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Annual Review of Biochemistry Jun 2017What happens inside an enzyme's active site to allow slow and difficult chemical reactions to occur so rapidly? This question has occupied biochemists' attention for a... (Review)
Review
What happens inside an enzyme's active site to allow slow and difficult chemical reactions to occur so rapidly? This question has occupied biochemists' attention for a long time. Computer models of increasing sophistication have predicted an important role for electrostatic interactions in enzymatic reactions, yet this hypothesis has proved vexingly difficult to test experimentally. Recent experiments utilizing the vibrational Stark effect make it possible to measure the electric field a substrate molecule experiences when bound inside its enzyme's active site. These experiments have provided compelling evidence supporting a major electrostatic contribution to enzymatic catalysis. Here, we review these results and develop a simple model for electrostatic catalysis that enables us to incorporate disparate concepts introduced by many investigators to describe how enzymes work into a more unified framework stressing the importance of electric fields at the active site.
Topics: Bacterial Proteins; Biocatalysis; Catalytic Domain; Gene Expression; Hydrolases; Ketosteroids; Kinetics; Models, Chemical; Molecular Dynamics Simulation; Mutation; Pseudomonas; Spectrophotometry, Infrared; Static Electricity; Steroid Isomerases; Thermodynamics
PubMed: 28375745
DOI: 10.1146/annurev-biochem-061516-044432 -
Protein Science : a Publication of the... Aug 2020New enzyme functions often evolve through the recruitment and optimization of latent promiscuous activities. How do mutations alter the molecular architecture of enzymes... (Review)
Review
New enzyme functions often evolve through the recruitment and optimization of latent promiscuous activities. How do mutations alter the molecular architecture of enzymes to enhance their activities? Can we infer general mechanisms that are common to most enzymes, or does each enzyme require a unique optimization process? The ability to predict the location and type of mutations necessary to enhance an enzyme's activity is critical to protein engineering and rational design. In this review, via the detailed examination of recent studies that have shed new light on the molecular changes underlying the optimization of enzyme function, we provide a mechanistic perspective of enzyme evolution. We first present a global survey of the prevalence of activity-enhancing mutations and their distribution within protein structures. We then delve into the molecular solutions that mediate functional optimization, specifically highlighting several common mechanisms that have been observed across multiple examples. As distinct protein sequences encounter different evolutionary bottlenecks, different mechanisms are likely to emerge along evolutionary trajectories toward improved function. Identifying the specific mechanism(s) that need to be improved upon, and tailoring our engineering efforts to each sequence, may considerably improve our chances to succeed in generating highly efficient catalysts in the future.
Topics: Directed Molecular Evolution; Enzymes; Evolution, Molecular; Protein Domains; Protein Engineering; Substrate Specificity
PubMed: 32557882
DOI: 10.1002/pro.3901 -
Clinical Microbiology and Infection :... Jan 2008The term 'extended-spectrum beta-lactamase' (ESBL), initially 'extended-broad-spectrum beta-lactamase', was first coined for derivatives of TEM and SHV enzymes able to... (Review)
Review
The term 'extended-spectrum beta-lactamase' (ESBL), initially 'extended-broad-spectrum beta-lactamase', was first coined for derivatives of TEM and SHV enzymes able to hydrolyse oxyimino-cephalosporins. These all belonged to beta-lactamase functional group 2be. Subsequently, the term has been stretched to include: (i) enzymes with spectra similar to those of TEM and SHV mutants but derived from other sources, e.g., the CTX-M and VEB types; (ii) TEM and SHV mutants with borderline ESBL activity, e.g., TEM-12; and (iii) various beta-lactamases conferring wider resistance than their parent types but not meeting the definition for group 2be, e.g., OXA derivatives and mutant AmpC types with increased activity against cefepime. It seems best-and pragmatic-that the term 'ESBL' retains its broad modern usage, but that should always be accompanied by mention of the enzyme's family as, e.g., in 'TEM ESBL' or 'OXA ESBL', not as a sole moniker.
Topics: Anti-Bacterial Agents; Carbapenems; Cephalosporins; Escherichia coli; Gram-Negative Bacteria; Kinetics; Microbial Sensitivity Tests; Mutation; beta-Lactam Resistance; beta-Lactamases; beta-Lactams
PubMed: 18154524
DOI: 10.1111/j.1469-0691.2007.01857.x -
The Biochemical Journal Dec 1996The enzymic stages of mammalian mitochondrial beta-oxidation were elucidated some 30-40 years ago. However, the discovery of a membrane-associated multifunctional enzyme... (Review)
Review
The enzymic stages of mammalian mitochondrial beta-oxidation were elucidated some 30-40 years ago. However, the discovery of a membrane-associated multifunctional enzyme of beta-oxidation, a membrane-associated acyl-CoA dehydrogenase and characterization of the carnitine palmitoyl transferase system at the protein and at the genetic level has demonstrated that the enzymes of the system itself are incompletely understood. Deficiencies of many of the enzymes have been recognized as important causes of disease. In addition, the study of these disorders has led to a greater understanding of the molecular mechanism of beta-oxidation and the import, processing and assembly of the beta-oxidation enzymes within the mitochondrion. The tissue-specific regulation, intramitochondrial control and supramolecular organization of the pathway is becoming better understood as sensitive analytical and molecular techniques are applied. This review aims to cover enzymological and organizational aspects of mitochondrial beta-oxidation together with the biochemical aspects of inherited disorders of beta-oxidation and the intrinsic control of beta-oxidation.
Topics: Acyl-CoA Dehydrogenases; Animals; Carnitine O-Palmitoyltransferase; Coenzyme A Ligases; Fatty Acids; Humans; Lipid Metabolism, Inborn Errors; Mammals; Mitochondria; Models, Biological; Oxidation-Reduction
PubMed: 8973539
DOI: 10.1042/bj3200345 -
The Journal of Organic Chemistry Jul 2020In continuous flow biocatalysis, chemical transformations can occur under milder, greener, more scalable, and safer conditions than conventional organic synthesis....
In continuous flow biocatalysis, chemical transformations can occur under milder, greener, more scalable, and safer conditions than conventional organic synthesis. However, the method typically involves extensive screening to optimize each enzyme's immobilization on its solid support material. The task of weighing solids for large numbers of experiments poses a bottleneck for screening enzyme immobilization conditions. For example, screening conditions often require multiple replicates exploring different support chemistries, buffer compositions, and temperatures. Thus, we report 3D-printed labware designed to measure and handle solids in multichannel format and expedite screening of enzyme immobilization conditions. To demonstrate the generality of these advances, alkaline phosphatase, glucose dehydrogenase, and laccase were screened for immobilization efficiency on seven resins. The results illustrate the requirements for optimization of each enzyme's loading and resin choice for optimal catalytic performance. Here, 3D-printed labware can decrease the requirements for an experimentalist's time by >95%. The approach to rapid optimization of enzyme immobilization is applicable to any enzyme and many solid support resins. Furthermore, the reported devices deliver precise and accurate aliquots of essentially any granular solid material.
Topics: Biocatalysis; Catalysis; Enzymes, Immobilized; Laccase; Printing, Three-Dimensional
PubMed: 32502347
DOI: 10.1021/acs.joc.0c00789 -
The Biochemical Journal Sep 19681. A number of yeast species were examined for the presence of beta-glucanases. Extracts obtained by cell disruption of Saccharomyces cerevisiae, Fabospora fragilis and...
1. A number of yeast species were examined for the presence of beta-glucanases. Extracts obtained by cell disruption of Saccharomyces cerevisiae, Fabospora fragilis and Hansenula anomala hydrolysed laminarin and pustulan with the production of glucose. Enzymic activities were also detected in the culture fluids of F. fragilis and H. anomala grown aerobically in buffered mineral medium with glucose as the carbon source. 2. F. fragilis and H. anomala possessed approximately sevenfold higher beta-(1-->3)-glucanase activity than S. cerevisiae. 3. Intracellular exo-beta-glucanase from baker's yeast was purified 344-fold from the dialysed cell extract. 4. Exo-beta-glucanase from F. fragilis was purified 114-fold from the dialysed culture fluid and 423-fold from the dialysed intracellular extract. The purified extracellular and intracellular enzymes had similar properties and essentially the same specific activity, 79 enzyme units/mg. of protein. 5. Extracellular exo-beta-glucanase of H. anomala was purified 600-fold. 6. The optimum pH of the enzymes from F. fragilis, S. cerevisiae and H. anomala was 5.5 in each case. Chromatographic evidence indicated that the three enzymes remove glucosyl units sequentially from laminarin as well as pustulan. 7. The ratio of activities towards laminarin and pustulan remained constant during purification of the exo-beta-glucanase obtained from the three species, suggesting a single enzyme. Additional evidence for its unienzymic nature are: (i) the two activities were destroyed at exactly the same rate on heating of the purified enzyme from F. fragilis at three different temperatures; (ii) the competitive inhibitor glucono-delta-lactone gave the same value of K(i) when tested with either substrate; (iii) quantitative application of the ;mixed-substrate' method with the purified enzyme of S. cerevisiae gave data that were in excellent agreement with those calculated on the assumption of a single enzyme. 8. The purified exo-beta-glucanases of the different species of yeast had different kinetic constants. The ratios of maximal velocities and K(m) values with laminarin and pustulan differed markedly. Comparison of V(max.) and K(m) values suggests that the rapid release of spores from asci in F. fragilis might be explained in terms of an enzyme with higher maximal velocity and higher affinity to the ascus wall than that present in baker's yeast. 9. The estimated molecular weights for exo-beta-glucanases from F. fragilis, S. cerevisiae and H. anomala were 22000, 40000 and 30000 respectively.
Topics: Chromatography; Chromatography, Gel; Glycoside Hydrolases; Molecular Weight; Polysaccharides; Yeasts
PubMed: 5685856
DOI: 10.1042/bj1090347 -
Cell Death and Differentiation Aug 2011Activities displaying caspase cleavage specificity have been well documented in various plant programmed cell death (PCD) models. However, plant genome analyses have not... (Review)
Review
Activities displaying caspase cleavage specificity have been well documented in various plant programmed cell death (PCD) models. However, plant genome analyses have not revealed clear orthologues of caspase genes, indicating that enzyme(s) structurally unrelated yet possessing caspase specificity have functions in plant PCD. Here, we review recent data showing that some caspase-like activities are attributable to the plant subtilisin-like proteases, saspases and phytaspases. These proteases hydrolyze a range of tetrapeptide caspase substrates following the aspartate residue. Data obtained with saspases implicate them in the proteolytic degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) during biotic and abiotic PCD, whereas phytaspase overproducing and silenced transgenics provide evidence that phytaspase regulates PCD during both abiotic (oxidative and osmotic stresses) and biotic (virus infection) insults. Like caspases, phytaspases and saspases are synthesized as proenzymes, which are autocatalytically processed to generate a mature enzyme. However, unlike caspases, phytaspases and saspases appear to be constitutively processed and secreted from healthy plant cells into the intercellular space. Apoplastic localization presumably prevents enzyme-mediated protein fragmentation in the absence of PCD. In response to death-inducing stimuli, phytaspase has been shown to re-localize to the cell interior. Thus, plant PCD-related proteases display both common (D-specific protein fragmentation during PCD) and distinct (enzyme structure and activity regulation) features with animal PCD-related proteases.
Topics: Animals; Apoptosis; Caspases; Catalytic Domain; Cysteine Proteinase Inhibitors; Isoenzymes; Models, Molecular; Phylogeny; Plant Proteins; Plants; Protein Conformation; Subtilisin
PubMed: 21546909
DOI: 10.1038/cdd.2011.49 -
Biomolecules Dec 2019Bacterial resistance to β-lactams, the most commonly used class of antibiotics, poses a global challenge. This resistance is caused by the production of bacterial... (Review)
Review
Bacterial resistance to β-lactams, the most commonly used class of antibiotics, poses a global challenge. This resistance is caused by the production of bacterial enzymes that are termed β-lactamases (βLs). The evolution of serine-class A β-lactamases from penicillin-binding proteins (PBPs) is related to the formation of the Ω-loop at the entrance to the enzyme's active site. In this loop, the Glu166 residue plays a key role in the two-step catalytic cycle of hydrolysis. This residue in TEM-type β-lactamases, together with Asn170, is involved in the formation of a hydrogen bonding network with a water molecule, leading to the deacylation of the acyl-enzyme complex and the hydrolysis of the β-lactam ring of the antibiotic. The activity exhibited by the Ω-loop is attributed to the positioning of its N-terminal residues near the catalytically important residues of the active site. The structure of the Ω-loop of TEM-type β-lactamases is characterized by low mutability, a stable topology, and structural flexibility. All of the revealed features of the Ω-loop, as well as the mechanisms related to its involvement in catalysis, make it a potential target for novel allosteric inhibitors of β-lactamases.
Topics: Bacteria; Biocatalysis; Drug Resistance, Bacterial; Protein Conformation; beta-Lactamases
PubMed: 31835662
DOI: 10.3390/biom9120854 -
Proceedings of the National Academy of... Mar 2021Metabolic engineering uses enzymes as parts to build biosystems for specified tasks. Although a part's working life and failure modes are key engineering performance...
Metabolic engineering uses enzymes as parts to build biosystems for specified tasks. Although a part's working life and failure modes are key engineering performance indicators, this is not yet so in metabolic engineering because it is not known how long enzymes remain functional in vivo or whether cumulative deterioration (wear-out), sudden random failure, or other causes drive replacement. Consequently, enzymes cannot be engineered to extend life and cut the high energy costs of replacement. Guided by catalyst engineering, we adopted catalytic cycles until replacement (CCR) as a metric for enzyme functional life span in vivo. CCR is the number of catalytic cycles that an enzyme mediates in vivo before failure or replacement, i.e., metabolic flux rate/protein turnover rate. We used estimated fluxes and measured protein turnover rates to calculate CCRs for ∼100-200 enzymes each from , yeast, and CCRs in these organisms had similar ranges (<10 to >10) but different median values (3-4 × 10 in and yeast versus 4 × 10 in ). In all organisms, enzymes whose substrates, products, or mechanisms can attack reactive amino acid residues had significantly lower median CCR values than other enzymes. Taken with literature on mechanism-based inactivation, the latter finding supports the proposal that 1) random active-site damage by reaction chemistry is an important cause of enzyme failure, and 2) reactive noncatalytic residues in the active-site region are likely contributors to damage susceptibility. Enzyme engineering to raise CCRs and lower replacement costs may thus be both beneficial and feasible.
Topics: Arabidopsis; Biocatalysis; Enzymes; Lactococcus lactis; Metabolic Engineering; Saccharomyces cerevisiae
PubMed: 33753504
DOI: 10.1073/pnas.2023348118 -
The FEBS Journal Jul 2013The chitinolytic machinery of Serratia marcescens is one of the best known enzyme systems for the conversion of insoluble polysaccharides. This machinery includes four... (Review)
Review
The chitinolytic machinery of Serratia marcescens is one of the best known enzyme systems for the conversion of insoluble polysaccharides. This machinery includes four chitin-active enzymes: ChiC, an endo-acting non-processive chitinase; ChiA and ChiB, two processive chitinases moving along chitin chains in opposite directions; and CBP21, a surface-active CBM33-type lytic polysaccharide monooxygenase that introduces chain breaks by oxidative cleavage. Furthermore, an N-acetylhexosaminidase or chitobiase converts the oligomeric products from the other enzymes to monomeric N-acetylglucosamine. Here we discuss the catalytic mechanisms of these enzymes as well as the structural basis of each enzyme's specific role in the chitin degradation process. We also discuss how knowledge of this enzyme system may be extrapolated to other enzyme systems for conversion of insoluble polysaccharides, in particular conversion of cellulose by cellulases and GH61-type lytic polysaccharide monooxygenases.
Topics: Bacterial Proteins; Biocatalysis; Carrier Proteins; Chitin; Chitinases; Fungal Proteins; Glycoside Hydrolases; Hydrolysis; Intracellular Signaling Peptides and Proteins; Isoenzymes; Models, Molecular; Molecular Conformation; Plant Proteins; Polysaccharides; Serratia marcescens; Substrate Specificity
PubMed: 23398882
DOI: 10.1111/febs.12181