Advances in Enzymology and related Areas of Molecular Biology

Ever since the era of recombinant DNA technology for natural product biosynthesis emerged (292), microorganisms are increasingly becoming common production platforms for many fine chemicals, including natural products and biofuels, that are currently being produced either through chemical methods or using plant and organ cell cultures. The rapid elucidation of biosynthetic pathways made possible through advanced genomic tools has made natural products again the molecules of choice for drug development. Indeed, half of the drugs currently in clinical use are natural products and it is expected that the market size of biotechnology-derived small molecules will exceed billion U...

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No abstract text is available yet for this article.

The generation of enzymes with new catalytic activities remains a major challenge. So far, several different strategies have been developed to tackle this problem, including site-directed mutagenesis, random mutagenesis (directed evolution), antibody catalysis, computational redesign, and de novo methods. Using these techniques, a broad array of novel enzymes has been created (aldolases, decarboxylases, dehydratases, isomerases, oxidases, reductases, and others), although their low efficiencies (10 to 100 M(-1) s(-l)) compared to those of the best natural enzymes (10(6) to 10(8) M(-1) s(-1)) remains a significant concern...

Protein engineering holds great promise for the development of new biosensors, diagnostics, therapeutics, and agents for bioremediation. Despite some remarkable successes in experimental and computational protein design, engineered proteins rarely achieve the efficiency or specificity of natural enzymes. Current protein design methods utilize evolutionary concepts, including mutation, recombination, and selection, but the inability to fully recapitulate the success of natural evolution suggests that some evolutionary principles have not been fully exploited...

Proteins, by virtue of their central role in most biological processes, represent one of the key subjects of the study of molecular evolution. Inherent in the indispensability of proteins for living cells is the fact that a given protein can adopt a specific three-dimensional shape that is specified solely by the protein's sequence of amino acids. Over the past several decades, structural biologists have demonstrated that the array of structures that proteins may adopt is quite astounding, and this has lead to a strong interest in understanding how protein structures change and evolve over time...

Experimental paleomolecular biology, paleobiochemistry, and paleogenetics are closely related emerging fields that infer the sequences of ancient genes and proteins from now-extinct organisms, and then resurrect them for study in the laboratory. The goal of paleogenetics is to use information from natural history to solve the conundrum of modern genomics: How can we understand deeply the function of biomolecular structures uncovered and described by modern chemical biology? Reviewed here are the first 20 cases where biomolecular resurrections have been achieved...

The enzyme L-aspartate ammonia-lyase (aspartase) catalyzes the reversible deamination of the amino acid L-aspartic acid, using a carbanion mechanism to produce fumaric acid and ammonium ion. Aspartase is among the most specific enzymes known with extensive studies failing, until recently, to identify any alternative amino acid substrates that can replace L-aspartic acid. Aspartases from different organisms show high sequence homology, and this homology extends to functionally related enzymes such as the class II fumarases, the argininosuccinate and adenylosuccinate lyases...

The enzymes phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute the family of pterin-dependent aromatic amino acid hydroxylases. Each enzyme catalyzes the hydroxylation of the aromatic side chain of its respective amino acid substrate using molecular oxygen and a tetrahydropterin as substrates. Recent advances have provided insights into the structures, mechanisms, and regulation of these enzymes. The eukaryotic enzymes are homotetramers comprised of homologous catalytic domains and discrete regulatory domains...

The 31P NMR data suggest slight differences in the structures around the 5'-P for the internal Schiff base and the lanthionine external Schiff base (both largely ketoeneamine) and a large difference for enolimine portion of the serine external Schiff base. Addition of cysteine or serine increase delayed fluorescence and triplet to singlet energy transfer. Addition of OAS exhibits a splitting of the 0,0 vibronic, the result of two distinct conformations, likely enolimine and ketoeneamine tautomers. Nonetheless, the alpha-amino-acrylate Schiff base conformation differs from either the internal or external Schiff base conformations...

The pyridoxal-5-phosphate-dependent enzymes (B6 enzymes) that act on amino acid substrates are of multiple evolutionary origin. The numerous common mechanistic features of B6 enzymes thus are not historical traits passed on from a common ancestor enzyme but rather reflect evolutionary or chemical necessities. Family profile analysis of amino acid sequences supported by comparison of the available three-dimensional (3-D) crystal structures indicates that the B6 enzymes known to date belong to four independent evolutionary lineages of homologous (or more precisely paralogous) proteins, of which the alpha family is by far the largest...

Phosphoribulokinase (PRK), an enzyme unique to the reductive pentose phosphate pathway of CO2 assimilation, exhibits distinctive contrasting properties when the proteins from eukaryotic and prokaryotic sources are compared. The eukaryotic PRKs are typically dimers of -39 kDa subunits while the prokaryotic PRKs are octamers of -32 kDa subunits. The enzymes from these two classes are regulated by different mechanisms. Thioredoxin of mediated thiol-disulfide exchange interconverts eukaryotic PRKs between reduced (active) and oxidized (inactive) forms...

The bacterial PTE is able to catalyze the hydrolysis of a wide range of organophosphate nerve agents. The active site has been shown to consist of a unique binuclear metal center that has evolved to deliver hydroxide to the site of bond cleavage. The reaction rate for the hydrolysis of activated substrates such as paraoxon is limited by product release or an associated protein conformational change.

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The metabolite glutathione fulfills many important and chemically complex roles in protecting cellular components from the deleterious effects of toxic species. GSH combines with hydroxyl radical, peroxynitrite, and hydroperoxides, as well as reactive electrophiles, including activated phosphoramide mustard. This thiol-containing reductant also maintains so-called thiol-enzymes in their catalytically active form, and maintains vitamins C and E in their biologically active forms. The key step in glutathione synthesis, namely the ATP-dependent synthesis of gamma-glutamylcysteine, is the topic of this review...

The MutT enzyme prevents errors in DNA replication by hydrolyzing mutagenic nucleotide substrates such as 8-oxo-dGTP. It does so by catalyzing nucleophilic attack at the electron rich P beta of nucleoside triphosphates. Members of this small mechanistic class of enzymes require two divalent cations per active site for activity--one coordinated by the enzyme and the other by the enzyme-bound NTP--and show low catalytic powers of 10(7)- to 10(9)-fold. The first structure of an enzyme of this class, obtained by NMR methods in solution, shows MutT to be a compact globular protein with an alpha + beta-fold...

Beyond its role as an essential coenzyme in numerous oxidoreductase reactions as well as respiration, there is growing recognition that NAD+ fulfills many other vital regulatory functions both as a substrate and as an allosteric effector. This review describes the enzymes involved in pyridine nucleotide metabolism, starting with a detailed consideration of the anaerobic and aerobic pathways leading to quinolinate, a key precursor of NAD+. Conversion of quinolinate and 5'-phosphoribosyl-1'-pyrophosphate to NAD+ and diphosphate by phosphoribosyltransferase is then explored before proceeding to a discussion the molecular and kinetic properties of NMN adenylytransferase...

The catalytic mechanisms of adenylate kinase, guanylate kinase, uridylate kinase, and cytidylate kinase are reviewed in terms of kinetic and structural information that has been obtained in recent years. All four kinases share a highly related tertiary structure, characterized by a central five-stranded parallel beta-sheet with helices on both sides, as well as the three regions designated as the CORE, NMPbind, and LID domains. The catalytic mechanism continues to be refined to higher levels of resolution by iterative structure-function studies, and the strengths and limitations of site-directed mutagenesis are well illustrated in the case of adenylate kinase...