Short-chain Dehydrogenase/reductase Superfamily Research Articles

Crystal structures and functional studies clarify substrate selectivity and catalytic residues for the unique orphan enzyme N-acetyl-D-mannosamine dehydrogenase.

NAMDH (N-acetyl-D-mannosamine dehydrogenase), from the soil bacteroidete Flavobacterium sp. 141-8, catalyses a rare NAD+-dependent oxidation of ManNAc (N-acetyl-D-mannosamine) into N-acetylmannosamino-lactone, which spontaneously hydrolyses into N-acetylmannosaminic acid. NAMDH belongs to the SDR (short-chain dehydrogenase/reductase) superfamily and is the only NAMDH characterized to date. Thorough functional, stability, site-directed mutagenesis and crystallographic studies have been carried out to understand better the structural and biochemical aspects of this unique enzyme. NAMDH exhibited a remarkable alkaline pH optimum (pH9.4) with a high thermal stability in glycine buffer (Tm=64°C) and a strict selectivity towards ManNAc and NAD+. Crystal structures of ligand-free and ManNAc- and NAD+-bound enzyme forms revealed a compact homotetramer having point 222 symmetry, formed by subunits presenting the characteristic SDR α3β7α3 sandwich fold. A highly developed C-terminal tail used as a latch connecting nearby subunits stabilizes the tetramer. A dense network of polar interactions with the substrate including the encasement of its acetamido group in a specific binding pocket and the hydrogen binding of the sugar 4OH atom ensure specificity for ManNAc. The NAMDH-substrate complexes and site-directed mutagenesis studies identify the catalytic tetrad and provide useful traits for identifying new NAMDH sequences.

Functional Mechanism of C-Terminal Tail in the Enzymatic Role of Porcine Testicular Carbonyl Reductase: A Combined Experiment and Molecular Dynamics Simulation Study of the C-Terminal Tail in the Enzymatic Role of PTCR

Porcine testicular carbonyl reductase, PTCR which is one of the short chain dehydrogenases/reductases (SDR) superfamily catalyzes the NADPH-dependent reduction of carbonyl compounds including steroids and prostaglandins. Previously we reported C- terminal tail of PTCR was deleted due to a nonsynonymous single nucleotide variation (nsSNV). Here we identified from kinetic studies that the enzymatic properties for 5α-dihydrotestosterone (5α-DHT) were different between wild-type and C-terminal-deleted PTCRs. Compared to wild-type PTCR, C-terminal-deleted PTCR has much higher reduction rate. To investigate structural difference between wild-type and C-terminal-deleted PTCRs upon 5α-DHT binding, we performed molecular dynamics simulations for two complexes. Using trajectories, molecular interactions including hydrogen bonding patterns, distance between 5α-DHT and catalytic Tyr193, and interaction energies are analyzed and compared. During the MD simulation time, the dynamic behavior of C-terminal tail in wild-type PTCR is also examined using essential dynamics analysis. The results of our simulations reveal that the binding conformation of 5α-DHT in C-terminal-deleted PTCR is more favorable for reduction reaction in PTCR, which shows strong agreement with kinetic data. These structural findings provide valuable information to understand substrate specificity of PTCR and further kinetic properties of enzymes belonging to the SDR superfamily.

Short‐chain dehydrogenase/reductase catalyzing the final step of noscapine biosynthesis is localized to laticifers in opium poppy

The final step in the biosynthesis of the phthalideisoquinoline alkaloid noscapine involves a purported dehydrogenation of the narcotinehemiacetal keto moiety. A short-chain dehydrogenase/reductase (SDR), designated noscapine synthase (NOS), that catalyzes dehydrogenation of narcotinehemiacetal to noscapine was identified in opium poppy and functionally characterized. The NOS gene was isolated using an integrated transcript and metabolite profiling strategy and subsequently expressed in Escherichia coli. Noscapine synthase is highly divergent from other characterized members of the NADPH-dependent SDR superfamily involved in benzylisoquinoline alkaloid metabolism, and it exhibits exclusive substrate specificity for narcotinehemiacetal. Kinetic analyses showed that NOS exhibits higher catalytic efficiency with NAD+ as the cofactor compared with NADP+. Suppression of NOS transcript levels in opium poppy plants subjected to virus-induced gene silencing resulted in a corresponding reduction in the accumulation of noscapine and an increase in narcotinehemiacetal levels in the latex. Noscapine and NOS transcripts were detected in all opium poppy organs, but both were most abundant in stems. Unlike other putative biosynthetic genes clustered in the opium poppy genome, and their corresponding proteins, NOS transcripts and the cognate enzyme were abundant in latex, indicating that noscapine metabolism is completed in a distinct cell type compared with the rest of the pathway.

Biochemical properties of human dehydrogenase/reductase (SDR family) member 7

Dehydrogenase/reductase (SDR family) member 7 (DHRS7, retSDR4, SDR34C1) is a previously uncharacterized member of the short-chain dehydrogenase/reductase (SDR) superfamily. While human SDR members are known to play an important role in various (patho)biochemical pathways including intermediary metabolism and biotransformation of xenobiotics, only 20% of them are considered to be well characterized. Based on phylogenetic tree and SDR sequence clusters analysis DHRS7 is a close relative to well-known SDR member 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) that participates in metabolism of endogenous and xenobiotic substances with carbonyl group. The aim of present study is to determine the basic biochemical properties of DHRS7 and its possible involvement in metabolism of substrates with carbonyl group. For the first time the computational predictions of this membrane protein and membrane topology were experimentally confirmed. DHRS7 has been demonstrated to be an integral protein facing the lumen of the endoplasmic reticulum with lack of posttranscriptional glycosylation modification. Subsequently, NADP(H) cofactor preference and enzymatic reducing activity of DHRS7 was determined towards endogenous substrates with a steroid structure (cortisone, 4-androstene-3,17-dion) and also toward relevant exogenous substances bearing a carbonyl group harmful to human health (1,2-naphtoquinone, 9,10-phenantrenequinone). In addition to 11β-HSD1, DHRS7 is another enzyme from SDR superfamily that have been proved, at least in vitro, to contribute to the metabolism of xenobiotics with carbonyl group.

Cloning, expression and characterization of a putative 7alpha-hydroxysteroid dehydrogenase in Comamonas testosteroni

The short-chain dehydrogenase/reductase (SDR) superfamily is a large and diverse group of genes with members found in all forms of life. Comamonas testosteroni (C. testosterone) ATCC11996 is a Gram-negative bacterium which can use steroids as carbon and energy source. In the present investigation, we found a novel SDR gene 7alpha-hydroxysteroid dehydrogenase (7α-HSD) which is located 11.9kb upstream from hsdA with the same transcription orientation in the C. testosteroni genome. The open reading frame of this putative 7alpha-hydroxysteroid dehydrogenase gene consists of 771bp and translates into a protein of 256 amino acids. Two consensus sequences of the SDR superfamily were found, an N-terminal Gly-X-X-X-Gly-X-Gly cofactor-binding motif and a Tyr-X-X-X-Lys segment (residues 161–165 in the 7α-HSD sequence) essential for catalytic activity of SDR proteins. To produce purified 7α-HSD protein, the 7α-HSD gene was cloned into plasmid pET-15b and the over expressed protein was purified by His-tag sequence on metal chelate chromatography. To prove that 7α-HSD is involved in the metabolic pathway of steroid compounds, we constructed a 7α-HSD knock-out mutant of C. testosteroni. Compared to the wild type C. testosteroni, degradation of testosterone, estradiol and cholesterol were decreased in the 7α-HSD knock-out mutant.Furthermore, growth in the medium with testosterone, estradiol and cholesterol was impaired in 7α-HSD knock-out mutant. The results showed that 7α-HSD is involved in steroid degradation.

Crystal structure of the capsular polysaccharide synthesizing protein CapE of Staphylococcus aureus

Enzymes synthesizing the bacterial CP (capsular polysaccharide) are attractive antimicrobial targets. However, we lack critical information about the structure and mechanism of many of them. In an effort to reduce that gap, we have determined three different crystal structures of the enzyme CapE of the human pathogen Staphylococcus aureus. The structure reveals that CapE is a member of the SDR (short-chain dehydrogenase/reductase) super-family of proteins. CapE assembles in a hexameric complex stabilized by three major contact surfaces between protein subunits. Turnover of substrate and/or coenzyme induces major conformational changes at the contact interface between protein subunits, and a displacement of the substrate-binding domain with respect to the Rossmann domain. A novel dynamic element that we called the latch is essential for remodelling of the protein–protein interface. Structural and primary sequence alignment identifies a group of SDR proteins involved in polysaccharide synthesis that share the two salient features of CapE: the mobile loop (latch) and a distinctive catalytic site (MxxxK). The relevance of these structural elements was evaluated by site-directed mutagenesis.

Classification and nomenclature of the superfamily of short-chain dehydrogenases/reductases (SDRs)

The short-chain dehydrogenases/reductases (SDRs) constitute one of the largest protein superfamilies known today. The members are distantly related with typically 20–30% residue identity in pair-wise comparisons. Still, all hitherto structurally known SDRs present a common three-dimensional structure consisting of a Rossmann fold with a parallel beta sheet flanked by three helices on each side. Using hidden Markov models (HMMs), we have developed a semi-automated subclassification system for this huge family. Currently, 75% of all SDR forms have been assigned to one of the 464 families totalling 122,940 proteins. There are 47 human SDR families, corresponding to 75 genes. Most human SDR families (35 families) have only one gene, while 12 have between 2 and 8 genes. For more than half of the human SDR families, the three-dimensional fold is known. The number of SDR members increases considerably every year, but the number of SDR families now starts to converge. The classification method has paved the ground for a sustainable and expandable nomenclature system. Information on the SDR superfamily is continuously updated at http://sdr-enzymes.org/.

Insights into subtle conformational differences in the substrate-binding loop of fungal 17β-hydroxysteroid dehydrogenase: a combined structural and kinetic approach

The 17β-HSD (17β-hydroxysteroid dehydrogenase) from the filamentous fungus Cochliobolus lunatus (17β-HSDcl) is a NADP(H)-dependent enzyme that preferentially catalyses the interconversion of inactive 17-oxo-steroids and their active 17β-hydroxy counterparts. 17β-HSDcl belongs to the SDR (short-chain dehydrogenase/reductase) superfamily. It is currently the only fungal 17β-HSD member that has been described and represents one of the model enzymes of the cP1 classical subfamily of NADPH-dependent SDR enzymes. A thorough crystallographic analysis has been performed to better understand the structural aspects of this subfamily and provide insights into the evolution of the HSD enzymes. The crystal structures of the 17β-HSDcl apo, holo and coumestrol-inhibited ternary complex, and the active-site Y167F mutant reveal subtle conformational differences in the substrate-binding loop that probably modulate the catalytic activity of 17β-HSDcl. Coumestrol, a plant-derived non-steroidal compound with oestrogenic activity, inhibits 17β-HSDcl [IC50 2.8μM; at 100μM substrate (4-oestrene-3,17-dione)] by occupying the putative steroid-binding site. In addition to an extensive hydrogen-bonding network, coumestrol binding is stabilized further by π-π stacking interactions with Tyr212. A stopped-flow kinetic experiment clearly showed the coenzyme dissociation as the slowest step of the reaction and, in addition to the low steroid solubility, it prevents the accumulation of enzyme-coenzyme-steroid ternary complexes.

Biochemical and structural analysis of Gox2181, a new member of the SDR superfamily from Gluconobacter oxydans

Gluconobacter oxydans enable to oxidize sugars and polyols incompletely to corresponding materials with potential industrial applications, containing around 75 putative dehydrogenases. One of these putative dehydrogenases, Gox2181, was cloned and expressed in Escherichia coli BL21 (DE3), and its X-ray crystal structure was determined to a resolution of 1.8Å. Gox2181 formed a homo-tetramer in the crystal that was coincident with the apparent molecular mass determined in the solution. Gox2181 displayed α/β-folding patterns, the conserved catalytic tetrad of Asn119-Ser147-Tyr162-Lys166, and the NAD-binding pocket, which aligned well with the ‘classical’ type of short-chain dehydrogenase/reductase (SDR) enzymes. Gox2181 was denoted SDR51C based on the SDR nomenclature system. The purified recombinant Gox2181 was demonstrated to be NAD(H)-dependent and active towards a wide range of substrates, including sugar alcohols, secondary alcohols, ketones, and ketoses. Among the substrates tested, Gox2181 displayed preference for secondary hydroxyl or carbonyl groups, showing low Km values with d-arabitol and butanedione.

A short‐chain dehydrogenase involved in terpene metabolism from Zingiber zerumbet

The rhizome oil of Zingiber zerumbet Smith contains an exceptionally high content of sesquiterpenoids with zerumbone, a predominating potential multi-anticancer agent. Biosynthetic pathways of zerumbone have been proposed, and two genes ZSS1 and CYP71BA1 that encode the enzymes catalyzing the first two steps have been cloned. In this paper, we isolated a cDNA clone (ZSD1) that encodes an alcohol dehydrogenase capable of catalyzing the final step of zerumbone biosynthesis. ZSD1 has an open reading frame of 804 bp that encodes a 267-residue enzyme with a calculated molecular mass of 28.7 kDa. After expression in Escherichia coli, the recombinant enzyme was found to catalyze 8-hydroxy-α-humulene into zerumbone. ZSD1 is a member of the short-chain dehydrogenase/reductase superfamily (SDR) and shares high identities with other plant SDRs involved in secondary metabolism, stress responses and phytosteroid biosynthesis. In contrast to the transcripts of ZSS1 and CYP71BA1, which are almost exclusively expressed in rhizomes, ZSD1 transcripts are detected in leaves, stems and rhizomes, suggesting that ZSD1 may also be involved in other biological processes. Consistent with its proposed flexible substrate-binding pocket, ZSD1 also converts borneol to camphor with K(m) and k(cat) values of 22.8 μm and 4.1 s(-1) , displaying its bisubstrate feature.

Cloning, expression and characterization of a novel short-chain dehydrogenase/reductase (SDRx) in Comamonas testosteroni

The short-chain dehydrogenase/reductase (SDR) superfamily is a large and diverse group of genes with members found in all forms of life. Comamonas testosteroni ATCC11996 is a Gram-negative bacterium which can use steroids as carbon and energy source. In previous investigations, we have identified 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) from C. testosteroni as a member of the SDR superfamily that catalyzes the reversible interconversion of hydroxyl and oxo groups at position 3 of the steroid nucleus of a great variety of C19–27 steroids. In addition, 3α-HSD/CR was shown to mediate the carbonyl reduction of non-steroidal aldehydes and ketones. Interestingly, the 3α-HSD/CR gene (hsdA) expression is induced by steroids such as testosterone and progesterone. In the present investigation, we found a novel SDR gene (SDRx) which is located 3.6kb downstream from hsdA with the same transcription orientation in the C. testosteroni genome. The open reading frame of this SDRx consists of 768bp and translates into a protein of 255 amino acids. Two consensus sequences of the SDR superfamily were found, an N-terminal Gly-X-X-X-Gly-X-Gly cofactor-binding motif and a Tyr-X-X-X-Lys segment (residues 160–164 in the SDRx sequence) essential for catalytic activity of SDR proteins. Phylogenetic analyses indicated that the novel SDRx gene codes for 7α-hydroxysteroid dehydrogenase (7α-HSD) in C. testosteroni which is active in steroid metabolism. To produce purified SDRx protein, the SDRx gene was cloned into plasmid pET-15b and the overexpressed protein was purified by its His-tag sequence on metal chelate chromatography. To prove that SDRx is involved in the metabolic pathway of steroid compounds, we constructed an SDRx knock-out mutant of C. testosteroni. Compared to wild type C. testosteroni, degradation of the steroids testosterone and estradiol decreased in the SDRx knock-out mutant. Furthermore, growth on the steroids cholic acid, estradiol and testosterone was impaired in the SDRx knock-out strain. Combined, the novel SDRx in C. testosteroni was identified as 7α-HSD that is involved in steroid degradation.Article from a special issue on steroids and microorganisms.

Human Carbonyl Reductases

Enzymatic carbonyl reduction means the formation of a hydroxy function out of a ketone or aldehyde moiety and applies for the metabolism of physiological (endogenous) or xenobiotic (exogenous) molecules. As for endogenous substrates, carbonyl reduction is often part of a reversible oxidoreductase process and involves the activation or inactivation of important signal molecules like steroids, prostaglandins, retinoids and biogenic amines. These reactions are carried out by NAD(P)(H)-dependent dehydrogenases belonging to two protein superfamilies, the aldo-keto reductases (AKR) and the short-chain dehydrogenases/reductases (SDR). With regard to exogenous substrates, carbonyl reduction of xenobiotics is generally a "one-way" detoxification reaction, since the resulting alcohol is easier to conjugate and to eliminate. Interestingly, the participating enzymes do also belong to the AKR and SDR superfamilies. Moreover, some enzymes from the two protein superfamilies exhibit pluripotency in that they are able to catalyze the oxidoreduction of endobiotics but do also function in the reductive metabolism of carbonyl group bearing xenobiotics. A special case are carbonyl reductases per se which belong to the SDR superfamily and whose substrates or physiological roles are not quite clear. Usually, carbonyl reductases have a broad and diverse substrate spectrum for xenobiotics, however, for some of them a specific physiological function has been speculated. In the human genome, three SDR genes have been identified to code for the carbonyl reductases CBR1 (SDR21C1), CBR3 (SDR21C2) and CBR4 (SDR45C1). The present review summarizes the current knowledge on these enzymes with special emphasis on their role as a defence system against toxicants, as well as their possible physiological function and medical application. In detail, we have screened the recent literature on these three enzymes with regard to endogenous and exogenous substrates, their three-dimensional structure, tissues specific expression, polymorphisms, transcriptional regulation, occurrence in pathological states, and their possible association with cancer. Combined, this review contributes to understanding the complex nature and biological roles(s) of the human carbonyl reductases CBR1, CBR3 and CBR4.

Biochemical characterization of a recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius

The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh gene was heterologously overexpressed in Escherichia coli, and the protein (SaADH) was purified to homogeneity and characterized. SaADH is a tetrameric enzyme consisting of identical 28,978-Da subunits, each composed of 264 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 75 degrees C and a 30-min half-inactivation temperature of ~90 degrees C, and shows good tolerance to common organic solvents. SaADH has a strict requirement for NAD(H) as the coenzyme, and displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and alpha-keto esters, but is poorly active on aliphatic, cyclic and aromatic alcohols, and shows no activity on aldehydes. The enzyme catalyses the reduction of alpha-methyl and alpha-ethyl benzoylformate, and methyl o-chlorobenzoylformate with 100% conversion to methyl (S)-mandelate [17% enantiomeric excess (ee)], ethyl (R)-mandelate (50% ee), and methyl (R)-o-chloromandelate (72% ee), respectively, with an efficient in situ NADH-recycling system which involves glucose and a thermophilic glucose dehydrogenase. This study provides further evidence supporting the critical role of the D37 residue in discriminating NAD(H) from NAD(P)H in members of the SDR superfamily.

A novel NADPH-dependent aldehyde reductase gene from Saccharomyces cerevisiae NRRL Y-12632 involved in the detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion

Aldehyde inhibitors such as furfural, 5-hydroxymethylfurfural, anisaldehyde, benzaldehyde, cinnamaldehyde, and phenylaldehyde are commonly generated during lignocellulosic biomass conversion process for low-cost cellulosic ethanol production that interferes with subsequent microbial growth and fermentation. In situ detoxification of the aldehyde inhibitors is possible by the tolerant ethanologenic yeast that involves multiple genes including numerous functional reductases. In this study, we report a novel aldehyde reductase gene clone Y63 from ethanologenic yeast Saccharomyces cerevisiae NRRL Y12632, representing the uncharacterized ORF YGL157W, which demonstrated NADPH-dependent reduction activities toward at least 14 aldehyde substrates. The identity of gene clone Y63 is the same with YGL157W of SGD since a variation of only 35 nucleotides in genomic sequence and three amino acid residues were observed between the two that share the same length of 347 residues in size. As one among the highly induced genes, YGL157W of Y-12632 showed significantly high levels of transcript abundance in response to furfural and HMF challenges. Based on the deduced amino acid sequence and the most conserved functional motif analyses including closely related reductases from five other yeast species to this date, YGL157W was identified as a member of the subclass ‘intermediate’ of the SDR (short-chain dehydrogenase/reductase) superfamily with the following typical characteristics: the most conserved catalytic site to lie at Tyr 169-X-X-X-Lys 173; an indispensable reduction catalytic triad at Ser 131, Tyr 169, and Lys 173, and an approved cofactor-binding motif at Gly 11-X-X-Gly 14-X-X-Ala 17 near the N-terminus. YGL039W, YDR541C, and YOL151W (GRE2) appeared to be the similar type of enzymes falling into the same category of the intermediate subfamily.

A new member of the short-chain dehydrogenases/reductases superfamily: Purification, characterization and substrate specificity of a recombinant carbonyl reductase from Pichia stipitis

A novel short-chain dehydrogenases/reductases superfamily (SDRs) reductase (PsCR) from Pichia stipitis that produced ethyl ( S)-4-chloro-3-hydroxybutanoate with greater than 99% enantiomeric excess, was purified to homogeneity using fractional ammonium sulfate precipitation followed by DEAE-Sepharose chromatography. The enzyme purified from recombinant Escherichia coli had a molecular mass of about 35 kDa on SDS–PAGE and only required NADPH as an electron donor. The K m value of PsCR for ethyl 4-chloro-3-oxobutanoate was 4.9 mg/mL and the corresponding V max was 337 μmol/mg protein/min. The catalytic efficiency value was the highest ever reported for reductases from yeasts. Moreover, PsCR exhibited a medium-range substrate spectrum toward various keto and aldehyde compounds, i.e., ethyl-3-oxobutanoate with a chlorine substitution at the 2 or 4-position, or α,β-diketones. In addition, the activity of the enzyme was strongly inhibited by SDS and β-mercaptoethanol, but not by ethylene diamine tetra acetic acid.

Identification and characterization of retinoid-active short-chain dehydrogenases/reductases in Drosophila melanogaster

Background In chordates, retinoid metabolism is an important target of short-chain dehydrogenases/reductases (SDRs). It is not known whether SDRs play a role in retinoid metabolism of protostomes, such as Drosophila melanogaster. Methods Drosophila genome was searched for genes encoding proteins with ∼ 50% identity to human retinol dehydrogenase 12 (RDH12). The corresponding proteins were expressed in Sf9 cells and biochemically characterized. Their phylogenetic relationships were analyzed using PHYLIP software. Results A total of six Drosophila SDR genes were identified. Five of these genes are clustered on chromosome 2 and one is located on chromosome X. The deduced proteins are 300 to 406 amino acids long and are associated with microsomal membranes. They recognize all- trans-retinaldehyde and all- trans-3-hydroxyretinaldehyde as substrates and prefer NADPH as a cofactor. Phylogenetically, Drosophila SDRs belong to the same branch of the SDR superfamily as human RDH12, indicating a common ancestry early in bilaterian evolution, before a protostome–deuterostome split. Conclusions Similarities in the substrate and cofactor specificities of Drosophila versus human SDRs suggest conservation of their function in retinoid metabolism throughout protostome and deuterostome phyla. General significance The discovery of Drosophila retinaldehyde reductases sheds new light on the conversion of β-carotene and zeaxantine to visual pigment and provides a better understanding of the evolutionary roots of retinoid-active SDRs.

Analysis of the substrate-binding site of human carbonyl reductases CBR1 and CBR3 by site-directed mutagenesis

Human carbonyl reductase is a member of the short-chain dehydrogenase/reductase (SDR) protein superfamily and is known to play an important role in the detoxification of xenobiotics bearing a carbonyl group. The two monomeric NADPH-dependent human isoforms of cytosolic carbonyl reductase CBR1 and CBR3 show a sequence similarity of 85% on the amino acid level, which is definitely high if compared to the low similarities usually observed among other members of the SDR superfamily (15–30%). Despite the sequence similarity and the similar features found in the available crystal structures of the two enzymes, CBR3 shows only low or no activity towards substrates that are metabolised by CBR1. This surprising substrate specificity is still not fully understood. In the present study, we introduced several point mutations and changed sequences of up to 17 amino acids of CBR3 to the corresponding amino acids of CBR1, to gather insight into the catalytic mechanism of both enzymes. Proteins were expressed in Escherichia coli and purified by Ni-affinity chromatography. Their catalytic properties were then compared using isatin and 9,10-phenanthrenequinone as model substrates. Towards isatin, wild-type CBR3 showed a catalytic efficiency of 0.018 μM −1 min −1, whereas wild-type CBR1 showed a catalytic efficiency of 13.5 μM −1 min −1. In particular, when nine residues (236–244) in the vicinity of the catalytic center and a proline (P230) in CBR3 were mutated to the corresponding residues of CBR1 a much higher k cat/ K m value (5.7 μM −1 min −1) towards isatin was observed. To gain further insight into the protein-ligand binding process, docking simulations were perfomed on this mutant and on both wild-type enzymes (CBR1 and CBR3). The theoretical model of the mutant was ad hoc built by means of standard comparative modelling.

The human short-chain dehydrogenase/reductase (SDR) superfamily: A bioinformatics summary

The short-chain dehydrogenase/reductase (SDR) superfamily represents one of the largest protein superfamilies known to date. Enzymes of this family usually catalyse NAD(P)(H) dependent reactions with a substrate spectrum ranging from polyols, retinoids, steroids and fatty acid derivatives to xenobiotics. We have currently identified 73 SDR superfamily members within the human genome. A status report of the human SDR superfamily is provided in terms of 3D structure determination, co-factor preferences, subcellular localisation and functional annotation. A simple scoring system for measuring structural and functional information (SFS score) has also been introduced to monitor the status of 5 key metrics. Currently there are 17 SDR members with an SFS score of zero indicating that almost a quarter of the human SDR superfamily lacks substantial functional annotation.

Purification and Characterization of a Novel Recombinant Highly Enantioselective Short-Chain NAD(H)-Dependent Alcohol Dehydrogenase from Thermus thermophilus

The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the short-chain dehydrogenase/reductase (SDR) superfamily was identified in the extremely thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27. The T. thermophilus ADH gene (adh(Tt)) was heterologously overexpressed in Escherichia coli, and the protein (ADH(Tt)) was purified to homogeneity and characterized. ADH(Tt) is a tetrameric enzyme consisting of identical 26,961-Da subunits composed of 256 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to approximately 73 degrees C and a 30-min half-inactivation temperature of approximately 90 degrees C, as well as good tolerance to common organic solvents. ADH(Tt) has a strict requirement for NAD(H) as the coenzyme, a preference for reduction of aromatic ketones and alpha-keto esters, and poor activity on aromatic alcohols and aldehydes. This thermophilic enzyme catalyzes the following reactions with Prelog specificity: the reduction of acetophenone, 2,2,2-trifluoroacetophenone, alpha-tetralone, and alpha-methyl and alpha-ethyl benzoylformates to (S)-(-)-1-phenylethanol (>99% enantiomeric excess [ee]), (R)-alpha-(trifluoromethyl)benzyl alcohol (93% ee), (S)-alpha-tetralol (>99% ee), methyl (R)-(-)-mandelate (92% ee), and ethyl (R)-(-)-mandelate (95% ee), respectively, by way of an efficient in situ NADH-recycling system involving 2-propanol and a second thermophilic ADH. This study further supports the critical role of the D37 residue in discriminating NAD(H) from NADP(H) in members of the SDR superfamily.

Vibrio cholerae FabV Defines a New Class of Enoyl-Acyl Carrier Protein Reductase

Enoyl-acyl carrier protein (ACP) reductase catalyzes the last step of the fatty acid elongation cycle. The paradigm enoyl-ACP reductase is the FabI protein of Escherichia coli that is the target of the antibacterial compound, triclosan. However, some Gram-positive bacteria are naturally resistant to triclosan due to the presence of the triclosan-resistant enoyl-ACP reductase isoforms, FabK and FabL. The genome of the Gram-negative bacterium, Vibrio cholerae lacks a gene encoding a homologue of any of the three known enoyl-ACP reductase isozymes suggesting that this organism encodes a novel fourth enoyl-ACP reductase isoform. We report that this is the case. The gene encoding the new isoform, called FabV, was isolated by complementation of a conditionally lethal E. coli fabI mutant strain and was shown to restore fatty acid synthesis to the mutant strain both in vivo and in vitro. Like FabI and FabL, FabV is a member of the short chain dehydrogenase reductase superfamily, although it is considerably larger (402 residues) than either FabI (262 residues) or FabL (250 residues). The FabV, FabI and FabL sequences can be aligned, but only poorly. Alignment requires many gaps and yields only 15% identical residues. Thus, FabV defines a new class of enoyl-ACP reductase. The native FabV protein has been purified to homogeneity and is active with both crotonyl-ACP and the model substrate, crotonyl-CoA. In contrast to FabI and FabL, FabV shows a very strong preference for NADH over NADPH. Expression of FabV in E. coli results in markedly increased resistance to triclosan and the purified enzyme is much more resistant to triclosan than is E. coli FabI.