Short-chain Dehydrogenase Research Articles

High substrate specificity of ipsdienol dehydrogenase (IDOLDH), a short-chain dehydrogenase from Ips pini bark beetles.

Ips spp. bark beetles use ipsdienol, ipsenol, ipsdienone and ipsenone as aggregation pheromone components and pheromone precursors. For Ips pini, the short-chain oxidoreductase ipsdienol dehydrogenase (IDOLDH) converts (-)-ipsdienol to ipsdienone, and thus likely plays a role in determining pheromone composition. In order to further understand the role of IDOLDH in pheromone biosynthesis, we compared IDOLDH to its nearest functionally characterized ortholog with a solved structure: human L-3-hydroxyacyl-CoA dehydrogenase type II/ amyloid-β binding alcohol dehydrogenase (hHADH II/ABAD), and conducted functional assays of recombinant IDOLDH to determine substrate and product ranges and structural characteristics. Although IDOLDH and hHADH II/ABAD had only 35% sequence identity, their predicted tertiary structures had high identity. We found IDOLDH is a functional homo-tetramer. In addition to oxidizing (-)-ipsdienol, IDOLDH readily converted racemic ipsenol to ipsenone, and stereo-specifically reduced both ketones to their corresponding (-)-alcohols. The (+)-enantiomers were never observed as products. Assays with various substrate analogs showed IDOLDH had high substrate specificity for (-)-ipsdienol, ipsenol, ipsenone and ipsdienone, supporting that IDOLDH functions as a pheromone-biosynthetic enzyme. These results suggest that different IDOLDH orthologs and or activity levels contribute to differences in Ips spp. pheromone composition.

The First Defect in Electron Transfer to Mitochondrial P450 Enzymes.

Cloning of the cDNAs and genes for the various steroidogenic enzymes and their cofactors permitted identificationoftheprecisegeneticcausesofalltheknown human lesions in steroid biosynthesis and the identification of new diseases (1). There are 2 major classes of steroidogenic enzymes, hydroxysteroid dehydrogenases and cytochromes P450. Each of these classes is divided into 2 maingroups.Thehydroxysteroiddehydrogenasesmaybe either short-chain dehydrogenases or aldo-keto reductases. The short-chain dehydrogenases are familiar to most endocrinologists, and include the 2 isozymes of 3-hydroxysteroid dehydrogenase, the 2 functionally distinct forms of 11-hydroxysteroid dehydrogenase, and the principal forms of 17-hydroxysteroid dehydrogenase. By contrast the aldo-keto reductase enzymes catalyze less familiar reactions, such as 3-hydroxysteroid dehydrogenation and 5-reduction, which are important in the “backdoor pathway” of androgen synthesis (2, 3) and in bile acid synthesis (4). Thus, the hydroxysteroid dehydrogenases are important regulators of steroid activity at the termini of steroidogenic pathways (5), but we will say no more about them here. The cytochrome P450 enzymes are generally more familiar, because they catalyze biosynthetic reactions in the production of steroids, activated vitamin D, leukotrienes, prostaglandins, and bile acids, as well as participating in the degradation of countless drugs and xenobiotic agents. There are 2 distinct groups of vertebrate P450 enzymes: type I enzymes, which are targeted to the mitochondria, andtypeIIenzymes,whicharetargetedtotheendoplasmic reticulum. Enzymes of both classes are about 500-amino acid long, contain a heme group, and undergo a characteristic shift in their absorption spectra at 450 nm when

Structure of iridoid synthase in complex with NADP(+)/8-oxogeranial reveals the structural basis of its substrate specificity.

Iridoid synthase (IS), as a vegetal enzyme belonging to the short-chain dehydrogenase/reductase (SDR) superfamily, produces the ring skeletons for downstream alkaloids with various pharmaceutical activities, including the commercially available antineoplastic agents, vinblastine and vincristine. Here, we present the crystal structures of IS in apo state and in complex with NADP(+)/8-oxogeranial, exhibiting an active center that lacks the classical Tyr/Lys/Ser triad spatially conserved in SDRs, with only the catalytically critical function of triad tyrosine remained in Tyr178. In consistent, mutation of Tyr178 to a phenylalanine residue significantly abolished the catalytic activity of IS. Within the substrate binding pocket, the linear-shaped 8-oxogeranial adopts an entirely extended conformation with its two aldehyde ends hydrogen-bonded to Tyr178-OH and Ser349-OH, respectively. In addition, the intermediate carbon chain of bound substrate is harbored by a well-ordered hydrophobic scaffold, involving residues Ile145, Phe149, Leu203, Met213, Phe342, Ile345 and Leu352. Mutagenesis studies showed that both Ser349 and the hydrophobic residues around are determinant to the substrate specificity and, consequently, the catalytic activity of IS. In contrast, the Gly150-Pro160 loop previously proposed as a factor involved in substrate binding might have very limited contribution, because the deletion of residues Ile151-His161 has only slight influence on the catalytic activity. We believe that the present work will help to elucidate the substrate specificity of IS and to integrate its detailed catalytic mechanism.

Large-scale proteome analysis of abscisic acid and ABSCISIC ACID INSENSITIVE3-dependent proteins related to desiccation tolerance in Physcomitrella patens

Desiccation tolerance is an ancestral feature of land plants and is still retained in non-vascular plants such as bryophytes and some vascular plants. However, except for seeds and spores, this trait is absent in vegetative tissues of vascular plants. Although many studies have focused on understanding the molecular basis underlying desiccation tolerance using transcriptome and proteome approaches, the critical molecular differences between desiccation tolerant plants and non-desiccation plants are still not clear. The moss Physcomitrella patens cannot survive rapid desiccation under laboratory conditions, but if cells of the protonemata are treated by the phytohormone abscisic acid (ABA) prior to desiccation, it can survive 24 h exposure to desiccation and regrow after rehydration. The desiccation tolerance induced by ABA (AiDT) is specific to this hormone, but also depends on a plant transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3). Here we report the comparative proteomic analysis of AiDT between wild type and ABI3 deleted mutant (Δabi3) of P. patens using iTRAQ (Isobaric Tags for Relative and Absolute Quantification). From a total of 1980 unique proteins that we identified, only 16 proteins are significantly altered in Δabi3 compared to wild type after desiccation following ABA treatment. Among this group, three of the four proteins that were severely affected in Δabi3 tissue were Arabidopsis orthologous genes, which were expressed in maturing seeds under the regulation of ABI3. These included a Group 1 late embryogenesis abundant (LEA) protein, a short-chain dehydrogenase, and a desiccation-related protein. Our results suggest that at least three of these proteins expressed in desiccation tolerant cells of both Arabidopsis and the moss are very likely to play important roles in acquisition of desiccation tolerance in land plants. Furthermore, our results suggest that the regulatory machinery of ABA- and ABI3-mediated gene expression for desiccation tolerance might have evolved in ancestral land plants before the separation of bryophytes and vascular plants.

Characterization of two novel alcohol short-chain dehydrogenases/reductases from Ralstonia eutropha H16 capable of stereoselective conversion of bulky substrates

Biocatalysis has significant advantages over organic synthesis in the field of chiral molecule production and several types of stereoselective enzymes are already in use in industrial biotechnology. However, there is still a high demand for new enzymes capable of transforming bulky molecules with sufficient operability. In order to reveal novel high-potential biocatalysts, the complete genome of the β-proteobacterium Ralstonia eutropha H16 was screened for potential short-chain dehydrogenases/reductases (SDRs). We were able to identify two (S)-enantioselective SDRs named A5 and B3. These showed clear preference towards long-chain and aromatic secondary alcohols, aldehydes and ketones, with diaryl diketone benzil as one of the best substrates. In addition the phylogenetic analysis of all enzyme types, which are known to facilitate benzil reduction, revealed at least two separate evolutionary clusters. Our results indicate the biotechnological potential of SDRs A5 and B3 for the production of chiral compounds with potential commercial value.

Structure-guided stereoselectivity inversion of a short-chain dehydrogenase/reductase towards halogenated acetophenones.

The structure-guided rational design of an NADH-dependent short-chain dehydrogenase/reductase (SDR) reversed the stereoselectivity towards halogenated acetophenones from Prelog to anti-Prelog. The enzyme-substrate interactions involving an aromatic ring and a halogen atom were proven to play critical roles in determining the stereoselectivity of these ketone reductions besides the steric repulsion.

Pharmacophore Models and Pharmacophore-Based Virtual Screening: Concepts and Applications Exemplified on Hydroxysteroid Dehydrogenases.

Computational methods are well-established tools in the drug discovery process and can be employed for a variety of tasks. Common applications include lead identification and scaffold hopping, as well as lead optimization by structure-activity relationship analysis and selectivity profiling. In addition, compound-target interactions associated with potentially harmful effects can be identified and investigated. This review focuses on pharmacophore-based virtual screening campaigns specifically addressing the target class of hydroxysteroid dehydrogenases. Many members of this enzyme family are associated with specific pathological conditions, and pharmacological modulation of their activity may represent promising therapeutic strategies. On the other hand, unintended interference with their biological functions, e.g., upon inhibition by xenobiotics, can disrupt steroid hormone-mediated effects, thereby contributing to the development and progression of major diseases. Besides a general introduction to pharmacophore modeling and pharmacophore-based virtual screening, exemplary case studies from the field of short-chain dehydrogenase/reductase (SDR) research are presented. These success stories highlight the suitability of pharmacophore modeling for the various application fields and suggest its application also in futures studies.

The effect of natural and artificial fruit dehiscence on floss properties, seed germination and protein expression in Calotropis procera

A method was established, based on artificial fruit dehiscence, to collect seeds and silk fibers of C.procera without any loss. The seeds and floss obtained by this method were compared with those collected naturally. Natural fruit dehiscence caused a marked decrease in seeds and fibers yielded from C. procera. The seeds and fibers obtained under artificial fruit dehiscence were approximately sixfold higher than those obtained under natural fruit dehiscence. Furthermore, the application of artificial fruit dehiscence treatment had further advantages, such as a decrease in fiber diameter, and an increase in fiber density and cellulose content. However, this treatment had a slight negative impact on seed biomass and fiber birefringence. Fiber length, lignin and ash content of fibers were not affected by artificial fruit dehiscence. Seed germinability and seedling biomass were slightly decreased following application of artificial fruit dehiscence in comparison with natural fruit dehiscence. A significant difference was observed between the two treatments in amylase activity, soluble sugars and protein content. Some variation in protein expression was also apparent. Artificial fruit dehiscence lowered the expression of the short chain dehydrogenase, elongation factor 1-alpha, and peptidyl-prolyl cis–trans isomerase in comparison with natural dehiscence. Conversely, the expression of chitinase was enhanced in response to the artificial fruit dehiscence. Two conserved protein regions (prrl and vvet) were also observed.

Origins of stereoselectivity in evolved ketoreductases

Mutants of Lactobacillus kefir short-chain alcohol dehydrogenase, used here as ketoreductases (KREDs), enantioselectively reduce the pharmaceutically relevant substrates 3-thiacyclopentanone and 3-oxacyclopentanone. These substrates differ by only the heteroatom (S or O) in the ring, but the KRED mutants reduce them with different enantioselectivities. Kinetic studies show that these enzymes are more efficient with 3-thiacyclopentanone than with 3-oxacyclopentanone. X-ray crystal structures of apo- and NADP(+)-bound selected mutants show that the substrate-binding loop conformational preferences are modified by these mutations. Quantum mechanical calculations and molecular dynamics (MD) simulations are used to investigate the mechanism of reduction by the enzyme. We have developed an MD-based method for studying the diastereomeric transition state complexes and rationalize different enantiomeric ratios. This method, which probes the stability of the catalytic arrangement within the theozyme, shows a correlation between the relative fractions of catalytically competent poses for the enantiomeric reductions and the experimental enantiomeric ratio. Some mutations, such as A94F and Y190F, induce conformational changes in the active site that enlarge the small binding pocket, facilitating accommodation of the larger S atom in this region and enhancing S-selectivity with 3-thiacyclopentanone. In contrast, in the E145S mutant and the final variant evolved for large-scale production of the intermediate for the antibiotic sulopenem, R-selectivity is promoted by shrinking the small binding pocket, thereby destabilizing the pro-S orientation.

Biochemical analyses and molecular modeling explain the functional loss of 17β-hydroxysteroid dehydrogenase 3 mutant G133R in three Tunisian patients with 46, XY Disorders of Sex Development

Mutations in the HSD17B3 gene resulting in 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) deficiency cause 46, XY Disorders of Sex Development (46, XY DSD). Approximately 40 different mutations in HSD17B3 have been reported; only few mutant enzymes have been mechanistically investigated. Here, we report novel compound heterozygous mutations in HSD17B3, composed of the nonsense mutation C206X and the missense mutation G133R, in three Tunisian patients from two non-consanguineous families. Mutants C206X and G133R were constructed by site-directed mutagenesis and expressed in HEK-293 cells. The truncated C206X enzyme, lacking part of the substrate binding pocket, was moderately expressed and completely lost its enzymatic activity. Wild-type 17β-HSD3 and mutant G133R showed comparable expression levels and intracellular localization. The conversion of Δ4-androstene-3,17-dione (androstenedione) to testosterone was almost completely abolished for mutant G133R compared with wild-type 17β-HSD3. To obtain further mechanistic insight, G133 was mutated to alanine, phenylalanine and glutamine. G133Q and G133F were almost completely inactive, whereas G133A displayed about 70% of wild-type activity. Sequence analysis revealed that G133 on 17β-HSD3 is located in a motif highly conserved in 17β-HSDs and other short-chain dehydrogenase/reductase (SDR) enzymes. A homology model of 17β-HSD3 predicted that arginine or any other bulky residue at position 133 causes steric hindrance of cofactor NADPH binding, whereas substrate binding seems to be unaffected. The results indicate an essential role of G133 in the arrangement of the cofactor binding pocket, thus explaining the loss-of-function of 17β-HSD3 mutant G133R in the patients investigated.

Human DHRS7, promising enzyme in metabolism of steroids and retinoids?

The metabolism of steroids and retinoids has been studied in detail for a long time, as these compounds are involved in a broad spectrum of physiological processes. Many enzymes participating in the conversion of such compounds are members of the short-chain dehydrogenase/reductase (SDR) superfamily. Despite great effort, there still remain a number of poorly characterized SDR proteins. According to various bioinformatics predictions, many of these proteins may play a role in the metabolism of steroids and retinoids. Dehydrogenase/reductase (SDR family) member 7 (DHRS7) is one such protein. In a previous study, we determined DHRS7 to be an integral membrane protein of the endoplasmic reticulum facing the lumen which has shown at least in vitro NADPH-dependent reducing activity toward several eobiotics and xenobiotics bearing a carbonyl moiety. In the present paper pure DHRS7 was used for a more detailed study of both substrate screening and an analysis of kinetics parameters of the physiologically important substrates androstene-3,17-dione, cortisone and all-trans-retinal. Expression patterns of DHRS7 at the mRNA as well as protein level were determined in a panel of various human tissue samples, a procedure that has enabled the first estimation of the possible biological function of this enzyme. DHRS7 is expressed in tissues such as prostate, adrenal glands, liver or intestine, where its activity could be well exploited. Preliminary indications show that DHRS7 exhibits dual substrate specificity recognizing not only steroids but also retinoids as potential substrates and could be important in the metabolism of these signalling molecules.

Ralstonia solanacearum fatty acid composition is determined by interaction of two 3-ketoacyl-acyl carrier protein reductases encoded on separate replicons.

BackgroundFabG is the only known enzyme that catalyzes reduction of the 3-ketoacyl-ACP intermediates of bacterial fatty acid synthetic pathways. However, there are two Ralstonia solanacearum genes, RSc1052 (fabG1) and RSp0359 (fabG2), annotated as encoding putative 3-ketoacyl-ACP reductases. Both FabG homologues possess the conserved catalytic triad and the N-terminal cofactor binding sequence of the short chain dehydrogenase/reductase (SDR) family. Thus, it seems reasonable to hypothesize that RsfabG1 and RsfabG2 both encode functional 3-ketoacyl-ACP reductases and play important roles in R. solanacearum fatty acid synthesis and growth.MethodsComplementation of Escherichia colifabG temperature-sensitive mutant with R. solanacearum fabGs encoded plasmids was carried out to test the function of RsfabGs in fatty acid biosynthesis. RsFabGs proteins were purified by nickel chelate chromatography and fatty acid biosynthetic reaction was reconstituted to investigate the 3-ketoacyl-ACP reductase activity of RsFabGs in vitro. Disruption of both RsfabG genes was done via DNA homologous recombination to test the function of both RsfabG in vivo. And more we also carried out pathogenicity tests on tomato plants using RsfabG mutant strains. ResultsWe report that expression of either of the two proteins (RsFabG1 and RsFabG2) restores growth of the E. coli fabG temperature-sensitive mutant CL104 under non-permissive conditions. In vitro assays demonstrate that both proteins restore fatty acid synthetic ability to extracts of the E. coli strain. The RsfabG1 gene carried on the R. solanacearum chromosome is essential for growth of the bacterium, as is the case for fabG in E. coli. In contrast, the null mutant strain with the megaplasmid-encoded RsfabG2 gene is viable but has a fatty acid composition that differs significantly from that of the wild type strain. Our study also shows that RsFabG2 plays a role in adaptation to high salt concentration and low pH, and in pathogenesis of disease in tomato plants.ConclusionR. solanacearum encodes two 3-ketoacyl-ACP reductases that both have functions in fatty acid synthesis. We supply the first evidence that, like other enzymes in the bacterial fatty acid biosynthetic pathway, one bacterium may simultaneously possess two or more 3-oxoacyl-ACP reductase isozymes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0554-x) contains supplementary material, which is available to authorized users.

Cloning, Expression, and Characterization of budC Gene Encoding meso-2,3-Butanediol Dehydrogenase from Bacillus licheniformis.

The budC gene encoding a meso-2,3-butanediol dehydrogenase (BlBDH) from Bacillus licheniformis was cloned and overexpressed in Escherichia coli BL21(DE3). Sequence analysis reveals that this BlBDH belongs to short-chain dehydrogenase/reductase (SDR) superfamily. In the presence of NADH, BlBDH catalyzes the reduction of diacetyl to (3S)-acetoin (97.3% ee), and further to (2S,3S)-2,3-butanediol (97.3% ee and 96.5% de). Similar to other meso-2,3-BDHs, it shows oxidative activity to racemic 2,3-butanediol whereas no activity toward racemic acetoin in the presence of NAD(+). For diacetyl reduction and 2,3-butanediol oxidation, the pH optimum of BlBDH is 5.0 and 10.0, respectively. Unusually, it shows relatively high activity over a wide pH range from 5.0 to 8.0 for racemic acetoin reduction. BlBDH shows lower K m and higher catalytic efficiency toward racemic acetoin (K m = 0.47 mM, k cat /K m = 432 s(-1)·mM(-1)) when compared with 2,3-butanediol (K m = 7.25 mM, k cat /K m = 81.5 s(-1)·mM(-1)), indicating its physiological role in favor of reducing racemic acetoin into 2,3-butanediol. The enzymatic characterization of BlBDH provides evidence for the directed engineering of B. licheniformis for producing enantiopure 2,3-butanediol.

Structural insights on mouse L-threonine dehydrogenase: A regulatory role of Arg180 in catalysis.

Mouse L-threonine dehydrogenase (mTDH), which belongs to the short-chain dehydrogenase/reductase (SDR) superfamily and mediates threonine catabolism, plays pivotal roles in both powerful biosynthesis and signaling in mouse stem cells and has a regulatory residue Arg180. Here we determined three crystal structures of mTDH: wild-type (WT) in the apo form; in complex with NAD(+) and a substrate analog, glycerol, or with only NAD(+); as well as the R180K variant with NAD(+). This is the first description of a structure for mammalian SDR-type TDH. Structural comparison revealed the structural basis for SDR-type TDH catalysis remains strictly conserved in bacteria and mammals. Kinetic enzyme assays, and isothermal titration calorimetry (ITC) measurements indicated the R180K mutation has little effect on NAD(+) binding affinity, whereas affects the substrate's affinity for the enzyme. The crystal structure of R180K with NAD(+), biochemical and spectroscopic studies suggested that the R180K mutant should bind NAD(+) in a similar way and have a similar folding to the WT. However, the R180K variant may have difficulty adopting the closed form due to reduced interaction of residue 180 with a loop which connects a key position for mTDH switching between the closed and open forms in mTDH catalysis, and thereby exhibited a significantly decreased kcat/Km value toward the substrate, L-Thr. In sum, our results suggest that activity of GalE-like TDH can be regulated by remote interaction, such as hydrogen bonding and hydrophobic interaction around the Arg180 of mTDH.

Human dehydrogenase/reductase (SDR family) member 8 (DHRS8): a description and evaluation of its biochemical properties.

Dehydrogenase/reductase (SDR family) member 8 (DHRS8, SDR16C2) belongs to the short-chain dehydrogenase/reductase (SDR) superfamily, one of the largest enzyme groups. In addition to the well-known members which participate in the metabolism of important eobiotics and xenobiotics, this superfamily contains many poorly characterized proteins. DHRS8 is a member of the Multisubstrate NADP(H)-dependent SDR16C family, which generally contains insufficiently described enzymes. Despite the limited knowledge about DHRS8, preliminary indicators have emerged regarding its significant function in the modulation of steroidal activity, at least in the case of 3α-adiol, lipid metabolism and detoxification. The aim of this study was to describe additional biochemical properties of DHRS8 and to unify knowledge about this enzyme. The DHRS8 was prepared in recombinant form and its membrane topology in the endoplasmic reticulum as an integral protein with cytosolic orientation was demonstrated. The enzyme participates in the NAD(+)-dependent oxidation of steroid hormones as β-estradiol and testosterone in vitro; apparent K m and V max values were 39.86µM and 0.80nmol×mg(-1)× min(-1) for β-estradiol and 1207.29µM and 3.45nmol×mg(-1)× min(-1) for testosterone. Moreover, synthetic steroids (methyltestosterone and nandrolone) used as anabolics as well as all-trans-retinol were for the first time identified as substrates of DHRS8. This knowledge of its in vitro activity together with a newly described expression pattern at the protein level in tissues involved in steroidogenesis (adrenal gland and testis) and detoxification (liver, lung, kidney and small intestine) could suggest a potential role of DHRS8 in vivo.

Disruptions in a cluster of computationally identified enhancers near FOXC1 and GMDS may influence brain development

Regulatory elements are more evolutionarily conserved and provide a larger mutational target than coding regions of the human genome, suggesting that mutations in non-coding regions contribute significantly to development and disease. Using a computational approach to predict gene regulatory enhancers, we found that many known and predicted embryonic enhancers cluster in genomic loci harboring development-associated genes. One of the densest clusters of predicted enhancers in the human genome is near the genes GMDS and FOXC1. GMDS encodes a short-chain mannose dehydrogenase enzyme involved in the regulation of hindbrain neural migration, and FOXC1 encodes a developmental transcription factor required for brain, heart, and eye development. We experimentally validate four novel enhancers in this locus and demonstrate that these enhancers show consistent activity during embryonic development in domains that overlap with the expression of FOXC1 and GMDS. These four enhancers contain binding motifs for several transcription factors, including the ZIC family of transcription factors. Removal of the ZIC binding sites significantly alters enhancer activity in three of these enhancers, reducing expression in the eye, hindbrain, and limb, suggesting a mechanism whereby ZIC family members may transcriptionally regulate FOXC1 and/or GMDS expression. Our findings uncover novel enhancer regions that may control transcription in a topological domain important for embryonic development.Electronic supplementary materialThe online version of this article (doi:10.1007/s10048-015-0458-9) contains supplementary material, which is available to authorized users.

Diversity in Reduction with Short‐Chain Dehydrogenases: Tetrahydroxynaphthalene Reductase, Trihydroxynaphthalene Reductase, and Glucose Dehydrogenase

AbstractNAD(P)H‐dependent oxidoreductases from the short‐chain dehydrogenases/reductases (SDRs) family possess high functional diversity. Three SDRs, namely, tetrahydroxy‐ and trihydroxynaphthalene reductases (T4HNR, T3HNR) involved in the dihydroxynaphthalene‐melanin biosynthesis of the phytopathogenic fungus Magnaporthe grisea, and glucose dehydrogenase (GDH) from Bacillus subtilis, were characterized regarding their substrate range and functional behavior. T4HNR and T3HNR share activities towards the stereoselective reduction of 2‐tetralone derivatives and 2,3‐dihydro‐1,4‐naphthoquinones and show distinct but different stereochemical outcome in the case of epoxy‐1,4‐napthoquinones as substrates. GDH shares the activity towards 2,3‐dihydro‐1,4‐naphthoquinones, however, with low stereocontrol. Moreover, GDH reduces 2‐hydroxy‐2,3‐dihydro‐1,4‐naphthoquinone into trans‐4‐hydroxyscytalone with a high diastereomeric excess (96 %), whereas T4HNR gave the cis diastereomer (diastereomeric excess>99 %). Thus, SDRs provide a much higher functional and stereochemical diversity than previously thought, already exemplified by many transformations of three members of this enzyme family.

Myxococcus CsgA, Drosophila Sniffer, and human HSD10 are cardiolipin phospholipases.

Myxococcus xanthus development requires CsgA, a member of the short-chain alcohol dehydrogenase (SCAD) family of proteins. We show that CsgA and SocA, a protein that can replace CsgA function in vivo, oxidize the 2'-OH glycerol moiety on cardiolipin and phosphatidylglycerol to produce diacylglycerol (DAG), dihydroxyacetone, and orthophosphate. A lipid extract enriched in DAGs from wild-type cells initiates development and lipid body production in a csgA mutant to bypass the mutational block. This novel phospholipase C-like reaction is widespread. SCADs that prevent neurodegenerative disorders, such as Drosophila Sniffer and human HSD10, oxidize cardiolipin with similar kinetic parameters. HSD10 exhibits a strong preference for cardiolipin with oxidized fatty acids. This activity is inhibited in the presence of the amyloid β peptide. Three HSD10 variants associated with neurodegenerative disorders are inactive with cardiolipin. We suggest that HSD10 protects humans from reactive oxygen species by removing damaged cardiolipin before it induces apoptosis.

Assessing the stereoselectivity of carbonyl reductases toward the reduction of OPBE and docking analysis.

The asymmetric reduction of prochiral carbonyl compounds by NAD(P)H-dependent carbonyl reductases represents a powerful method for the production of optically active alcohols. The stereoselectivity of a series of carbonyl reductases were evaluated toward the reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE). A majority of reductases produced the ethyl (R)-2-hydroxy-4-phenylbutyrate ((R)-HPBE) with low to excellent enantiomeric excess (e.e.), whereas about 30% reductases catalyzed OPBE to form (S)-HPBE. Among them, the carbonyl reductase from Saccharomyces cerevisiae (SeCR) and short-chain dehydrogenase from Gluconobacter oxydans (GoKR) exhibited 100% e.e., yielding the corresponding (R) and (S)-HPBE, respectively. However, the SeCR showed relative higher activity (29.0U/mg) and affinity (Km of 0.22mM) than those of GoKR. Docking analysis found that the interaction of OPBE with enzyme-NADPH complex determined the NADPH-provided hydrogen transfer and the configuration of reductive product. These results indicated that the three-dimensional (3D) structure of enzymes controlled the stereoselectivity of the reductive product based on the geometry of the substrate and cofactor.

Intramolecular hydrogen bonding in the polyextremophilic short-chain dehydrogenase from the archaeon Thermococcus sibiricus and its close structural homologs.

The short-chain alcohol dehydrogenase from the archaeon Thermococcus sibiricus (TsAdh319) exhibits adaptation to different kinds of stress: high temperature, high salinity, and the presence of organic solvents and denaturants. Previously a comparison of TsAdh319 with close structural homologs revealed an abnormally large number of charged residues on the surface of TsAdh319 tetramer. We further focused on the analysis of hydrogen bonding of TsAdh319 and its structural homologs from thermophilic and mesophilic organisms as a structural factor of adaptation to extreme environment. The calculation and analysis of the dynamics of hydrogen bonds of different kind were performed. In particular, the intramolecular hydrogen bonds of different kind according to their location and the type of a.a. residues involved in the bond were analyzed. TsAdh319 showed the greatest contribution of charged residues to the formation of surface hydrogen bonds, inner hydrogen bonding, and the bonds between different subunits compared to its structural homologs. Molecular dynamics simulations revealed that, of three enzyme molecules analyzed, TsAdh319 shows the least change in the number of hydrogen bonds of different kinds upon a temperature shift from 27 to 85°C. The greatest changes were observed for a homologous enzyme from a mesophilic host. Only guanidine hydrochloride being a charged agent was able to deactivate TsAdh319. We suggest that the percentage of charged residues plays a key role in the resistance of TsAdh319 to environmental stress. The analysis shows that salt bridges in TsAdh319 serve as a universal instrument of stabilization under different extreme conditions.