Recent advances in the biocatalytic reduction of ketones and oxidation of sec-alcohols

To improve the efficiency and applicability of biocatalytic redox-reactions for asymmetric ketone-reduction and enantioselective alcohol-oxidation catalyzed by nicotinamide-dependent dehydrogenases/reductases, several achievements for cofactor-recycling have been made during the last two years. First, the use of hydrogenases for NADPH recycling in a two enzyme system. Second, preparative transformations with alcohol dehydrogenases coupled with NADH oxidases for NAD+/NADP+ recycling. Third, an exceptional chemo-stable alcohol dehydrogenase can efficiently use i-propanol and acetone as cosubstrates for reduction and oxidation, respectively, in a single-enzyme system. Novel carbonyl reductases and dehydrogenases derived from plant cells are particularly suited for sterically demanding substrates.     

Evolutionary Aspects of Enzyme Dynamics

The role of evolutionary pressure on the chemical step catalyzed by enzymes is somewhat enigmatic, in part because chemistry is not rate-limiting for many optimized systems. Herein, we present studies that examine various aspects of the evolutionary relationship between protein dynamics and the chemical step in two paradigmatic enzyme families, dihydrofolate reductases and alcohol dehydrogenases. Molecular details of both convergent and divergent evolution are beginning to emerge. The findings suggest that protein dynamics across an entire enzyme can play a role in adaptation to differing physiological conditions. The growing tool kit of kinetics, kinetic isotope effects, molecular biology, biophysics, and bioinformatics provides means to link evolutionary changes in structure-dynamics function to the vibrational and conformational states of each protein.     

Medium-chain dehydrogenases/reductases (MDR). Family characterizations including genome comparisons and active site modeling.

Completed eukaryotic genomes were screened for medium-chain dehydrogenases/reductases (MDR). In the human genome, 23 MDR forms were found, a number that probably will increase, because the genome is not yet fully interpreted. Partial sequences already indicate that at least three further members exist. Within the MDR superfamily, at least eight families were distinguished. Three families are formed by dimeric alcohol dehydrogenases (ADH; originally detected in animals/plants), cinnamyl alcohol dehydrogenases (originally detected in plants) and tetrameric alcohol dehydrogenases (originally detected in yeast). Three further families are centred around forms initially detected as mitochondrial respiratory function proteins, acetyl-CoA reductases of fatty acid synthases, and leukotriene B4 dehydrogenases. The two remaining families with polyol dehydrogenases (originally detected as sorbitol dehydrogenase) and quinone reductases (originally detected as zeta-crystallin) are also distinct but with variable sequences. The most abundant families in the human genome are the dimeric ADH forms and the quinone oxidoreductases. The eukaryotic patterns are different from those of Escherichia coli. The different families were further evaluated by molecular modelling of their active sites as to geometry, hydrophobicity and volume of substrate-binding pockets. Finally, sequence patterns were derived that are diagnostic for the different families and can be used in genome annotations.     

Electron redistribution on binding of a substrate to an enzyme: folate and dihydrofolate reductase

The migration of electron density of a substrate (folate) on binding to an enzyme (dihydrofolate reductase) is studied by a quantum-mechanical method originally developed in solid state physics. A significant polarization of the substrate is induced by the enzyme, toward the transition state of the enzymatic reaction, at the same time giving rise to "electronic strain energy" in the substrate and enhanced protein-ligand interactions. The spatial arrangement of protein charges that induces the polarization is identified and found to be structurally conserved for bacterial and vertebrate dihydrofolate reductases.     

A new mechanism of plasmid trimethoprim resistance. Characterization of an inducible dihydrofolate reductase

The dihydrofolate reductase encoded by plasmid pUK1123, which confers only a moderate level of trimethoprim resistance on its host, has been isolated and characterized. This enzyme, designated type IV, differs markedly from all previously described plasmid dihydrofolate reductases. It has a relatively high molecular weight of 46,700 as measured by gel filtration and, unlike previous plasmid dihydrofolate reductases, its synthesis is induced in the presence of increasing concentrations of trimethoprim. It is only slightly resistant to trimethoprim but is competitively inhibited by this drug with an inhibitor binding constant of 63 nM. In addition, the enzyme has a relatively low affinity for the substrate, dihydrofolate (Km = 37 microM). This is the first report of a plasmid trimethoprim resistance mechanism resulting from the induced synthesis of a large molecular weight dihydrofolate reductase which is only slightly resistant to trimethoprim. The possible origins of the type IV enzyme are discussed.     

Variations and constant patterns in eukaryotic MDR enzymes. Conclusions from novel structures and characterized genomes

Medium-chain dehydrogenases/reductases (MDR) alcohol dehydrogenases exhibit multiple forms through a number of gene duplications. A crucial duplication was the one leading from the glutathione-dependent formaldehyde dehydrogenase line to the liver alcohol dehydrogenase (ADH) lines of vertebrates, the first duplication of which can now be further positioned at early vertebrate times. Similarly, screening of MDR forms in recently completed eukaryotic genomes of Caenorhabditis elegans and Drosophila melanogaster suggest that the MDR family may constitute a moderately sized protein family centered around a limited number of enzyme activities of five different structural types.     

Active contraction of cardiac cells: a reduced model for sarcomere dynamics with cooperative interactions

We propose a reduced ODE model for the mechanical activation of cardiac myofilaments, which is based on explicit spatial representation of nearest-neighbour interactions. Our model is derived from the cooperative Markov Chain model of Washio et al. (Cell Mol Bioeng 5(1):113–126, 2012), under the assumption of conditional independence of specific sets of events. This physically motivated assumption allows to drastically reduce the number of degrees of freedom, thus resulting in a significantly large computational saving. Indeed, the original Markov Chain model involves a huge number of degrees of freedom (order of \(10^{21}\)) and is solved by means of the Monte Carlo method, which notoriously reaches statistical convergence in a slow fashion. With our reduced model, instead, numerical simulations can be carried out by solving a system of ODEs, reducing the computational time by more than 10, 000 times. Moreover, the reduced model is accurate with respect to the original Markov Chain model. We show that the reduced model is capable of reproducing physiological steady-state force–calcium and force–length relationships with the observed asymmetry in apparent cooperativity near the calcium level producing half activation. Finally, we also report good qualitative and quantitative agreement with experimental measurements under dynamic conditions.     

Variations and constant patterns in eukaryotic MDR enzymes: Conclusions from novel structures and characterized genomes

Medium-chain dehydrogenases/reductases (MDR) alcohol dehydrogenases exhibit multiple forms through a number of gene duplications. A crucial duplication was the one leading from the glutathione-dependent formaldehyde dehydrogenase line to the liver alcohol dehydrogenase (ADH) lines of vertebrates, the first duplication of which can now be further positioned at early vertebrate times. Similarly, screening of MDR forms in recently completed eukaryotic genomes of Caenorhabditis elegans and Drosophila melanogaster suggest that the MDR family may constitute a moderately sized protein family centered around a limited number of enzyme activities of five different structural types.     

Characterization of Candida albicans dihydrofolate reductase

Dihydrofolate reductase from Candida albicans was purified 31,000-fold and characterized. In addition, the C. albicans dihydrofolate reductase gene was cloned into a plasmid vector and expressed in Escherichia coli, and the enzyme was purified from this source. Both preparations showed a single protein-staining band with a molecular weight of about 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymes were stable and had an isoelectric point of pH 7.1 on gel isoelectric focusing. Kinetic characterization showed that the enzymes from each source had similar turnover numbers (about 11,000 min-1) and Km values for NADPH and dihydrofolate of 3-4 microM. Like other eukaryotic dihydrofolate reductases, the C. albicans enzyme exhibited weak binding affinity for the antibacterial agent trimethoprim (Ki = 4 microM), but further characterization showed that the inhibitor binding profile of the yeast and mammalian enzymes differed. Methotrexate was a tight binding inhibitor of human but not C. albicans dihydrofolate reductase; the latter had a relatively high methotrexate Ki of 150 pM. The yeast and vertebrate enzymes also differed in their interactions with KCl and urea. These two agents activate vertebrate dihydrofolate reductases but inhibited the C. albicans enzyme. The sequence of the first 36 amino-terminal amino acids of the yeast enzyme was also determined. This portion of the C. albicans enzyme was more similar to human than to E. coli dihydrofolate reductases (50% and 30% identity, respectively). Some key amino acid residues in the C. albicans sequence, such as E-30 (human enzyme numbering), were "vertebrate-like" whereas others, such as I-31, were not. These results indicate that there are physical and kinetic differences between the eukaryotic mammalian and yeast enzymes.     

Dihydropteridine reductase as an alternative to dihydrofolate reductase for synthesis of tetrahydrofolate in Thermus thermophilus

A strategy devised to isolate a gene coding for a dihydrofolate reductase from Thermus thermophilus DNA delivered only clones harboring instead a gene (the T. thermophilus dehydrogenase [DH(Tt)] gene) coding for a dihydropteridine reductase which displays considerable dihydrofolate reductase activity (about 20% of the activity detected with 6,7-dimethyl-7,8-dihydropterine in the quinonoid form as a substrate). DH(Tt) appears to account for the synthesis of tetrahydrofolate in this bacterium, since a classical dihydrofolate reductase gene could not be found in the recently determined genome nucleotide sequence (A. Henne, personal communication). The derived amino acid sequence displays most of the highly conserved cofactor and active-site residues present in enzymes of the short-chain dehydrogenase/reductase family. The enzyme has no pteridine-independent oxidoreductase activity, in contrast to Escherichia coli dihydropteridine reductase, and thus appears more similar to mammalian dihydropteridine reductases, which do not contain a flavin prosthetic group. We suggest that bifunctional dihydropteridine reductases may be responsible for the synthesis of tetrahydrofolate in other bacteria, as well as archaea, that have been reported to lack a classical dihydrofolate reductase but for which possible substitutes have not yet been identified.     

Cytochemical research on cysts of the sarcosporidian Sarcocystis bovicanis. II. Oxidation-reduction enzymes

By the means of light-microscopic cytological enzymatic methods, the presence of several enzymes (NAD.H and NADP.H-tetrazolium reductases, in addition to alcohol, succinate, isocitrate, glucose-6-phosphate, beta-hydroxybutyrate and glutamate dehydrogenases) has been studied in the tissue cysts of S. bovicanis. A mixed character of oxidative metabolism in the cyst stages is suggested, the involvement of gluconeogenesis being proposed. Neither beta-hydroxybutyrate nor alcohol dehydrogenase activity was demonstrated indicating the absence or a very low rate of lipid metabolism, and suggesting that the process of glycolysis may end with malate formation. From the low activity level of succinate dehydrogenases it is concluded that the citric acid cycle plays presumably a secondary role, if at all, in the energy supply of the cyst stages. Also, a low activity of glucose-6-phosphate dehydrogenases is pointed out. Thus, it is proposed that glycolysis may be primary, if not the only, oxidative pathway in the cyst stages.     

Structural evidence for lack of inhibition of fish goose-type lysozymes by a bacterial inhibitor of lysozyme

It is known that bacteria contain inhibitors of lysozyme activity. The recently discovered Escherichia coli inhibitor of vertebrate lysozyme (Ivy) and its potential interactions with several goose-type (g-type) lysozymes from fish were studied using functional enzyme assays, comparative homology modelling, protein–protein docking, and molecular dynamics simulations. Enzyme assays carried out on salmon g-type lysozyme revealed a lack of inhibition by Ivy. Detailed analysis of the complexes formed between Ivy and both hen egg white lysozyme (HEWL) and goose egg white lysozyme (GEWL) suggests that electrostatic interactions make a dominant contribution to inhibition. Comparison of three dimensional models of aquatic g-type lysozymes revealed important insertions in the β domain, and specific sequence substitutions yielding altered electrostatic surface properties and surface curvature at the protein–protein interface. Thus, based on structural homology models, we propose that Ivy is not effective against any of the known fish g-type lysozymes. Docking studies suggest a weaker binding mode between Ivy and GEWL compared to that with HEWL, and our models explain the mechanistic necessity for conservation of a set of residues in g-type lysozymes as a prerequisite for inhibition by Ivy.     

1,3-丙二醇氧化还原酶结构与功能关系的探讨

用生物信息学方法对来源于klebsiella pneumonide的1,3-丙二醇氧化还原酶(即1,3-丙二醇氧化还原酶,PDOR)进行高级结构模建,并搜索其功能位点,以所得三维结构为对象,定位其铁信号结合位点、辅酶NADP大致位置以及可能的底物结合部位;在此基础上模拟PDOR活性部位,探讨该酶的构效关系。     

Theoretical calculations of the catalytic triad in short-chain alcohol dehydrogenases/reductases

Three highly conserved active site residues (Ser, Tyr, and Lys) of the family of short-chain alcohol dehydrogenases/reductases (SDRs) were demonstrated to be essential for catalytic activity and have been denoted the catalytic triad of SDRs. In this study computational methods were adopted to study the ionization properties of these amino acids in SDRs from Drosophila melanogaster and Drosophila lebanonensis. Three enzyme models, with different ionization scenarios of the catalytic triad that might be possible when inhibitors bind to the enzyme cofactor complex, were constructed. The binding of the two alcohol competitive inhibitors were studied using automatic docking by the Internal Coordinate Mechanics program, molecular dynamic (MD) simulations with the AMBER program package, calculation of the free energy of ligand binding by the linear interaction energy method, and the hydropathic interactions force field. The calculations indicated that deprotonated Tyr acts as a strong base in the binary enzyme-NAD⁺ complex. Molecular dynamic simulations for 5 ns confirmed that deprotonated Tyr is essential for anchoring and orientating the inhibitors at the active site, which might be a general trend for the family of SDRs. The findings here have implications for the development of therapeutically important SDR inhibitors.     

X-ray structures of NADPH-dependent carbonyl reductase from Sporobolomyces salmonicolor provide insights into stereoselective reductions of carbonyl compounds

The X-ray structures of red yeast Sporobolomyces salmonicolor carbonyl reductase (SSCR) and its complex with a coenzyme, NADPH, have been determined at a resolution of 1.8A and 1.6A, respectively. SSCR was crystallized in an orthorhombic system with the space group P2(1)2(1)2(1) and cell dimensions of a=54.86 A, b=83.49 A, and c=148.72 A. On its cocrystallization with NADPH, isomorphous crystals of the SSCR/NADPH complex were obtained. The structure of SSCR was solved by a single wavelength anomalous diffraction measurement using a selenomethionine-substituted enzyme, and that of the SSCR/NADPH complex was solved by a molecular replacement method using the solved structure of SSCR. The structures of SSCR and the SSCR/NADPH complex were refined to an R-factor of 0.193 (R(free)=0.233) and 0.211 (R(free)=0.238), respectively. SSCR has two domains, an NADPH-binding domain and a substrate-binding domain, and belongs to the short-chain dehydrogenases/reductases family. The structure of the NADPH-binding domain and the interaction between the enzyme and NADPH are very similar to those found in other structure-solved enzymes belonging to the short-chain dehydrogenases/reductases family, while the structure of the substrate-binding domain is unique. SSCR has stereoselectivity in its catalytic reaction, giving rise to excessive production of (S)-alcohols from ethyl 4-chloro-3-oxobutanoate. The X-ray structure of the SSCR/NADPH complex and preliminary modeling show that the formation of the hydrophobic channel induced by the binding of NADPH is closely related to the stereoselective reduction by SSCR.     

Bioinformatic and biochemical characterization of DCXR and DHRS2/4 from Caenorhabditis elegans

Several reductases belonging to the large enzyme superfamily of the short-chain dehydrogenases/reductases (SDR) are involved in the reductive metabolism of carbonyl containing xenobiotics. In order to characterize the human enzymes dicarbonyl/l-xylulose reductase (DCXR), and dehydrogenase/reductase members 2 and 4 (DHRS2, DHRS4) in terms of metabolism of xenobiotics, orthologues from the model organism Caenorhabditis elegans (C. elegans) were identified by using hidden Markov models that were developed in the present study. Accordingly, we describe the characterization of proteins from C. elegans as orthologous to the human enzymes DCXR and DHRS2/4 using a combined approach of bioinformatic and biochemical methods. With the hidden Markov model based system we identified the C. elegans proteins SDR20C18, SDR25C21 and SDR25C22 as being homologous to the human enzymes DCXR, and DHRS2 or DHRS4, respectively. After cloning and overexpression of these three C. elegans genes in Escherichia coli we could purify SDR20C18 and SDR25C22 as soluble proteins by Ni-affinity chromatography, whereas recombinant SDR25C21 was only found in inclusion bodies. Both SDR20C18 (UniProtAcc: Q21929) and SDR25C22 (UniProtAcc: Q93790) were tested with a variety of xenobotic carbonyl compounds as substrates. A comparison of the catalytic activities of SDR20C18 and SDR25C22 with well-known substrates of the human forms revealed that SDR20C18 is the DCXR-orthologue enzyme to the human enzyme and that SDR25C22 might be a DHRS2/4 homologue. Due to their high sequence identity, it was so far not possible to distinguish between SDR25C22 and the human DHRS2/4 proteins by means of sequence analysis alone. However, the study of homologue genes in the model organism C. elegans can provide valuable information on the putative physiological role of the corresponding human form.     

Superfamilies SDR and MDR: From early ancestry to present forms. Emergence of three lines, a Zn-metalloenzyme, and distinct variabilities

Two large gene and protein superfamilies, SDR and MDR (short- and medium-chain dehydrogenases/reductases), were originally defined from analysis of alcohol and polyol dehydrogenases. The superfamilies contain minimally 82 and 25 genes, respectively, in humans, minimally 324 and 86 enzyme families when known lines in other organisms are also included, and over 47,000 and 15,000 variants in existing sequence data bank entries. SDR enzymes have one-domain subunits without metal and MDR two-domain subunits without or with zinc, and these three lines appear to have emerged in that order from the universal cellular ancestor. This is compatible with their molecular architectures, present multiplicity, and overall distribution in the kingdoms of life, with SDR also of viral occurrence. An MDR-zinc, when present, is often, but not always, catalytic. It appears also to have a structural role in inter-domain interactions, coenzyme binding and substrate pocket formation, as supported by domain variability ratios and ligand positions. Differences among structural and catalytic zinc ions may be relative and involve several states. Combined, the comparisons trace evolutionary properties of huge superfamilies, with partially redundant enzymes in cellular redox functions.     

Quinone-dependent alcohol dehydrogenases and fad-dependent alcohol oxidases

This review considers quinone-dependent alcohol dehydrogenases and FAD-dependent alcohol oxidases, enzymes that are present in numerous methylotrophic eu- and prokaryotes and significantly differ in their primary and quaternary structure. The cofactors of the enzymes are bound to the protein polypeptide chain through ionic and hydrophobic interactions. Microorganisms containing these enzymes are described. Methods for purification of the enzymes, their physicochemical properties, and spatial structures are considered. The supposed mechanism of action and practical application of these enzymes as well as their producers are discussed.