Laccases: a never-ending story

Laccases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) are blue multicopper oxidases that catalyze the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. In fungi, laccases carry out a variety of physiological roles during their life cycle. These enzymes are being increasingly evaluated for a variety of biotechnological applications due to their broad substrate range. In this review, the most recent studies on laccase structural features and catalytic mechanisms along with analyses of their expression are reported and examined with the aim of contributing to the discussion on their structure–function relationships. Attention has also been paid to the properties of enzymes endowed with unique characteristics and to fungal laccase multigene families and their organization.     

Laccases of prokaryotic origin: enzymes at the interface of protein science and protein technology

The ubiquitous members of the multicopper oxidase family of enzymes oxidize a range of aromatic substrates such as polyphenols, methoxy-substituted phenols, amines and inorganic compounds, concomitantly with the reduction of molecular dioxygen to water. This family of enzymes can be broadly divided into two functional classes: metalloxidases and laccases. Several prokaryotic metalloxidases have been described in the last decade showing a robust activity towards metals, such as Cu(I), Fe(II) or Mn(II) and have been implicated in the metal metabolism of the corresponding microorganisms. Many laccases, with a superior efficiency for oxidation of organic compounds when compared with metals, have also been identified and characterized from prokaryotes, playing roles that more closely conform to those of intermediary metabolism. This review aims to present an update of current knowledge on prokaryotic multicopper oxidases, with a special emphasis on laccases, anticipating their enormous potential for industrial and environmental applications.     

Sugar-mimicking glycosidase inhibitors: bioactivity and application

A large number of compounds mimicking the structures of monosaccharides or oligosaccharides have been discovered from natural sources. Such sugar mimics inhibit carbohydrate-degrading enzymes because of a structural resemblance to the sugar moiety of the natural substrate. Carbohydrate-degrading enzymes are involved in a wide range of important biological processes, such as intestinal digestion, posttranslational processing of the sugar chain of glycoproteins, their quality control mechanisms, lysosomal catabolism of glycoconjugates, and some viral infections. It has now been realized that inhibitors of the enzymes have enormous therapeutic potential in diabetes and lysosomal storage disorders. In this review, the general bioactivity, current applications, and the prospects for new therapeutic applications are described. Received 27 August 2008; received after revision 08 November 2008; accepted 03 December 2008     

Molecular modeling of mammalian cytochrome P450s

The cytochrome P450s are a superfamily of hemoprotein enzymes responsible for the metabolism of a wide variety of xenobiotic and endogenous compounds. The individual P450s exhibit unique substrate specificity and stereoselectivity profiles which reflect corresponding differences in primary sequence and tertiary structure. In the absence of an experimental structure, models for mammalian P450s have been generated by their homology with bacterial P450s of known structure. The rather low sequence identity between target and template proteins renders P450 modeling a challenging task. However, the substrate recognition properties of several P450s are consistent with recently developed working models. This review summarizes the major concepts and current approaches of molecular modeling of P450s. Received 28 September 1999; received after revision 25 November 1999; accepted 31 December 1999     

Stereochemistry of enzymic processes in the biosynthesis of pyrrolizidine alkaloids.

The harmonization of biosynthetic pathways with organic reaction mechanisms has relied heavily on stereochemical studies. The field of biosynthesis of pyrrolizidine alkaloids exemplifies these connections through a wide range of common organic reactions including oxidation condensation, and decarboxylation. Further, the applications of biogenetic concepts and enzyme-catalysed reactions to synthesis are illustrated. The results are exciting, not only for their intrinsic scientific interest, but because they point the way to using plant enzymes to recognise structurally modified biosynthetic intermediates and hence open routes to the synthesis of new compounds that would otherwise be difficult to obtain.     

Bacterial nonspecific acid phosphohydrolases: physiology, evolution and use as tools in microbial biotechnology

Bacterial nonspecific acid phosphohydrolases (NSAPs) are secreted enzymes, produced as soluble periplasmic proteins or as membrane-bound lipoproteins, that are usually able to dephosphorylate a broad array of structurally unrelated substrates and exhibit optimal catalytic activity at acidic to neutral pH values. Bacterial NSAPs are monomeric or oligomeric proteins containing polypeptide components with an M _r of 25 – 30 kDa. On the basis of amino acid sequence relatedness, three different molecular families of NSAPs can be distinguished, indicated as molecular class A, B and C, respectively. Members of each class share some common biophysical and functional features, but may also exhibit functional differences. NSAPs have been detected in several microbial taxa, and enzymes of different classes can be produced by the same bacterial species. Structural and phyletic relationships exist among the various bacterial NSAPs and some other bacterial and eucaryotic phosphohydrolases. Current knowledge on bacterial NSAPs is reviewed, together with analytical tools that may be useful for their characterization. An overview is also presented concerning the use of bacterial NSAPs in biotechnology. Received 21 November 1997; received after revision 10 March 1998; accepted 10 March 1998     

Direct observation of enzyme substrate complexes by stopped-flow fluorescence: mathematical analyses

The fluorescence changes which occur upon the interaction of enzyme and substrate under stopped-flow conditions can provide a sensitive means to directly observe ES complexes. The interconversion of the intermediates during catalysis causes changes in fluorescence, signaling directly their existence, and allowing their quantitation. We have studied extensively an approach which measures radiationless energy transfer (RET) between enzyme tryptophanyl residues and a fluorescent peptide or ester substrate. Our studies of a number of proteolytic enzymes have validated the approach, which is sensitive and applicable to a variety of enzymes under a wide range of experimental conditions, including subzero temperatures. Direct excitation of fluorescent substrates can also be used to observe ES complex formation and breakdown and is complementary to the RET approach. Here we review both the RET and direct excitation kinetic approaches, with particular emphasis on the mathematical foundations we have developed which are critical to the successful interpretation of these or any other spectroscopic approach which yields a signal that is unique to the ES complex.     

Stereochemistry of enzymic processes in the biosynthesis of pyrrolizidine alkaloids

The harmonization of biosynthetic pathways with organic reaction mechanisms has relied heavily on stereochemical studies. The field of biosynthesis of pyrrolizidine alkaloids exemplifies these connections through a wide range of common organic reactions including oxidation, condensation, and decarboxylation. Further, the applications of biogenetic concepts and enzyme-catalysed reactions to synthesis are illustrated. The results are exciting, not only for their intrinsic scientific interest, but because they point the way to using plant enzymes to recognise structurally modified biosynthetic intermediates and hence open routes to the synthesis of new compounds that would otherwise be difficult to obtain.     

Complexing agents from microorganisms

Summary The majority of extracellular complexing ligands produced by microorganisms are summarized as being of low molecular mass (<10,000 daltons) and are usually released as part of metal detoxification processes. These exudates appear to exhibit strong metal-binding characteristics, often reducing metal toxicity. Under certain conditions microbes produce metal-specific compounds of low molecular mass called siderophores; although these are normally specific for iron they also have relatively high affinities for radionuclides such as Pu and facilitate their uptake into cells. The occurrence of specific actinide complexing agents has been recorded.The breakdown of lignins and cellulosic material produces large macromolecular compounds called humates. These contain multiligand sites and display a wide range of complexing abilities. They form both soluble and insoluble complexes with toxic elements with various results. Humates also considerably influence adsorption of metals to substrate surfaces and at high pH may compete with OH-ions for metal binding.As well as with extracellular ligands, metals can interact directly with microorganisms by accumulation in subcellular compartments or by adsorption on bacterial surfaces.     

Substrate ambiguity among the nudix hydrolases: biologically significant, evolutionary remnant, or both?

Many members of the nudix hydrolase family exhibit considerable substrate multispecificity and ambiguity, which raises significant issues when assessing their functions in vivo and gives rise to errors in database annotation. Several display low antimutator activity when expressed in bacterial tester strains as well as some degree of activity in vitro towards mutagenic, oxidized nucleotides such as 8-oxo-dGTP. However, many of these show greater activity towards other nucleotides such as ADP-ribose or diadenosine tetraphosphate (Ap₄A). The antimutator activities have tended to gain prominence in the literature, whereas they may in fact represent the residual activity of an ancestral antimutator enzyme that has become secondary to the more recently evolved major activity after gene duplication. Whether any meaningful antimutagenic function has also been retained in vivo requires very careful assessment. Then again, other examples of substrate ambiguity may indicate as yet unexplored regulatory systems. For example, bacterial Ap₄A hydrolases also efficiently remove pyrophosphate from the 5′ termini of mRNAs, suggesting a potential role for Ap₄A in the control of bacterial mRNA turnover, while the ability of some eukaryotic mRNA decapping enzymes to degrade IDP and dIDP or diphosphoinositol polyphosphates (DIPs) may also be indicative of new regulatory networks in RNA metabolism. DIP phosphohydrolases also degrade diadenosine polyphosphates and inorganic polyphosphates, suggesting further avenues for investigation. This article uses these and other examples to highlight the need for a greater awareness of the possible significance of substrate ambiguity among the nudix hydrolases as well as the need to exert caution when interpreting incomplete analyses.     

Bacterial antibiotic efflux systems of medical importance

Multidrug efflux systems endow on bacterial cells the ability to limit the access of antimicrobial agents to their targets. By actively pumping out antibiotic molecules, these systems prevent the intracellular accumulation necessary for antibiotics to exert their lethal activity. Drug efflux appears to be one of the most widespread antibiotic resistance mechanisms among microorganisms, since it has been demonstrated to occur in many Gram-positive and Gram-negative bacteria including medically important species like staphylococci, streptococci, enterobacteria and opportunistic pathogens like Pseudomonas aeruginosa. Efflux pumps can be specific for only one substrate or accommodate a more or less wide range of noxious products. Export of structurally unrelated compounds confers a multidrug-resistance phenotype on bacterial cells. Therapeutically critical levels of resistance can be achieved by overexpression of efflux systems, especially in those species such as P. aeruginosa which possess a low outer membrane permeability. It is suspected that the dual physiological function of active efflux systems is both the secretion of intracellular metabolites and the protection against a variety of harmful substances that the microorganism may encounter in its natural environment.     

Direct observation of enzyme substrate complexes by stopped-flow fluorescence: Mathematical analyses

Summary The fluorescence changes which occur upon the interaction of enzyme and substrate under stopped-flow conditions can provide a sensitive means to directly observe ES complexes. The interconversion of the intermediates during catalysis causes changes in fluorescence, signaling directly their existence, and allowing their quantitation. We have studied extensively and approach which measures radiationless energy transfer (RET) between enzyme tryptophanyl residues and a fluorescent peptide or ester substrate. Our studies of a number of proteolytic enzymes have validated the approach, which is sensitive and applicable to a variety of enzymes under a wide range of experimental conditions, including subzero temperatures. Direct excitation of fluorescent substrates can also be used to observe ES complex formation and breakdown and is complementary to the RET approach. Here we review both the RET and direct excitation kinetic approaches, with particular emphasis on the mathematical foundations we have developed which are critical to the successful interpretation of these or any other spectroscopic approach which yields a signal that is unique to ES complex.Acknowledgment. This work was supported by Grants-in-Aid GM-24967 and 24968 from the National Institute of Health of the Department of Health, Education and Welfare to Harvard Medical School. The continued advice and support of Dr B.L. Vallee is gratefully acknowledged, as is the help of Dr L. Bethune on theoretical aspects of the steady-state assumption. The excellent technical assistance of P. Maiorana is acknowledged. The abbreviations used are: RET; radiationless energy transfer, Dns, or dansyl; 5-dimethylaminonaphthalene-1-sulfonyl; DED; dansylethylenediamine, Mes; 2-(N-morpholino)ethane sulfonic acid, OMe; methoxide, mansyl; 6-(N-methylanilino)-naphthalene-2-sulfonyl.     

Substrate specificity of bacterial DD-peptidases (penicillin-binding proteins)

The DD-peptidase enzymes (penicillin-binding proteins) catalyze the final transpeptidation reaction of bacterial cell wall (peptidoglycan) biosynthesis. Although there is now much structural information available about these enzymes, studies of their activity as enzymes lag. It is now established that representatives of two low-molecular-mass classes of DD-peptidases recognize elements of peptidoglycan structure and rapidly react with substrates and inhibitors incorporating these elements. No members of other DD-peptidase classes, including the high-molecular-mass enzymes, essential for bacterial growth, appear to interact strongly with any particular elements of peptidoglycan structure. Rational design of inhibitors for these enzymes is therefore challenging.     

Enzymatic in vitro synthesis of I-branches of mammalian polylactosamines: generation of scaffolds for multiple selectin-binding saccharide determinants

Polylactosamines are covalent monosaccharide assemblies of the animal kingdom and some bacteria, and are characterized by backbones of interlinked N-acetyllactosamine units (Galbeta1-4GlcNAc, LacNAc). The mammalian LacNAc arrays are linear (blood group i-type) and branched (blood group I-type), and are linked to the core elements of glycolipids as well as O- and N-glycans of glycoproteins and keratan sulfate proteoglycans. Generation of I-branches to linear i-type polylactosamines is initiated by two kinds of beta6GlcNAc transferases. One type of the enzymes transfers to Glc-NAcbeta 1-3Galbeta 1-OR of growing i-chains at the peridistal (underlined) Gal; these enzymes are called dIGnT (d for 'distally acting'). The other enzymes transfer to internal Gal units of preformed i-chains; they are called cIGnT (c for 'centrally acting'). Purified natural and recombinant enzymes of both types have been described. The structures of I-type polylactosamines result from a collaboration of dIGnTs, cIGnTs, beta4GalTs and the i-chain-elongating iGnTs. At present, the interplay of these enzymes in vivo is poorly understood. By contrast, enzyme-assisted in vitro synthesis of branched polylactosamines representing distinct LacNAc arrays that are multiply capped by a variety of decorations is possible. Some of the synthetic polylactosamines reduce the lymphocyte-endothelium adhesion in a tissue-specific mode, raising the possibility of achieving local immunosuppression in the future. Useful applications of multiply decorated I-type polylactosamines may also be found in prevention of mammalian gamete adhesion and in inhibition of bacterial and viral adhesion to host tissues.     

从文献计量分析看世界合成生物学研究现状

合成生物学是生物学的一个新兴分支学科.以美国科学引文索引数据库扩展版数据库(SCI-EXPANDED)收录入库,发表于1990～2010年的3 949篇与合成生物学相关的论文为研究对象,通过对论文的产出规模、地域分布、机构分布、研究热点、学科关联性、知识基础等方面的计量分析,在一定程度上揭示出近年来世界合成生物学的发展是一种成果产出不断丰富,理论研究与应用研究均得到积极推进,与众多学科发展联系紧密,各国科研投入力量在显著加强以及美国在全球一枝独秀的局面.     

Bacterial chemoreceptors and chemoeffectors

Bacteria use chemotaxis signaling pathways to sense environmental changes. Escherichia coli chemotaxis system represents an ideal model that illustrates fundamental principles of biological signaling processes. Chemoreceptors are crucial signaling proteins that mediate taxis toward a wide range of chemoeffectors. Recently, in deep study of the biochemical and structural features of chemoreceptors, the organization of higher-order clusters in native cells, and the signal transduction mechanisms related to the on–off signal output provides us with general insights to understand how chemotaxis performs high sensitivity, precise adaptation, signal amplification, and wide dynamic range. Along with the increasing knowledge, bacterial chemoreceptors can be engineered to sense novel chemoeffectors, which has extensive applications in therapeutics and industry. Here we mainly review recent advances in the E. coli chemotaxis system involving structure and organization of chemoreceptors, discovery, design, and characterization of chemoeffectors, and signal recognition and transduction mechanisms. Possible strategies for changing the specificity of bacterial chemoreceptors to sense novel chemoeffectors are also discussed.     

Insights into acylphosphatase structure and catalytic mechanism

Acylphosphatase is one of the smallest enzymes known (about 98 amino acid residues). It is present in organs and tissues of vertebrate species as two isoenzymes sharing over 55% of sequence homology; these appear highly conserved in differing species. The two isoenzymes can be involved in a number of physiological processes, though their effective biological function is not still certain. The solution and crystal structures of different isoenzymes are known, revealing a close packed protein with a fold similar to that shown by other phosphate-bind ing proteins. The structural data, together with an extended site-directed mutagenesis investigation, led to the identification of the residues involved in enzyme catalysis. However, it appears unlikely that these residues are able to perform the full catalytic cycle: a substrate-assisted catalytic mechanism has therefore been proposed, in which the phosphate moiety of the substrate could act as a nucleophile activating the catalytic water molecule. Received 12 November 1996; accepted 27 November 1996     

Multiple roles of class I HDACs in proliferation, differentiation, and development

Class I Histone deacetylases (HDACs) play a central role in controlling cell cycle regulation, cell differentiation, and tissue development. These enzymes exert their function by deacetylating histones and a growing number of non-histone proteins, thereby regulating gene expression and several other cellular processes. Class I HDACs comprise four members: HDAC1, 2, 3, and 8. Deletion and/or overexpression of these enzymes in mammalian systems has provided important insights about their functions and mechanisms of action which are reviewed here. In particular, unique as well as redundant functions have been identified in several paradigms. Studies with small molecule inhibitors of HDACs have demonstrated the medical relevance of these enzymes and their potential as therapeutic targets in cancer and other pathological conditions. Going forward, better understanding the specific role of individual HDACs in normal physiology as well as in pathological settings will be crucial to exploit this protein family as a useful therapeutic target in a range of diseases. Further dissection of the pathways they impinge on and of their targets, in chromatin or otherwise, will form important avenues of research for the future.     

Deoxyribozymes: useful DNA catalysts in vitro and in vivo

Deoxyribozymes (DNA enzymes; DNAzymes) are catalytic DNA sequences. Using the technique of in vitro selection, individual deoxyribozymes have been identified that catalyze RNA cleavage, RNA ligation, and a growing range of other chemical reactions. DNA enzymes have been used in vitro for applications such as biochemical RNA manipulation and analytical assays for metal ions, small organic compounds, oligonucleotides, and proteins. Deoxyribozymes have also been utilized as in vivo therapeutic agents to destroy specific mRNA targets. Although many conceptual and practical challenges remain to be addressed, deoxyribozymes have substantial promise to contribute meaningfully for applications both in vitro and in vivo.     

Mechanisms and structures of crotonase superfamily enzymes – How nature controls enolate and oxyanion reactivity

Structural and mechanistic studies on the crotonase superfamily (CS) are reviewed with the aim of illustrating how a conserved structural platform can enable catalysis of a very wide range of reactions. Many CS reactions have precedent in the 'carbonyl' chemistry of organic synthesis; they include alkene hydration/isomerization, aryl-halide dehalogenation, (de)carboxylation, CoA ester and peptide hydrolysis, fragmentation of beta-diketones and C-C bond formation, cleavage and oxidation. CS enzymes possess a canonical fold formed from repeated betabetaalpha units that assemble into two approximately perpendicular beta-sheets surrounded by alpha-helices. CS enzymes often, although not exclusively, oligomerize as trimers or dimers of trimers. Two conserved backbone NH groups in CS active sites form an oxyanion 'hole' that can stabilize enolate/oxyanion intermediates. The range and efficiency of known CS-catalyzed reactions coupled to their common structural platforms suggest that CS variants may have widespread utility in biocatalysis.