were therefore classified as secreted proteins. The GH families were the CAZyme class with the greatest number of expressed genes, including GH18 (23 genes), GH3 (17 genes), GH16 (16 genes), GH2 (13 genes) and GH5 (12 genes). A phylogenetic analysis of the proteins in the AA9/GH61, CE5 and GH55 families showed high functional variation among the proteins. Identifying the main proteins used by T. harzianum for biomass degradation can ensure new advances in the biofuel production field. Herein, we annotated and characterized the expression levels of all of the CAZymes from T. harzianum, which may contribute to future studies focusing on the functional and structural characterization of the identified proteins.

Protein Kinase C Enzymes in the Hematopoietic and Immune Systems.

PubMed

Altman, Amnon; Kong, Kok-Fai

2016-05-20

The protein kinase C (PKC) family, discovered in the late 1970s, is composed of at least 10 serine/threonine kinases, divided into three groups based on their molecular architecture and cofactor requirements. PKC enzymes have been conserved throughout evolution and are expressed in virtually all cell types; they represent critical signal transducers regulating cell activation, differentiation, proliferation, death, and effector functions. PKC family members play important roles in a diverse array of hematopoietic and immune responses. This review covers the discovery and history of this enzyme family, discusses the roles of PKC enzymes in the development and effector functions of major hematopoietic and immune cell types, and points out gaps in our knowledge, which should ignite interest and further exploration, ultimately leading to better understanding of this enzyme family and, above all, its role in the many facets of the immune system.

Quantitative RT-PCR Comparison of the Urea and Nitric Oxide Cycle Gene Transcripts in Adult Human Tissues

PubMed Central

Neill, Meaghan Anne; Aschner, Judy; Barr, Frederick; Summar, Marshall L.

2009-01-01

The urea cycle and nitric oxide cycle play significant roles in complex biochemical and physiologic reactions. These cycles have distinct biochemical goals including the clearance of waste nitrogen; the production of the intermediates ornithine, citrulline, and arginine for the urea cycle; and the production of nitric oxide for the nitric oxide pathway. Despite their disparate functions, the two pathways share two enzymes, argininosuccinic acid synthase and argininosuccinic acid lyase, and a transporter, citrin. Studying the gene expression of these enzymes is paramount in understanding these complex biochemical pathways. Here, we examine the expression of genes involved in the urea cycle and the nitric oxide cycle in a panel of eleven different tissue samples obtained from individual adults without known inborn errors of metabolism. In this study, the pattern of co-expressed enzymes provides a global view of the metabolic activity of the urea and nitric oxide cycles in human tissues. Our results show that these transcripts are differentially expressed in different tissues. The pattern of co-expressed enzymes provides a global view of the metabolic activity of the urea and nitric oxide cycles in human tissues. Using the co-expression profiles, we discovered that the combination of expression of enzyme transcripts as detected in our study, might serve to fulfill specific physiologic function(s) in tissue including urea production/nitrogen removal, arginine/citrulline production, nitric oxide production, and ornithine production. Our study reveals the importance of studying not only the expression profile of an enzyme of interest, but also studying the expression profiles of the other enzymes involved in a particular pathway so as to better understand the context of expression. The tissue patterns we observed highlight the variety of important functions they conduct and provide insight into many of the clinical observations from their disruption. PMID:19345634

Probing the Complex Architecture of Multimodular Carbohydrate-Active Enzymes Using a Combination of Small Angle X-Ray Scattering and X-Ray Crystallography.

PubMed

Czjzek, Mirjam; Ficko-Blean, Elizabeth

2017-01-01

The various modules in multimodular carbohydrate-active enzymes (CAZymes) may function in catalysis, carbohydrate binding, protein-protein interactions or as linkers. Here, we describe how combining the biophysical techniques of Small Angle X-ray Scattering (SAXS) and macromolecular X-ray crystallography (XRC) provides a powerful tool for examination into questions related to overall structural organization of ultra multimodular CAZymes.

Self-assembled monolayers of 1-alkenes on oxidized platinum surfaces as platforms for immobilized enzymes for biosensing

NASA Astrophysics Data System (ADS)

Alonso, Jose Maria; Bielen, Abraham A. M.; Olthuis, Wouter; Kengen, Servé W. M.; Zuilhof, Han; Franssen, Maurice C. R.

2016-10-01

Alkene-based self-assembled monolayers grafted on oxidized Pt surfaces were used as a scaffold to covalently immobilize oxidase enzymes, with the aim to develop an amperometric biosensor platform. NH2-terminated organic layers were functionalized with either aldehyde (CHO) or N-hydroxysuccinimide (NHS) ester-derived groups, to provide anchoring points for enzyme immobilization. The functionalized Pt surfaces were characterized by X-ray photoelectron spectroscopy (XPS), static water contact angle (CA), infrared reflection absorption spectroscopy (IRRAS) and atomic force microscopy (AFM). Glucose oxidase (GOX) was covalently attached to the functionalized Pt electrodes, either with or without additional glutaraldehyde crosslinking. The responses of the acquired sensors to glucose concentrations ranging from 0.5 to 100 mM were monitored by chronoamperometry. Furthermore, lactate oxidase (LOX) and human hydroxyacid oxidase (HAOX) were successfully immobilized onto the PtOx surface platform. The performance of the resulting lactate sensors was investigated for lactate concentrations ranging from 0.05 to 20 mM. The successful attachment of active enzymes (GOX, LOX and HAOX) on Pt electrodes demonstrates that covalently functionalized PtOx surfaces provide a universal platform for the development of oxidase enzyme-based sensors.

Enzyme Mimics: Advances and Applications.

PubMed

Kuah, Evelyn; Toh, Seraphina; Yee, Jessica; Ma, Qian; Gao, Zhiqiang

2016-06-13

Enzyme mimics or artificial enzymes are a class of catalysts that have been actively pursued for decades and have heralded much interest as potentially viable alternatives to natural enzymes. Aside from having catalytic activities similar to their natural counterparts, enzyme mimics have the desired advantages of tunable structures and catalytic efficiencies, excellent tolerance to experimental conditions, lower cost, and purely synthetic routes to their preparation. Although still in the midst of development, impressive advances have already been made. Enzyme mimics have shown immense potential in the catalysis of a wide range of chemical and biological reactions, the development of chemical and biological sensing and anti-biofouling systems, and the production of pharmaceuticals and clean fuels. This Review concerns the development of various types of enzyme mimics, namely polymeric and dendrimeric, supramolecular, nanoparticulate and proteinic enzyme mimics, with an emphasis on their synthesis, catalytic properties and technical applications. It provides an introduction to enzyme mimics and a comprehensive summary of the advances and current standings of their applications, and seeks to inspire researchers to perfect the design and synthesis of enzyme mimics and to tailor their functionality for a much wider range of applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression of a Cellobiose Phosphorylase from Thermotoga maritima in Caldicellulosiruptor bescii Improves the Phosphorolytic Pathway and Results in a Dramatic Increase in Cellulolytic Activity

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

Kim, Sun-Ki; Himmel, Michael E.; Bomble, Yannick J.

Members of the genusCaldicellulosiruptorhave the ability to deconstruct and grow on lignocellulosic biomass without conventional pretreatment. A genetically tractable species,Caldicellulosiruptor bescii, was recently engineered to produce ethanol directly from switchgrass.C. besciicontains more than 50 glycosyl hydrolases and a suite of extracellular enzymes for biomass deconstruction, most prominently CelA, a multidomain cellulase that uses a novel mechanism to deconstruct plant biomass. Accumulation of cellobiose, a product of CelA during growth on biomass, inhibits cellulase activity. Here, we show that heterologous expression of a cellobiose phosphorylase from Thermotoga maritimaimproves the phosphorolytic pathway inC. besciiand results in synergistic activity with endogenous enzymes, includingmore » CelA, to increase cellulolytic activity and growth on crystalline cellulose. CelA is the only known cellulase to function well on highly crystalline cellulose and it uses a mechanism distinct from those of other cellulases, including fungal cellulases. Also unlike fungal cellulases, it functions at high temperature and, in fact, outperforms commercial cellulase cocktails. Factors that inhibit CelA during biomass deconstruction are significantly different than those that impact the performance of fungal cellulases and commercial mixtures. Here, this work contributes to understanding of cellulase inhibition and enzyme function and will suggest a rational approach to engineering optimal activity.« less

Expression of a Cellobiose Phosphorylase from Thermotoga maritima in Caldicellulosiruptor bescii Improves the Phosphorolytic Pathway and Results in a Dramatic Increase in Cellulolytic Activity

DOE PAGES

Kim, Sun-Ki; Himmel, Michael E.; Bomble, Yannick J.; ...

2017-11-03

Members of the genusCaldicellulosiruptorhave the ability to deconstruct and grow on lignocellulosic biomass without conventional pretreatment. A genetically tractable species,Caldicellulosiruptor bescii, was recently engineered to produce ethanol directly from switchgrass.C. besciicontains more than 50 glycosyl hydrolases and a suite of extracellular enzymes for biomass deconstruction, most prominently CelA, a multidomain cellulase that uses a novel mechanism to deconstruct plant biomass. Accumulation of cellobiose, a product of CelA during growth on biomass, inhibits cellulase activity. Here, we show that heterologous expression of a cellobiose phosphorylase from Thermotoga maritimaimproves the phosphorolytic pathway inC. besciiand results in synergistic activity with endogenous enzymes, includingmore » CelA, to increase cellulolytic activity and growth on crystalline cellulose. CelA is the only known cellulase to function well on highly crystalline cellulose and it uses a mechanism distinct from those of other cellulases, including fungal cellulases. Also unlike fungal cellulases, it functions at high temperature and, in fact, outperforms commercial cellulase cocktails. Factors that inhibit CelA during biomass deconstruction are significantly different than those that impact the performance of fungal cellulases and commercial mixtures. Here, this work contributes to understanding of cellulase inhibition and enzyme function and will suggest a rational approach to engineering optimal activity.« less

[Over-expression of uracil DNA glycosylase 2 (UNG2) enhances the resistance to oxidative damage in HepG2 cells].

PubMed

Cao, Liyan; Cheng, Shan; Du, Juan; Guo, Yanhai; Huang, Xiaofeng

2017-04-01

Objective To investigate the uracil glycosidic enzyme activity of uracil DNA glycosylase 2 (UNG2) and study the role of UNG2 in the resistance of antioxidant stress of HepG2 cells. Methods The UNG2-expressing vector was built. Western blotting was used to detect the expression of UNG2. Immunofluorescence staining was performed to observe the cellular location of UNG2. Oligonucleotide was used as substrate for the determination of the UNG2 glycosidic enzyme activity. H 2 O 2 toxicity assay was done to study the function of UNG2 in the antioxidant resistance of hepatocellular carcinoma HepG2 cells. Results UNG2 was successfully over-expressed in HEK293FT cells, and UNG2 was found to be mainly located in nucleus. Enzyme activity assay showed that UNG2 had significant oligonucleotide dU glycosidic enzyme activity. H 2 O 2 toxicity assay showed that over-expressed UNG2 could remarkably increase the survival of HepG2 cells after exposed to H 2 O 2 . Conclusion UNG2 possesses specific DNA glycosidic enzyme activity, and it can protect HepG2 cells against oxidative stress damage.

Metagenomic insights into the rumen microbial fibrolytic enzymes in Indian crossbred cattle fed finger millet straw.

PubMed

Jose, V Lyju; Appoothy, Thulasi; More, Ravi P; Arun, A Sha

2017-12-01

The rumen is a unique natural habitat, exhibiting an unparalleled genetic resource of fibrolytic enzymes of microbial origin that degrade plant polysaccharides. The objectives of this study were to identify the principal plant cell wall-degrading enzymes and the taxonomic profile of rumen microbial communities that are associated with it. The cattle rumen microflora and the carbohydrate-active enzymes were functionally classified through a whole metagenomic sequencing approach. Analysis of the assembled sequences by the Carbohydrate-active enzyme analysis Toolkit identified the candidate genes encoding fibrolytic enzymes belonging to different classes of glycoside hydrolases(11,010 contigs), glycosyltransferases (6366 contigs), carbohydrate esterases (4945 contigs), carbohydrate-binding modules (1975 contigs), polysaccharide lyases (480 contigs), and auxiliary activities (115 contigs). Phylogenetic analysis of CAZyme encoding contigs revealed that a significant proportion of CAZymes were contributed by bacteria belonging to genera Prevotella, Bacteroides, Fibrobacter, Clostridium, and Ruminococcus. The results indicated that the cattle rumen microbiome and the CAZymes are highly complex, structurally similar but compositionally distinct from other ruminants. The unique characteristics of rumen microbiota and the enzymes produced by resident microbes provide opportunities to improve the feed conversion efficiency in ruminants and serve as a reservoir of industrially important enzymes for cellulosic biofuel production.

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

PubMed

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

2006-12-01

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

'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.

Separation of periportal and perivenous rat hepatocytes by fluorescence-activated cell sorting: confirmation with colloidal gold as an exogenous marker.

PubMed

Braakman, I; Keij, J; Hardonk, M J; Meijer, D K; Groothuis, G M

1991-01-01

Periportal and perivenous hepatocytes are known to display various functional differences. In this study we present a new method to separate periportal and perivenous cells: after selectively loading zone 1 or zone 3 with the fluorescent label acridine orange in an antegrade or retrograde perfusion, respectively, we separated the isolated hepatocytes on a fluorescence-activated cell sorter. The common way to check on proper separation is to estimate activities of enzymes known to exhibit a heterogeneous acinar distribution. Using enzyme histochemistry, however, we found that already on short collagenase perfusion, some enzymes displayed a more shallow gradient than in vivo, making enzyme activities less suitable as zonal markers. We therefore used colloidal gold granules (17 nm) injected intravenously (2.5 mg) into the rat 2 to 3 hr before cell isolation. The gold is taken up predominantly by perivenous hepatocytes, probably because of the efficient removal of gold granules in zone 1 by competing Kupffer cells. We compared acridine orange fluorescence, presence of gold particles and activities of six marker enzymes, three biochemically and three histochemically determined. Acridine orange and gold both pointed to a high enrichment of the fractions, whereas most enzyme activities were more randomly distributed among the cells as a result of the isolation procedure. Our separation procedure yielded fractions highly enriched in either viable periportal or perivenous cells, both from one liver. The use of colloidal gold as a marker to monitor separation is a valuable alternative to the more risky estimation of enzyme activities.

Effect of mutations in the qa gene cluster of Neurospora crassa on the enzyme catabolic dehydroquinase.

PubMed Central

Jacobson, J W; Hautala, J A; Case, M E; Giles, N H

1975-01-01

Catabolic dehydroquinase, which functions in the inducible quinic acid catabolic pathway of Neurospora crassa, has been purified from wild type (74-A) and three mutants in the qa gene cluster. The mutant strains were: 105c, a temperature-sensitive constitutive mutant in the qa-1 regulatory locus; M-16, a qa-3 mutant deficient in quinate dehydrogenase activity; and 237, a leaky qa-2 mutant which possess very low levels of catabolic dehydroquinase activity. The enzymes purified from strains 74-A, 105c, and M-16 are identical with respect to behavior during purification, specific activity, electrophoretic behavior, stability, molecular weight, subunit structure, immunological cross-reactivity, and amino acid content. The mutant enzyme from strain 237 is 1,500-fold less active and appears to have a slightly different amino acid content. It is identical by a number of the other criteria listed above and is presumed to be a mutant at or near the enzyme active site. These data demonstrate that the qa-1 gene product is not involved in the posttranslational expression of enzyme activity. The biochemical identity of catabolic dehydroquinase isolated from strains 105c and M-16 with that from wild type also demonstrates that neither the inducer, quinic acid, nor other enzymes encoded in the qa gene cluster are necessary for the expression of activity. Therefore the combined genetic and biochemical data on the qa system continue to support the hypothesis that the qa-1 regulatory protein acts as a positive initiator of qa enzyme synthesis. Images PMID:126226

Functional metagenomic selection of RubisCOs from uncultivated bacteria

USGS Publications Warehouse

Varaljay, Vanessa A; Satagopan, Sriram; North, Justin A.; Witteveen, Briana; Dourado, Manuella N.; Anantharaman, Karthik; Arbing, Mark A.; McCann, Shelley; Oremland, Ronald S.; Banfield, Jillian F.; Wrighton, Kelly C.; Tabita, F. Robert

2016-01-01

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a critical yet severely inefficient enzyme that catalyses the fixation of virtually all of the carbon found on Earth. Here, we report a functional metagenomic selection that recovers physiologically active RubisCO molecules directly from uncultivated and largely unknown members of natural microbial communities. Selection is based on CO2-dependent growth in a host strain capable of expressing environmental deoxyribonucleic acid (DNA), precluding the need for pure cultures or screening of recombinant clones for enzymatic activity. Seventeen functional RubisCO-encoded sequences were selected using DNA extracted from soil and river autotrophic enrichments, a photosynthetic biofilm and a subsurface groundwater aquifer. Notably, three related form II RubisCOs were recovered which share high sequence similarity with metagenomic scaffolds from uncultivated members of theGallionellaceae family. One of the Gallionellaceae RubisCOs was purified and shown to possessCO2/O2 specificity typical of form II enzymes. X-ray crystallography determined that this enzyme is a hexamer, only the second form II multimer ever solved and the first RubisCO structure obtained from an uncultivated bacterium. Functional metagenomic selection leverages natural biological diversity and billions of years of evolution inherent in environmental communities, providing a new window into the discovery of CO2-fixing enzymes not previously characterized.

Mesoporous silica nanoparticles supported copper(II) and nickel(II) Schiff base complexes: Synthesis, characterization, antibacterial activity and enzyme immobilization

NASA Astrophysics Data System (ADS)

Tahmasbi, Leila; Sedaghat, Tahereh; Motamedi, Hossein; Kooti, Mohammad

2018-02-01

Mesoporous silica nanoparticles (MSNs) were prepared by sol-gel method and functionalized with 3-aminopropyltriethoxysilane. Schiff base grafted mesoporous silica nanoparticle was synthesized by the condensation of 2-hydroxy-3-methoxybenzaldehyde and amine-functionalized MSNs. The latter material was then treated with Cu(II) and Ni(II) salts separately to obtain copper and nickel complexes anchored mesoporous composites. The newly prepared hybrid organic-inorganic nanocomposites have been characterized by several techniques such as FT-IR, LA-XRD, FE-SEM, TEM, EDS, BET and TGA. The results showed all samples have MCM-41 type ordered mesoporous structure and functionalization occurs mainly inside the mesopore channel. The presence of all elements in synthesized nanocomposites and the coordination of Schiff base via imine nitrogen and phenolate oxygen were confirmed. MSNs and all functionalized MSNs have uniform spherical nanoparticles with a mean diameter less than 100 nm. The as-synthesized mesoporous nanocomposites were investigated for antibacterial activity against Gram-positive (B. subtilis and S. aureus) and Gram-negative (E. coli and P. aeruginosa) bacteria, as carrier for gentamicin and also for immobilization of DNase, coagulase and amylase enzymes. MSN-SB-Ni indicated bacteriocidal effect against S.aureus and all compounds were found to be good carrier for gentamicin. Results of enzyme immobilization for DNase and coagulase and α-amylase revealed that supported metal complexes efficiently immobilized enzymes.

Strategies for an enzyme immobilization on electrodes: Structural and electrochemical characterizations

NASA Astrophysics Data System (ADS)

Ganesh, V.; Muthurasu, A.

2012-04-01

In this paper, we propose various strategies for an enzyme immobilization on electrodes (both metal and semiconductor electrodes). In general, the proposed methodology involves two critical steps viz., (1) chemical modification of substrates using functional monolayers [Langmuir - Blodgett (LB) films and/or self-assembled monolayers (SAMs)] and (2) anchoring of a target enzyme using specific chemical and physical interactions by attacking the terminal functionality of the modified films. Basically there are three ways to immobilize an enzyme on chemically modified electrodes. First method consists of an electrostatic interaction between the enzyme and terminal functional groups present within the chemically modified films. Second and third methods involve the introduction of nanomaterials followed by an enzyme immobilization using both the physical and chemical adsorption processes. As a proof of principle, in this work we demonstrate the sensing and catalytic activity of horseradish peroxidase (HRP) anchored onto SAM modified indium tin oxide (ITO) electrodes towards hydrogen peroxide (H2O2). Structural characterization of such modified electrodes is performed using X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and contact angle measurements. The binding events and the enzymatic reactions are monitored using electrochemical techniques mainly cyclic voltammetry (CV).

Two new gene clusters involved in the degradation of plant cell wall from the fecal microbiota of Tunisian dromedary

PubMed Central

Ameri, Rihab; Laville, Elisabeth; Potocki-Véronèse, Gabrielle; Trabelsi, Sahar; Mezghani, Monia; Elgharbi, Fatma

2018-01-01

Dromedaries are capable of digesting plant cell wall with high content of lignocellulose of poor digestibility. Consequently, their intestinal microbiota can be a source of novel carbohydrate-active enzymes (CAZymes). To the best of our knowledge, no data are available describing the biochemical analysis of enzymes in dromedary intestinal microbiota. To investigate new hydrolytic enzymes from the dromedary gut, a fosmid library was constructed using metagenomic DNA from feces of non-domestic adult dromedary camels living in the Tunisian desert. High-throughput functional screening of 13756 clones resulted in 47 hit clones active on a panel of various chromogenic and non-chromogenic glycan substrates. Two of them, harboring multiple activities, were retained for further analysis. Clone 26H3 displayed activity on AZO-CM-cellulose, AZCL Carob galactomannan and Tween 20, while clone 36A23 was active on AZCL carob galactomannan and AZCL barley β-glucan. The functional annotation of their sequences highlighted original metagenomic loci originating from bacteria of the Bacteroidetes/Chlorobi group, involved in the metabolization of mannosides and β-glucans thanks to a complete battery of endo- and exo-acting glycoside hydrolases, esterases, phosphorylases and transporters. PMID:29601586

Angiotensin converting enzyme (ACE) inhibitory and antihypertensive activities of protein hydrolysate from meat of Kacang goat (Capra aegagrus hircus).

PubMed

Mirdhayati, Irdha; Hermanianto, Joko; Wijaya, Christofora H; Sajuthi, Dondin; Arihara, Keizo

2016-08-01

The meat of Kacang goat has potential for production of a protein hydrolysate. Functional ingredients from protein hydrolysate of Kacang goat meat were determined by the consistency of angiotensin-converting enzyme (ACE) inhibitory activity and antihypertensive effect. This study examined the potency of Kacang goat protein hydrolysate in ACE inhibition and antihypertensive activity. Protein hydrolysates of Kacang goat meat were prepared using sequential digestion of endo-proteinase and protease complex at several concentrations and hydrolysis times. The highest ACE inhibitory activity resulted from a hydrolysate that was digested for 4 h with 5 g kg(-1) of both enzymes. An ACE inhibitory peptide was purified and a novel peptide found with a sequence of Phe-Gln-Pro-Ser (IC50 value of 27.0 µmol L(-1) ). Both protein hydrolysates and a synthesised peptide (Phe-Gln-Pro-Ser) demonstrated potent antihypertensive activities in spontaneously hypertensive rats. Protein hydrolysate of Kacang goat meat produced by sequential digestion with endo-proteinase and protease complex has great potential as a functional ingredient, particularly as an antihypertensive agent. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Structure and function of polyketide biosynthetic enzymes: various strategies for production of structurally diverse polyketides.

PubMed

Miyanaga, Akimasa

2017-12-01

Polyketides constitute a large family of natural products that display various biological activities. Polyketides exhibit a high degree of structural diversity, although they are synthesized from simple acyl building blocks. Recent biochemical and structural studies provide a better understanding of the biosynthetic logic of polyketide diversity. This review highlights the biosynthetic mechanisms of structurally unique polyketides, β-amino acid-containing macrolactams, enterocin, and phenolic lipids. Functional and structural studies of macrolactam biosynthetic enzymes have revealed the unique biosynthetic machinery used for selective incorporation of a rare β-amino acid starter unit into the polyketide skeleton. Biochemical and structural studies of cyclization enzymes involved in the biosynthesis of enterocin and phenolic lipids provide mechanistic insights into how these enzymes diversify the carbon skeletons of their products.

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.

Production of Delta(1)-tetrahydrocannabinolic acid by the biosynthetic enzyme secreted from transgenic Pichia pastoris.

PubMed

Taura, Futoshi; Dono, Emi; Sirikantaramas, Supaart; Yoshimura, Kohji; Shoyama, Yukihiro; Morimoto, Satoshi

2007-09-28

Delta(1)-Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes the oxidative cyclization of cannabigerolic acid into THCA, the acidic precursor of Delta(1)-tetrahydrocannabinol. We developed a novel expression system for THCA synthase using a methylotrophic yeast Pichia pastoris as a host. Under optimized conditions, the transgenic P. pastoris secreted approximately 1.32nkat/l of THCA synthase activity, and the culture medium, from which the cells were removed, effectively synthesized THCA from cannabigerolic acid with a approximately 98% conversion rate. The secreted THCA synthase was readily purified to homogeneity. Interestingly, endoglycosidase treatment afforded a deglycosylated THCA synthase with more catalytic activity than that of the glycosylated form. The non-glycosylated THCA synthase should be suitable for structure-function studies because it displayed much more activity than the previously reported native enzyme from Cannabis sativa as well as the recombinant enzyme from insect cell cultures.

[Effect of components and some protocols of anti-ulcer therapy on content and activity of monooxigenase system enzymes of the stomach mucosa in experimental stomach ulcer].

PubMed

Iakubov, A V; Pattakhova, M Kh

2009-01-01

The influence of components and some schemata of antiulcerous therapy on content and activity of monooxigenase system's enzymes in mucous membrane of stomach are studied on the model of experimental stomach ulcer in rats. It is established, that among components of antiulcerous therapy such as omeprazole, clarithromycin and metronidazole inhibit content and activity of MOS enzymes. Tinidazol, amoxicillin and azithromycin do not affect the function of MOS. Rifampicin and pantoprazole induce enzyme system of monooxigenase. In triple therapy with omeprazole, clarithromycin and metronidazole the inhibit effect of preparations to system of MOS is exponentiated and it leads to suppression of mucous cytoprotaction of gastro duodenal zone. Triple therapy of ulcerous disease with pantoprazole, rifampicin and azithromycin is effective planning to stimulate defense mechanisms of the organism.