Short-chain Dehydrogenase/reductases Research Articles

Deciphering the structure of a multi-drug resistant Acinetobacter baumannii short-chain dehydrogenase reductase.

The rapidly increasing threat of multi-drug-resistant Acinetobacter baumannii infections globally, encompassing a range of clinical manifestations from skin and soft tissue infections to life-threatening conditions like meningitis and pneumonia, underscores an urgent need for novel therapeutic strategies. These infections, prevalent in both hospital and community settings, present a formidable challenge to the healthcare system due to the bacterium's widespread nature and dwindling effective treatment options. Against this backdrop, the exploration of bacterial short-chain dehydrogenase reductases (SDRs) emerges as a promising avenue. These enzymes play pivotal roles in various critical bacterial processes, including fatty acid synthesis, homeostasis, metabolism, and contributing to drug resistance mechanisms. In this study, we present the first examination of the X-ray crystallographic structure of an uncharacterized SDR enzyme from A. baumannii. The tertiary structure of this SDR is distinguished by a central parallel β-sheet, consisting of seven strands, which is flanked by eight α-helices. This configuration exhibits structural parallels with other enzymes in the SDR family, underscoring a conserved architectural theme within this enzyme class. Despite the current ambiguity regarding the enzyme's natural substrate, the importance of many SDR enzymes as targets in anti-bacterial agent design is well-established. Therefore, the detailed structural insights provided in this study open new pathways for the in-silico design of therapeutic agents. By offering a structural blueprint, our findings may provide a platform for future research aimed at developing targeted treatments against this and other multi-drug-resistant infections.

Exploring the catalytic diversity of two short-chain dehydrogenases/reductases from Stachybotrys chartarum

Short-chain dehydrogenase/reductases (SDRs) belong to the NAD(P)(H)-dependent oxidoreductase superfamily, which have various functions of catalyzing oxidation/reduction reactions and have been generally used as powerful biocatalysts in the production of pharmaceuticals. In this study, ScSDR1 and ScSDR2, two new SDRs have been identified and characterized from Stachybotrys chartarum 3.5365. Substrate scope investigation revealed that both of the enzymes possessed the ability to oxidize β-OH to ketone specifically, and exhibited substrate promiscuity and high stereo-selectivity for efficiently catalyzing the structurally different prochiral ketones to chiral alcohols. These findings not only suggest that ScSDR1 and ScSDR2 might be potent synthetic tools in drug research and development, but also provide good examples for further engineered enzymes with higher efficiency and stereo-selectivity.

Reducing the vicissitudes of heterologous prochiral substrate catalysis by alcohol dehydrogenases through machine learning algorithms

Alcohol dehydrogenases (ADHs) are popular catalysts for synthesizing chiral synthons a vital step for active pharmaceutical intermediate (API) production. They are grouped into three superfamilies namely, medium-chain (MDRs), short-chain dehydrogenase/reductases (SDRs), and iron-containing alcohol dehydrogenases. The former two are used extensively for producing various chiral synthons. Many studies screen multiple enzymes or engineer a specific enzyme for catalyzing a substrate of interest. These processes are resource-intensive and intricate. The current study attempts to decipher the ability to match different ADHs with their ideal substrates using machine learning algorithms. We explore the catalysis of 284 antibacterial ketone intermediates, against MDRs and SDRs to demonstrate a unique pattern of activity. To facilitate machine learning we curated a dataset comprising 33 features, encompassing 4 descriptors for each compound. Subsequently, an ensemble of machine learning techniques viz. Partial Least Squares (PLS) regression, k-Nearest Neighbors (kNN) regression, and Support Vector Machine (SVM) regression, was harnessed. Moreover, the assimilation of Principal Component Analysis (PCA) augmented precision and accuracy, thereby refining and demarcating diverse compound classes. As such, this classification is useful for discerning substrates amenable to diverse alcohol dehydrogenases, thereby mitigating the reliance on high-throughput screening or engineering in identifying the optimal enzyme for specific substrate.

Enzyme Substrate Prediction from Three-Dimensional Feature Representations Using Space-Filling Curves.

Compact and interpretable structural feature representations are required for accurately predicting properties and function of proteins. In this work, we construct and evaluate three-dimensional feature representations of protein structures based on space-filling curves (SFCs). We focus on the problem of enzyme substrate prediction, using two ubiquitous enzyme families as case studies: the short-chain dehydrogenase/reductases (SDRs) and the S-adenosylmethionine-dependent methyltransferases (SAM-MTases). Space-filling curves such as the Hilbert curve and the Morton curve generate a reversible mapping from discretized three-dimensional to one-dimensional representations and thus help to encode three-dimensional molecular structures in a system-independent way and with only a few adjustable parameters. Using three-dimensional structures of SDRs and SAM-MTases generated using AlphaFold2, we assess the performance of the SFC-based feature representations in predictions on a new benchmark database of enzyme classification tasks including their cofactor and substrate selectivity. Gradient-boosted tree classifiers yield binary prediction accuracy of 0.77-0.91 and area under curve (AUC) characteristics of 0.83-0.92 for the classification tasks. We investigate the effects of amino acid encoding, spatial orientation, and (the few) parameters of SFC-based encodings on the accuracy of the predictions. Our results suggest that geometry-based approaches such as SFCs are promising for generating protein structural representations and are complementary to the existing protein feature representations such as evolutionary scale modeling (ESM) sequence embeddings.

The 3-Oxoacyl-(Acyl-Carrier-Protein) Reductase HSD-X1 of Pseudomonas Citronellolis SJTE-3 Catalyzes the Conversion of 17β-estradiol to Estrone.

Pseudomonas citronellolis SJTE-3 can efficiently degrade 17β-estradiol (E2) and other estrogenic chemicals. However, the enzyme responsible for E2 metabolism within strain SJTE-3 has remained unidentified. Here, a novel 3-oxoacyl-(acyl-carrier protein) (ACP) reductase, HSD-X1 (WP_ 009617962.1), was identified in SJTE-3 and its enzymatic characteristics for the transformation of E2 were investigated. Multiple sequence alignment and homology modelling were used to predict the protein structure of HSD-X1. The concentrations of different steroids in the culture of recombinant strains expressing HSD-X1 were determined by high performance liquid chromatography. Additionally, the transcription of hsd-x1 gene was investigated using reverse transcription and quantitative PCR analysis. Heterologous expression and affinity purification were used to obtain recombinant HSD- X1. The transcription of hsd-x1 gene in P. citronellolis SJTE-3 was induced by E2. Multiple sequence alignment (MSA) indicated that HSD-X1 contained the two consensus regions and conserved residues of short-chain dehydrogenase/reductases (SDRs) and 17β-hydroxysteroid dehydrogenases (17β-HSDs). Over-expression of hsd-x1 gene allowed the recombinant strain to degrade E2. Recombinant HSD-X1 was purified with a yield of 22.15 mg/L and used NAD+ as its cofactor to catalyze the oxidization of E2 into estrone (E1) while exhibiting a Km value of 0.025 ± 0.044 mM and a Vmax value of 4.92 ± 0.31 mM/min/mg. HSD-X1 could tolerate a wide range of temperature and pH, while the presence of divalent ions exerted little influence on its activity. Further, the transformation efficiency of E2 into E1 was over 98.03% across 15 min. Protein HSD-X1 efficiently catalyzed the oxidization of E2 and participated in estrogen degradation by P. citronellolis SJTE-3.

Identification of a fish short-chain dehydrogenase/reductase associated with bone metabolism

Although human and mouse genetics have largely contributed to the better understanding of the mechanisms underlying skeletogenesis, much more remains to be uncovered. In this regard alternative and complementary systems have been sought and cell systems capable of in vitro calcification have been developed to study the mechanisms underlying bone formation. In gilthead seabream (Sparus aurata), a gene coding for an unknown protein that is strongly up-regulated during extracellular matrix (ECM) mineralization of a pre-osteoblast cell line was recently identified as a potentially important player in bone formation. In silico analysis of the deduced protein revealed the presence of domains typical of short-chain dehydrogenase/reductases (SDR). Closely related to carbonyl reductase 1, seabream protein belongs to a novel subfamily of SDR proteins with no orthologs in mammals. Analysis of gene expression by qPCR confirmed the strong up-regulation of sdr-like expression during in vitro mineralization but also revealed high expression levels in calcified tissues. A possible role for Sdr-like in osteoblast and bone metabolism was further evidenced through (i) the localization by in situ hybridization of sdr-like transcript in pre-osteoblasts of the operculum and (ii) the regulation of sdr-like gene transcription by Runx2 and retinoic acid receptor, two regulators of osteoblast differentiation and mineralization. Expression data also indicated a role for Sdr-like in gastrointestinal tract homeostasis and during gilthead seabream development at gastrulation and metamorphosis. This study reports a new subfamily of short-chain dehydrogenases/reductases in vertebrates and, for the first time, provides evidence of a role for SDRs in bone metabolism, osteoblast differentiation and/or tissue mineralization.

Novel patient missense mutations in the HSD17B10 gene affect dehydrogenase and mitochondrial tRNA modification functions of the encoded protein

MRPP2 (also known as HSD10/SDR5C1) is a multifunctional protein that harbours both catalytic and non-catalytic functions. The protein belongs to the short-chain dehydrogenase/reductases (SDR) family and is involved in the catabolism of isoleucine in vivo and steroid metabolism in vitro. MRPP2 also moonlights in a complex with the MRPP1 (also known as TRMT10C) protein for N1-methylation of purines at position 9 of mitochondrial tRNA, and in a complex with MRPP1 and MRPP3 (also known as PRORP) proteins for 5′-end processing of mitochondrial precursor tRNA. Inherited mutations in the HSD17B10 gene encoding MRPP2 protein lead to a childhood disorder characterised by progressive neurodegeneration, cardiomyopathy or both. Here we report two patients with novel missense mutations in the HSD17B10 gene (c.34G>C and c.526G>A), resulting in the p.V12L and p.V176M substitutions. Val12 and Val176 are highly conserved residues located at different regions of the MRPP2 structure. Recombinant mutant proteins were expressed and characterised biochemically to investigate their effects towards the functions of MRPP2 and associated complexes in vitro. Both mutant proteins showed significant reduction in the dehydrogenase, methyltransferase and tRNA processing activities compared to wildtype, associated with reduced stability for protein with p.V12L, whereas the protein carrying p.V176M showed impaired kinetics and complex formation. This study therefore identified two distinctive molecular mechanisms to explain the biochemical defects for the novel missense patient mutations.

Mechanism and Stereochemistry of Polyketide Chain Elongation and Methyl Group Epimerization in Polyether Biosynthesis.

The polyketide synthases responsible for the biosynthesis of the polyether antibiotics nanchangmycin (1) and salinomycin (4) harbor a number of redox-inactive ketoreductase (KR0) domains that are implicated in the generation of C2-epimerized (2S)-2-methyl-3-ketoacyl-ACP intermediates. Evidence that the natural substrate for the polyether KR0 domains is, as predicted, a (2R)-2-methyl-3-ketoacyl-ACP intermediate, came from a newly developed coupled ketosynthase (KS)-ketoreductase (KR) assay that established that the decarboxylative condensation of methylmalonyl-CoA with S-propionyl-N-acetylcysteamine catalyzed by the Nan[KS1][AT1] didomain from module 1 of the nanchangmycin synthase generates exclusively the corresponding (2R)-2-methyl-3-ketopentanoyl-ACP (7a) product. In tandem equilibrium isotope exchange experiments, incubation of [2-2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-ACP (6a) with redox-active, epimerase-inactive EryKR6 from module 6 of the 6-deoxyerythronolide B synthase and catalytic quantities of NADP+ in the presence of redox-inactive, recombinant NanKR10 or NanKR50, from modules 1 and 5 of the nanchangmycin synthase, or recombinant SalKR70 from module 7 of the salinomycin synthase, resulted in first-order, time-dependent washout of deuterium from 6a. Control experiments confirmed that this washout was due to KR0-catalyzed isotope exchange of the reversibly generated, transiently formed oxidation product [2-2H]-(2R)-2-methyl-3-ketopentanoyl-ACP (7a), consistent with the proposed epimerase activity of each of the KR0 domains. Although they belong to the superfamily of short chain dehydrogenase-reductases, the epimerase-active KR0 domains from polyether synthases lack one or both residues of the conserved Tyr-Ser dyad that has previously been implicated in KR-catalyzed epimerizations.

Delineating Substrate Diversity of Disparate Short-Chain Dehydrogenase Reductase from Debaryomyces hansenii.

Short-chain dehydrogenase reductases (SDRs) have been utilized for catalyzing the reduction of many aromatic/aliphatic prochiral ketones to their respective alcohols. However, there is a paucity of data that elucidates their innate biological role and diverse substrate space. In this study, we executed an in-depth biochemical characterization and substrate space mapping (with 278 prochiral ketones) of an unannotated SDR (DHK) from Debaryomyces hansenii and compared it with structurally and functionally characterized SDR Synechococcus elongatus. PCC 7942 FabG to delineate its industrial significance. It was observed that DHK was significantly more efficient than FabG, reducing a diverse set of ketones albeit at higher conversion rates. Comparison of the FabG structure with a homology model of DHK and a docking of substrate to both structures revealed the presence of additional flexible loops near the substrate binding site of DHK. The comparative elasticity of the cofactor and substrate binding site of FabG and DHK was experimentally substantiated using differential scanning fluorimetry. It is postulated that the loop flexibility may account for the superior catalytic efficiency of DHK although the positioning of the catalytic triad is conserved.

Alternative Hydride Sources for Ene‐Reductases: Current Trends

Asymmetric hydrogenations are important reactions in industrial synthesis, as up to two stereogenic centres can be generated. Given the current trend towards more “green” or sustainable chemistry, biocatalytic approaches are becoming more important in the production of fine chemicals, pharmaceuticals, and agrochemical products. There are at least four known classes of enzymes that have been explored for their biocatalytic applicability, collectively known as ene-reductases (ERs). Each enzyme requires NAD(P)H as the hydride donor, whereas the mechanism, substrate scope and product stereoand/or enantiospecificity differs between biocatalyst classes. The most widely investigated class is the old yellow enzyme (OYE; EC 1.6.99.1) family and many recent reviews have summarised the (potential) applications of these enzymes for industrial syntheses, for example, Refs. [1–3] . These flavin mononucleotide (FMN)-containing enzymes catalyse the C=C reduction of a wide variety of a,b-unsaturated aldehydes, ketones, nitroalkenes, maleimides, dicarboxylic acids and their esters, and nitriles. A second less well-characterised enzyme class catalysing biocatalytic reductions are enoate reductases (EC 1.3.1.31). These oxygen-sensitive enzymes contain both FAD and [4Fe-4S], and are members of the NADH:flavin oxidoreductase/NADH oxidase family. They have been shown to catalyse the reduction of a variety of a,b-unsaturated monoacids and esters. 5] A more recent class of enzymes investigated are the medium-chain dehydrogenase/reductase (MDR) family of oxidoreductases (1.3.1.-). For example, the flavin-independent double-bond reductase from Nicotiana tabacum (NtDBR) catalyses the reduction of a wide variety of a,b-unsaturated aldehydes, ketones and nitroalkenes. There are a few examples in the literature of potential applications of a fourth class of enzymes, namely the flavin-independent short-chain dehydrogenase/reductases (SDR) from plants. Examples include two menthol dehydrogenases, ( )-menthol dehydrogenase (EC 1.1.1.207) and (+)-neomenthol dehydrogenase (EC 1.1.1.208), which catalyse the reduction of specific menthone isomers to menthol. Unfortunately, the high cost of NAD(P)H nicotinamide coenzymes makes them uneconomical for industrial-scale syntheses. Therefore, alternative methodologies have been adopted to supply the necessary hydride equivalents for alkene reduction. One such method is to include a coenzyme recycling system, in which catalytic levels of NAD(P) are constantly regenerated to NAD(P)H. Several systems have been developed and routinely employed for ER biocatalytic reactions, such as glucose dehydrogenase, glucose-6-phosphate dehydrogenase (G6PDH/glucose-6-phosphate), formate dehydrogenase and phosphite dehydrogenase, and reviewed elsewhere. In each case, only catalytic levels of NAD(P) are needed but stoichiometric levels of a co-substrate are required to drive the recycling enzyme. Faber and coworkers have performed comparative biotransformations by using multiple cofactor recycling systems, substrates and ERs, to determine the ideal hydride source. Product yield and/or enantioselectivity can vary considerably, depending on which coenzyme recycling system has been employed. Additionally, the inclusion of a second enzyme system into large-scale (bio)synthesis may present problems, for example in maintenance of the activity and stability of two enzymes and the high cost of some co-substrates. Electrochemical regeneration of nicotinamide coenzymes has also been investigated, for example in the direct cathodic reduction of NAD(P) . Unfortunately, this simple regeneration method is hampered by low selectivity and the formation of undesired NAD(P) dimeric side products. Several techniques have been developed to bypass the need for nicotinamide coenzymes by using alternative hydride donors to reduce the flavin cofactor of some ERs. Reetz and coworkers described a method whereby the FMN cofactor of the OYE homologue YqjM from Bacillus subtilis was photoreduced, employing free flavin and a sacrificial electron donor. The YqjM bound oxidised FMN was reduced by the free photoreduced FMN, thereby generating the active enzyme. This NAD(P)H-free system was successful in converting ketoisophorone to (R)-levodione, albeit with lower product enantiopurity. Similarly, Hollmann and co-workers described the photoenzymatic flavin reduction of YqjM and NEMR (N-ethylmalemide reductase from E. coli) by using alternative sacrificial electron donors formate and phosphite. These methods required strict anaerobic conditions to prevent the rapid reoxidation of reduced FMN by molecular oxygen. Early attempts at NAD(P)H-independent alkene reduction focussed on the use of artificial mediators, such as N,N-dimethyl4-4-bipyridinium [methyl viologen (MV)] , as a means of reducing the flavin in clostridial enoate reductases. A variety of reduced mediator recycling systems were employed, including direct electron transfer from a cathode and enzymatic-based recycling systems. Successful enzyme-coupled mediator recycling was achieved used the following systems: 1) hydroge[a] Dr. H. S. Toogood, Dr. T. Knaus, Prof. N. S. Scrutton Manchester Institute of Biotechnology, Faculty of Life Sciences University of Manchester 131 Princess Street, Manchester M1 7DN (UK) Fax: (+44)01613068918 E-mail : nigel.scrutton@manchester.ac.uk

Biochemical and structural characterization of a short-chain dehydrogenase/reductase of Thermus thermophilus HB8 : A hyperthermostable aldose-1-dehydrogenase with broad substrate specificity

Thermus thermophilus HB8 is a hyperthermophilic bacterium, thriving at environmental temperature near 80 °C. The genomic analysis of this bacterium predicted 18 genes for proteins belonging to the short-chain dehydrogenase/reductases (SDR) superfamily, but their functions remain unknown. A SDR encoded in a gene (TTHA0369) was chosen for functional and structural characterization. Enzymatic assays revealed that the recombinant tetrameric protein has a catalytic activity as NAD +-dependent aldose 1-dehydroganse, which accepts various aldoses such as d-fucose, d-galactose, d-glucose, l-arabinose, cellobiose and lactose. The enzyme also oxidized non-sugar alicyclic alcohols, and was competitively inhibited by hexestrol, 1,10-phenanthroline, 2,3-benzofuran and indole. The enzyme was stable at pH 2–13 and up to 85 °C. We have determined the crystal structure of the enzyme–NAD + binary complex at 1.65 Å resolution. The structure provided evidence for the strict coenzyme specificity and broad substrate specificity of the enzyme. Additionally, it has unusual features, aromatic–aromatic interactions among Phe141 and Phe249 in the subunit interface and hydrogen networks around the C-terminal Asp–Gly–Gly sequence at positions 242–244. Stability analysis of the mutant D242N, F141A and F249A enzymes indicated that the two unique structural features contribute to the hyperthermostability of the enzyme. This study demonstrates that aldose 1-dehydrogenase is a member of the SDR superfamily, and provides a novel structural basis of thermostability.

Structure and Reaction Mechanism of Basil Eugenol Synthase

Phenylpropenes, a large group of plant volatile compounds that serve in multiple roles in defense and pollinator attraction, contain a propenyl side chain. Eugenol synthase (EGS) catalyzes the reductive displacement of acetate from the propenyl side chain of the substrate coniferyl acetate to produce the allyl-phenylpropene eugenol. We report here the structure determination of EGS from basil (Ocimum basilicum) by protein x-ray crystallography. EGS is structurally related to the short-chain dehydrogenase/reductases (SDRs), and in particular, enzymes in the isoflavone-reductase-like subfamily. The structure of a ternary complex of EGS bound to the cofactor NADP(H) and a mixed competitive inhibitor EMDF ((7S,8S)-ethyl (7,8-methylene)-dihydroferulate) provides a detailed view of the binding interactions within the EGS active site and a starting point for mutagenic examination of the unusual reductive mechanism of EGS. The key interactions between EMDF and the EGS-holoenzyme include stacking of the phenyl ring of EMDF against the cofactor's nicotinamide ring and a water-mediated hydrogen-bonding interaction between the EMDF 4-hydroxy group and the side-chain amino moiety of a conserved lysine residue, Lys132. The C4 carbon of nicotinamide resides immediately adjacent to the site of hydride addition, the C7 carbon of cinnamyl acetate substrates. The inhibitor-bound EGS structure suggests a two-step reaction mechanism involving the formation of a quinone-methide prior to reduction. The formation of this intermediate is promoted by a hydrogen-bonding network that favors deprotonation of the substrate's 4-hydroxyl group and disfavors binding of the acetate moiety, akin to a push-pull catalytic mechanism. Notably, the catalytic involvement in EGS of the conserved Lys132 in preparing the phenolic substrate for quinone methide formation through the proton-relay network appears to be an adaptation of the analogous role in hydrogen bonding played by the equivalent lysine residue in other enzymes of the SDR family.

Structural basis of the multispecificity demonstrated by 17β-hydroxysteroid dehydrogenase types 1 and 5

17β-Hydroxysteroid dehydrogenases/ketosteroid reductases (17β-HSDs/KSRs) catalyze the last step of sex steroid synthesis or the first step of their degradation, and are thus critical for many physiological processes. The multispecificity demonstrated by 17β-HSDs is important for steroid metabolism in gonadal and peripheral tissues, and is a consequence of the architecture of their binding and catalytic sites. Structurally, most of the family members are short chain dehydrogenase-reductases (SDRs) except the type 5 enzyme, which is an aldo-keto reductase (AKR). 17β-HSD type 1, a representative of the SDR family, has been studied extensively since the 1950s. However, its structure was not determined until the 1990s. It has always been considered as estrogen specific, in accord with the narrow binding tunnel that has been structurally determined and has been found to be complementary to estrogens. A recent study revealed that, in spite of the enzyme's narrow binding tunnel, the pseudo-symmetry of C19 steroids leads to its alternative binding, resulting in the multispecificity of the enzyme. Expressed in ovary, breast and placenta, the enzyme catalyzes the formation of another estrogen A-diol from DHEA in addition to the biosynthesis of estradiol; it also inactivates the most active androgen DHT by both 17β-hydroxysteroid oxidation and 3-ketosteroid reduction. Type 5 17β-HSD (AKR1C3) differs significantly from the type 1 enzyme by possessing a spacious and flexible steroid-binding site. This is estimated to be about 960 or 470 Å 3 in ternary complex with testosterone or 4-dione, respectively, whereas the binding site volume of 17β-HSD1 is only about 340 Å 3. This characteristic of the 17β-HSD5 binding site permits the docking of various steroids in different orientations, which encompasses a wider range of activities from 20α-, 17β- and 3α-HSD/KSR to prostaglandin 11-ketoreductase. The in vitro activities of the enzyme are significantly lower than the type 1 enzyme. In the ternary complex with testosterone, the steroid C3-C17 position is quasi-reversed as compared to the complex with 4-dione. The multi-specificity contributes significantly to steroid metabolism in peripheral tissues, due to the high levels of 17β-HSD5 mRNA in both breast and prostate tissues.

Molecular determinants of steroid recognition and catalysis in aldo-keto reductases. Lessons from 3α-hydroxysteroid dehydrogenase

Hydroxysteroid Dehydrogenases (HSDs) regulate the occupancy of steroid hormone receptors by converting active steroid hormones into their cognate inactive metabolites. HSDs belong to either the Short-chain Dehydrogenase/Reductases (SDRs) or the Aldo-Keto Reductases (AKRs). The AKRs include virtually all mammalian 3α-HSDs, Type 5 17β-HSD, ovarian 20α-HSDs as well as the steroid 5β-reductases. Selective inhibitors of 3α-HSD isoforms could control occupancy of the androgen and GABA A receptors, while broader based AKR inhibitors targeting 3α-HSD, 20α-HSD and prostaglandin F 2α synthase could maintain pregnancy. We have determined three X-ray crystal structures of rat liver 3α-HSD, a representative AKR. These structures are of the apoenzyme (E), the binary-complex (E.NADP +), and the ternary complex (E.NADP +.testosterone). These structures are being used with site-directed mutagenesis to define the molecular determinants of steroid recognition and catalysis as a first step in rational inhibitor design. A conserved catalytic tetrad ( Tyr55, Lys84, His117 and Asp50) participates in a ‘proton-relay’ in which Tyr55 acts as general acid/base catalyst. Its bifunctionality relies on contributions from His117 and Lys84 which alter the p K b and p K a, respectively of this residue. Point mutation of the tetrad results in different enzymatic activities. H117E mutants display 5β-reductase activity while Y55F and Y55S mutants retain quinone reductase activity. Our results suggest that different transition states are involved in these reaction mechanisms. The ternary complex structure shows that the mature steroid binding pocket is comprised of ten residues recruited from five loops, and that there is significant movement of a C-terminal loop on binding ligand. Mutagenesis of pocket tryptophans shows that steroid substrates and classes of nonsteroidal inhibitors exhibit different binding modes which may reflect ligand-induced loop movement. Exploitation of these findings using steroidal and nonsteroidal mechanism based inactivators may lead to selective and broad based AKR inhibitors.

GDP-4-keto-6-deoxy-D-mannose epimerase/reductase from Escherichia coli, a key enzyme in the biosynthesis of GDP-L-fucose, displays the structural characteristics of the RED protein homology superfamily.

The process of guanosine 5'-diphosphate L-fucose (GDP-L-fucose) biosynthesis is conserved throughout evolution from prokaryotes to man. In animals, GDP-L-fucose is the substrate of fucosyltransferases that participate in the biosynthesis and remodeling of glycoconjugates, including ABH blood group and Lewis-system antigens. The 'de novo' pathway of GDP-L-fucose biosynthesis from GDP-D-mannose involves a GDP-D-mannose 4,6 dehydratase (GMD) and a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (GMER). Neither of the catalytic mechanisms nor the three-dimensional structures of the two enzymes has been elucidated yet. The severe leukocyte adhesion deficiency (LAD) type II genetic syndrome is known to result from deficiencies in this de novo pathway. The crystal structures of apo- and holo-GMER have been determined at 2.1 A and 2.2 A resolution, respectively. Each subunit of the homodimeric (2 x 34 kDa) enzyme is composed of two domains. The N-terminal domain, a six-stranded Rossmann fold, binds NADP+; the C-terminal domain (about 100 residues) displays an alpha/beta topology. NADP+ interacts with residues Arg12 and Arg36 at the adenylic ribose phosphate; moreover, a protein loop based on the Gly-X-X-Gly-X-X-Gly motif (where X is any amino acid) stabilizes binding of the coenzyme diphosphate bridge. The nicotinamide and the connected ribose ring are located close to residues Ser107, Tyr136 and Lys140, the putative GMER active-site center. The GMER fold is reminiscent of that observed for UDP-galactose epimerase (UGE) from Escherichia coli. Consideration of the enzyme fold and of its main structural features allows assignment of GMER to the reductase-epimerase-dehydrogenase (RED) enzyme homology superfamily, to which short-chain dehydrogenase/reductases (SDRs) also belong. The location of the NADP+ nicotinamide ring at an interdomain cleft is compatible with substrate binding in the C-terminal domain.