Short-chain Dehydrogenase/reductase Family Research Articles

Rv0687 a Putative Short-Chain Dehydrogenase Is Required for In Vitro and In Vivo Survival of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), a successful human pathogen, resides in host sentinel cells and combats the stressful intracellular environment induced by reactive oxygen and nitrogen species during infection. Mtb employs several evasion mechanisms in the face of the host as a survival strategy, including detoxifying enzymes as short-chain dehydrogenases/reductases (SDRs) to withstand host-generated insults. In this study, using specialized transduction, we have generated a Rv0687 deletion mutant and its complemented strain and investigated the functional role of Rv0687, a member of SDRs family genes in Mtb pathogenesis. A wildtype (WT) and a mutant Mtb strain lacking Rv0687 (RvΔ0687) were tested for the in vitro stress response and in vivo survival in macrophages and mice models of infection. The study demonstrates that the deletion of Rv0687 elevated the sensitivity of Mtb to oxidative and nitrosative stress-inducing agents. Furthermore, the lack of Rv0687 compromised the survival of Mtb in primary bone marrow macrophages and led to an increase in the levels of the secreted proinflammatory cytokines TNF-α and MIP-1α. Interestingly, the growth of WT and RvΔ0687 was similar in the lungs of infected immunocompromised mice; however, a significant reduction in RvΔ0687 growth was observed in the spleen of immunocompromised Rag-/- mice at 4 weeks post-infection. Moreover, Rag-/- mice infected with RvΔ0687 survived longer compared to those infected with the WT Mtb strain. Additionally, we observed a significant reduction in the bacterial burden in the spleens and lungs of immunocompetent C57BL/6 mice infected with RvΔ0687 compared to those infected with complemented and WT Mtb strains. Collectively, this study reveals that Rv0687 plays a role in Mtb pathogenesis.

Raising NAD+ Level Stimulates Short-Chain Dehydrogenase/Reductase Proteins to Alleviate Heart Failure Independent of Mitochondrial Protein Deacetylation.

Strategies to increase cellular NAD+ (oxidized nicotinamide adenine dinucleotide) level have prevented cardiac dysfunction in multiple models of heart failure, but molecular mechanisms remain unclear. Little is known about the benefits of NAD+-based therapies in failing hearts after the symptoms of heart failure have appeared. Most pretreatment regimens suggested mechanisms involving activation of sirtuin, especially Sirt3 (sirtuin 3), and mitochondrial protein acetylation. We induced cardiac dysfunction by pressure overload in SIRT3-deficient (knockout) mice and compared their response with nicotinamide riboside chloride treatment with wild-type mice. To model a therapeutic approach, we initiated the treatment in mice with established cardiac dysfunction. We found nicotinamide riboside chloride improved mitochondrial function and blunted heart failure progression. Similar benefits were observed in wild-type and knockout mice. Boosting NAD+ level improved the function of NAD(H) redox-sensitive SDR (short-chain dehydrogenase/reductase) family proteins. Upregulation of Mrpp2 (mitochondrial ribonuclease P protein 2), a multifunctional SDR protein and a subunit of mitochondrial ribonuclease P, improves mitochondrial DNA transcripts processing and electron transport chain function. Activation of SDRs in the retinol metabolism pathway stimulates RXRα (retinoid X receptor α)/PPARα (proliferator-activated receptor α) signaling and restores mitochondrial oxidative metabolism. Downregulation of Mrpp2 and impaired mitochondrial ribonuclease P were found in human failing hearts, suggesting a shared mechanism of defective mitochondrial biogenesis in mouse and human heart failure. These findings identify SDR proteins as important regulators of mitochondrial function and molecular targets of NAD+-based therapy. Furthermore, the benefit is observed regardless of Sirt3-mediated mitochondrial protein deacetylation, a widely held mechanism for NAD+-based therapy for heart failure. The data also show that NAD+-based therapy can be useful in pre-existing heart failure.

The Molecular Basis of Catalysis by SDR Family Members Ketoacyl-ACP Reductase FabG and Enoyl-ACP Reductase FabI in Type-II Fatty Acid Biosynthesis.

The short-chain dehydrogenase/reductase (SDR) superfamily members acyl-ACP reductases FabG and FabI are indispensable core enzymatic modules and catalytic orientation controllers in type-II fatty acid biosynthesis. Herein, we report their distinct substrate allosteric recognition and enantioselective reduction mechanisms. FabG achieves allosteric regulation of ACP and NADPH through ACP binding across two adjacent FabG monomers, while FabI follows an irreversible compulsory order of substrate binding in that NADH binding must precede that of ACP on a discrete FabI monomer. Moreover, FabG and FabI utilize a backdoor residue Phe187 or a "rheostat" α8 helix for acyl chain length selection, and their corresponding triad residues Ser142 or Tyr145 recognize the keto- or enoyl-acyl substrates, respectively, facilitating initiation of nucleophilic attack by NAD(P)H. The other two triad residues (Tyr and Lys) mediate subsequent proton transfer and (R)-3-hydroxyacyl- or saturated acyl-ACP production.

Two C18 hydroxy-cyclohexenone fatty acids from mammalian epidermis: Potential relation to 12R-lipoxygenase and covalent binding of ceramides

A key requirement in forming the water permeability barrier in the mammalian epidermis is the oxidation of linoleate esterified in a skin-specific acylceramide by the sequential actions of 12R-lipoxygenase, epidermal lipoxygenase-3, andthe epoxyalcohol dehydrogenase SDR9C7 (short-chain dehydrogenase-reductase family 7 member 9). By mechanisms that remain unclear, this oxidation pathway promotes the covalent binding of ceramides to protein, forming a critical structure of the epidermal barrier, the corneocyte lipid envelope. Here, we detected, in porcine, mouse, and human epidermis, two novel fatty acid derivatives formed by KOH treatment from precursors covalently bound to protein: a "polar" lipid chromatographing on normal-phase HPLC just before omega-hydroxy ceramide and a "less polar" lipid nearer the solvent front. Approximately 100μg of the novel lipids were isolated from porcine epidermis, and the structures were established by UV-spectroscopy, LC-MS, GC-MS, and NMR. Each is a C18 fatty acid and hydroxy-cyclohexenone with the ring on carbons C9-C14 in the polar lipid and C8-C13 in the less polar lipid. Overnight culture of [14C]linoleic acid with whole mouse skin exvivo led to recovery of the 14C-labeled hydroxy-cyclohexenones. We deduce they are formed from covalently bound precursors during the KOH treatment used to release esterified lipids. KOH-induced intramolecular aldol reactions from a common precursor can account for their formation. Discovery of these hydroxy-cyclohexenones presents an opportunity for a reverse pathway analysis, namely to work back from these structures to identify their covalently bound precursors and relationship to the linoleate oxidation pathway.

Short-chain dehydrogenases in Haemonchus contortus: changes during life cycle and in relation to drug-resistance

Short-chain dehydrogenases/reductases (SDRs) regulate the activities of many hormones and other signaling molecules and participate in the deactivation of various carbonyl-bearing xenobiotics. Nevertheless, knowledge about these important enzymes in helminths remains limited. The aim of our study was to characterize the SDR superfamily in the parasitic nematode Haemonchus contortus. Genome localization of SDRs was explored, and phylogenetic analysis in comparison with SDRs from free-living nematode Caenorhabditis elegans and the domestic sheep (Ovis aries, a typical host of H. contortus) was constructed. The expression profile of selected SDRs during the life cycle along with differences between the drug-susceptible and drug-resistant strains, were also studied. Genome sequencing enabled the identification of 46 members of the SDR family in H. contortus. A number of genes have no orthologue in the sheep genome. In all developmental stages of H. contortus, SDR1, SDR3, SDR5, SDR6, SDR14, and SDR18 genes were the most expressed, although in individual stages, huge differences in expression levels were observed. A comparison of SDRs expression between the drug-susceptible and drug-resistant strains of H. contortus revealed several SDRs with changed expression in the resistant strain. Specifically, SDR1, SDR12, SDR13, SDR16 are SDR candidates related to drug-resistance, as the expression of these SDRs is consistently increased in most stages of the drug-resistant H. contortus. These findings revealing several SDR enzymes of H. contortus warrant further investigation.

OsCCRL1 is Essential for Phenylpropanoid Metabolism in Rice Anthers

Phenylpropanoid metabolism and timely tapetal degradation are essential for anther and pollen development, but the underlying mechanisms are unclear. In the current study, to investigate this, we identified and analyzed the male-sterile mutant, osccrl1 (cinnamoyl coA reductase-like 1), which exhibited delayed tapetal programmed cell death (PCD) and defective mature pollen. Map-based cloning, genetic complementation, and gene knockout revealed that OsCCRL1 corresponds to the gene LOC_Os09g32020.2, a member of SDR (short-chain dehydrogenase/reductase) family enzyme. OsCCRL1 was preferentially expressed in the tapetal cells and microspores, and localized to the nucleus and cytoplasm in both rice protoplasts and Nicotiana benthamiana leaves. The osccrl1 mutant exhibited reduced CCRs enzyme activity, less lignin accumulation, delayed tapetum degradation, and disrupted phenylpropanoid metabolism. Furthermore, an R2R3 MYB transcription factor OsMYB103/OsMYB80/OsMS188/BM1, involved in tapetum and pollen development, regulates the expression of OsCCRL1. Finally, the osmyb103 osccrl1 double mutants, exhibited the same phenotype as the osmyb103 single mutant, further indicating that OsMYB103/OsMYB80/OsMS188/BM1 functions upstream of OsCCRL1. These findings help to clarify the role of phenylpropanoid metabolism in male sterility and the regulatory network underlying the tapetum degradation.

DHRS2 inhibits cell growth and metastasis in ovarian cancer by downregulation of CHKα to disrupt choline metabolism

The short-chain dehydrogenase/reductase (SDR) superfamily has essential roles in lipid metabolism and redox sensing. In recent years, accumulating evidence highlights the emerging association between SDR family enzymes and cancer. Dehydrogenase/reductase member 2(DHRS2) belongs to the NADH/NADPH-dependent SDR family, and extensively participates in the regulation of the proliferation, migration, and chemoresistance of cancer cells. However, the underlying mechanism has not been well defined. In the present study, we have demonstrated that DHRS2 inhibits the growth and metastasis of ovarian cancer (OC) cells in vitro and in vivo. Mechanistically, the combination of transcriptome and metabolome reveals an interruption of choline metabolism by DHRS2. DHRS2 post-transcriptionally downregulates choline kinase α (CHKα) to inhibit AKT signaling activation and reduce phosphorylcholine (PC)/glycerophosphorylcholine (GPC) ratio, impeding choline metabolism reprogramming in OC. These actions mainly account for the tumor-suppressive role of DHRS2 in OC. Overall, our findings establish the mechanistic connection among metabolic enzymes, metabolites, and the malignant phenotype of cancer cells. This could result in further development of novel pharmacological tools against OC by the induction of DHRS2 to disrupt the choline metabolic pathway.

Nitric oxide enhances resistance of Pleurotus eryngii to cadmium stress by alleviating oxidative damage and regulating of short-chain dehydrogenase/reductase family.

The function and mechanism of nitric oxide (NO) in regulating Pleurotus eryngii biological response to cadmium (Cd) stress was evaluated by using anti-oxidation and short-chain dehydrogenase/reductase (SDR) family analysis. The fresh biomass of P. eryngii mycelia sharply decreased after treatment with 50µM Cd; the lipid peroxidation and H2O2 accumulation in P. eryngii were found responsible for it. Proper exogenous supply of NO (150µM SNP) alleviated the oxidative damage induced by Cd stress in P. eryngii, which reduced the accumulation of thiobarbituric acid reactive substances (TBARS) and H2O2. The activities of antioxidant enzymes (superoxide dismutase, peroxidase) were significantly increased to deal with Cd stress when treated with SNP (150µM), and the content of proline was also closely related to NO-mediated reduction of Cd toxicity. Moreover, SDR family members were widely involved in the response to Cd stress, especially PleSCH70 gene was observed for the first time in participating in NO-mediated enhancement of Cd tolerance in P. eryngii. Taken together, this study provides new insights in understanding the tolerance mechanisms of P. eryngii to heavy metal and lays a foundation for molecular breeding of P. eryngii to improve its tolerance to environmental stress.

Characterization of Pyridomycin B Reveals the Formation of Functional Groups in Antimycobacterial Pyridomycin.

Pyridomycin, a cyclodepsipeptide with potent antimycobacterial activity, specifically inhibits the InhA enoyl reductase of Mycobacterium tuberculosis. Structure-activity relationship studies indicated that the enolic acid moiety in the pyridomycin core system is an important pharmacophoric group, and the natural configuration of the C-10 hydroxyl contributes to the bioactivity of pyridomycin. The ring structure of pyridomycin was generated by the nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) hybrid system (PyrE-PyrF-PyrG). Bioinformatics analysis reveals that short-chain dehydrogenase/reductase (SDR) family protein Pyr2 functions as a 3-oxoacyl acyl carrier protein (ACP) reductase in the pyridomycin pathway. Inactivation of pyr2 resulted in accumulation of pyridomycin B, a new pyridomycin analogue featured with enol moiety in pyridyl alanine moiety and a saturated 3-methylvaleric acid group. The elucidated structure of pyridomycin B suggests that rather than functioning as a post-tailoring enzyme, Pyr2 catalyzes ketoreduction to form the C-10 hydroxyl group in pyridyl alanine moiety and the double bond formation of the enolic acid moiety derived from isoleucine when the intermediate assembled by PKS-NRPS machinery is still tethered to the last NRPS module in a special energy-saving manner. Ser-His-Lys residues constitute the active site of Pyr2, which is different from the typically conserved Tyr-based catalytic triad in the majority of SDRs. Site-directed mutation identified that His154 in the active site is a critical residue for pyridomycin B production. These findings will improve our understanding of pyridomycin biosynthetic logic, identify the missing link for the double bound formation of enol ester in pyridomycin, and enable the creation of chemical diversity of pyridomycin derivatives. IMPORTANCE Tuberculosis (TB) is one of the world's leading causes of death by infection. Recently, pyridomycin, the antituberculous natural product from Streptomyces has garnered considerable attention for being determined as a target inhibitor of InhA enoyl reductase of Mycobacterium tuberculosis. In this study, we report a new pyridomycin analogue from mutant HTT12, demonstrate the essential role of a previously ignored gene pyr2 in pyridomycin biosynthetic pathway, and imply that Pyr2 functions as a trans ketoreductase (KR) contributing to the formation of functional groups of pyridomycin utilizing a distinct catalytic mechanism. As enol moiety are important for pharmaceutical activities of pyridomycin, our work would expand our understanding of the mechanism of SDR family proteins and set the stage for future bioengineering of new pyridomycin derivatives.

A new atypical short-chain dehydrogenase is required for interfungal combat and conidiation in Trichoderma guizhouense.

Hypocrealean Trichoderma are the most extensively studied facultative mycoparasites against phytopathogenic fungi. Aerial hyphae of Trichoderma guizhouense can rapidly proliferate over Fusarium oxysporum hyphae, cause sporadic cell death and arrest the growth of the host. The results of the present study demonstrated that a unique short-chain dehydrogenase/reductase (SDR), designated as TgSDR1, was expressed at a high level in T. guizhouense challenged by the hosts. Similar to other SDRs family members, the TgSDR1 protein contains a cofactor-binding motif and a catalytic site. The subcellular localization assay revealed that the TgSDR1::GFP fusion protein translocated to lipid droplets in mycelia and conidia. The data obtained using reverse genetic approach indicated that TgSDR1 is associated with antifungal ability, plays an important role in providing reducing equivalents in the form of NADPH and regulates the amino sugar and nucleotide sugar metabolism in T. guizhouense upon encountering a host. Moreover, the TgSDR1 deletion mutant was defective in conidiation. Thus, TgSDR1 functions as a key metabolic enzyme in T. guizhouense to regulate mycotrophic interactions, defence against other fungi, such as F. oxysporum, and conidiation.

Crossing the Border: From Keto- to Imine Reduction in Short-Chain Dehydrogenases/Reductases.

The family of NAD(P)H‐dependent short‐chain dehydrogenases/reductases (SDRs) comprises numerous biocatalysts capable of C=O or C=C reduction. The highly homologous noroxomaritidine reductase (NR) from Narcissus sp. aff. pseudonarcissus and Zt_SDR from Zephyranthes treatiae, however, are SDRs with an extended imine substrate scope. Comparison with a similar SDR from Asparagus officinalis (Ao_SDR) exhibiting keto‐reducing activity, yet negligible imine‐reducing capability, and mining the Short‐Chain Dehydrogenase/Reductase Engineering Database indicated that NR and Zt_SDR possess a unique active‐site composition among SDRs. Adapting the active site of Ao_SDR accordingly improved its imine‐reducing capability. By applying the same strategy, an unrelated SDR from Methylobacterium sp. 77 (M77_SDR) with distinct keto‐reducing activity was engineered into a promiscuous enzyme with imine‐reducing activity, thereby confirming that the ability to reduce imines can be rationally introduced into members of the “classical” SDR enzyme family. Thus, members of the SDR family could be a promising starting point for protein approaches to generate new imine‐reducing enzymes.

Metabolic engineering Saccharomyces cerevisiae for de novo production of the sesquiterpenoid (+)-nootkatone

Background(+)-Nootkatone is a highly valued sesquiterpenoid compound, exhibiting a typical grapefruit aroma and various desired biological activities for use as aromatics and pharmaceuticals. The high commercial demand of (+)-nootkatone is predominately met by chemical synthesis, which entails the use of environmentally harmful reagents. Efficient synthesis of (+)-nootkatone via biotechnological approaches is thus urgently needed to satisfy its industrial demand. However, there are only a limited number of studies that report the de novo synthesis of (+)-nootkatone from simple carbon sources in microbial cell factories, and with relatively low yield.ResultsAs the direct precursor of (+)-nootkatone biosynthesis, (+)-valencene was first produced in large quantities in Saccharomyces cerevisiae by overexpressing (+)-valencene synthase CnVS of Callitropsis nootkatensis in combination with various mevalonate pathway (MVA) engineering strategies, including the expression of CnVS and farnesyl diphosphate synthase (ERG20) as a fused protein, overexpression of a truncated form of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (tHMG1), and downregulating the squalene synthase enzyme (ERG9). These approaches altogether brought the production of (+)-valencene to 217.95 mg/L. Secondly, we addressed the (+)-valencene oxidation by overexpressing the Hyoscyamus muticus premnaspirodiene oxygenase (HPO) variant (V482I/A484I) and cytochrome P450 reductase (ATR1) from Arabidopsis thaliana. However, (+)-valencene was predominantly oxidized to β-nootkatol and only minor amounts of (+)-nootkatone (9.66 mg/L) were produced. We further tackled the oxidation of β-nootkatol to (+)-nootkatone by screening various dehydrogenases. Our results showed that the short-chain dehydrogenase/reductase (SDR) superfamily dehydrogenases ZSD1 of Zingiber zerumbet and ABA2 of Citrus sinensis were capable of effectively catalyzing β-nootkatol oxidation to (+)-nootkatone. The yield of (+)-nootkatone increased to 59.78 mg/L and 53.48 mg/L by additional overexpression of ZSD1 and ABA2, respectively.ConclusionWe successfully constructed the (+)-nootaktone biosynthesis pathway in S. cerevisiae by overexpressing the (+)-valencene synthase CnVS, cytochrome P450 monooxygenase HPO, and SDR family dehydrogenases combined with the MVA pathway engineering, providing a solid basis for the whole-cell production of (+)-nootkatone. The two effective SDR family dehydrogenases tested in this study will serve as valuable enzymatic tools in further optimizing (+)-nootkatone production.

Biochemical and Structural Insights Concerning Triclosan Resistance in a Novel YX7K Type Enoyl-Acyl Carrier Protein Reductase from Soil Metagenome

Enoyl-acyl carrier protein reductase (ENR) catalyzes the last reduction step in the bacterial type II fatty acid biosynthesis cycle. ENRs include FabI, FabL, FabL2, FabK, and FabV. Previously, we reported a unique triclosan (TCL) resistant ENR homolog that was predominant in obligate intracellular pathogenic bacteria and Apicomplexa. Herein, we report the biochemical and structural basis of TCL resistance in this novel ENR. The purified protein revealed NADH-dependent ENR activity and shared similarity to prototypic FabI. Thus, this metagenome-derived ENR was designated FabI2. Unlike other prototypic bacterial ENRs with the YX6K type catalytic domain, FabI2 possessed a unique YX7K type catalytic domain. Computational modeling followed by site-directed mutagenesis revealed that mild resistance (20 µg/ml of minimum inhibitory concentration) of FabI2 to TCL was confined to the relatively less bulky side chain of A128. Substitution of A128 in FabI2 with bulky valine (V128) elevated TCL resistance. Phylogenetic analysis further suggested that the novel FabI2 and prototypical FabI evolved from a common short-chain dehydrogenase reductase family. To our best knowledge, FabI2 is the only known ENR shared by intracellular pathogenic prokaryotes, intracellular pathogenic lower eukaryotes, and a few higher eukaryotes. This suggests that the ENRs of prokaryotes and eukaryotes diverged from a common ancestral ENR of FabI2.

Crystal Structure of NADPH-Dependent Methylglyoxal Reductase Gre2 from Candida Albicans

Gre2 is a key enzyme in the methylglyoxal detoxification pathway; it uses NADPH or NADH as an electron donor to reduce the cytotoxic methylglyoxal to lactaldehyde. This enzyme is a member of the short-chain dehydrogenase/reductase (SDR) superfamily whose members catalyze this type of reaction with a broad range of substrates. To elucidate the structural features, we determined the crystal structures of the NADPH-dependent methylglyoxal reductase Gre2 from Candida albicans (CaGre2) for both the apo-form and NADPH-complexed form at resolutions of 2.8 and 3.02 Å, respectively. The CaGre2 structure is composed of two distinct domains: the N-terminal cofactor-binding domain and the C-terminal substrate-binding domain. Extensive comparison of CaGre2 with its homologous structures reveals conformational changes in α12 and β3′ of the NADPH-complex forms. This study may provide insights into the structural and functional variation of SDR family proteins.

In silico Prediction, Characterization, Molecular Docking, and Dynamic Studies on Fungal SDRs as Novel Targets for Searching Potential Fungicides Against Fusarium Wilt in Tomato.

Vascular wilt of tomato caused by Fusarium oxysporum f.sp. lycopersici (FOL) is one of the most devastating diseases, that delimits the tomato production worldwide. Fungal short-chain dehydrogenases/reductases (SDRs) are NADP(H) dependent oxidoreductases, having shared motifs and common functional mechanism, have been demonstrated as biochemical targets for commercial fungicides. The 1,3,6,8 tetra hydroxynaphthalene reductase (T4HNR) protein, a member of SDRs family, catalyzes the naphthol reduction reaction in fungal melanin biosynthesis. We retrieved an orthologous member of T4HNR, (complexed with NADP(H) and pyroquilon from Magnaporthe grisea) in the FOL (namely; FOXG_04696) based on homology search, percent identity and sequence similarity (93% query cover; 49% identity). The hypothetical protein FOXG_04696 (T4HNR like) had conserved T-G-X-X-X-G-X-G motif (cofactor binding site) at N-terminus, similar to M. grisea (1JA9) and Y-X-X-X-K motif, as a part of the active site, bearing homologies with two fungal keto reductases T4HNR (M. grisea) and 17-β-hydroxysteroid dehydrogenase from Curvularia lunata (teleomorph: Cochliobolus lunatus PDB ID: 3IS3). The catalytic tetrad of T4HNR was replaced with ASN115, SER141, TYR154, and LYS158 in the FOXG_04696. The structural alignment and superposition of FOXG_04696 over the template proteins (3IS3 and 1JA9) revealed minimum RMSD deviations of the C alpha atomic coordinates, and therefore, had structural conservation. The best protein model (FOXG_04696) was docked with 37 fungicides, to evaluate their binding affinities. The Glide XP and YASARA docked complexes showed discrepancies in results, for scoring and ranking the binding affinities of fungicides. The docked complexes were further refined and rescored from their docked poses through 50 ns long MD simulations, and binding free energies (ΔGbind) calculations, using MM/GBSA analysis, revealed Oxathiapiprolin and Famoxadone as better fungicides among the selected one. However, Famoxadone had better interaction of the docked residues, with best protein ligand contacts, minimum RMSD (high accuracy of the docking pose) and RMSF (structural integrity and conformational flexibility of docking) at the specified docking site. The Famoxadone was found to be acceptable based on in silico toxicity and in vitro growth inhibition assessment. We conclude that the FOXG_04696, could be employed as a novel candidate protein, for structure-based design, and screening of target fungicides against the FOL pathogen.

Exploiting the Catalytic Diversity of Short-Chain Dehydrogenases/Reductases: Versatile Enzymes from Plants with Extended Imine Substrate Scope.

Numerous short-chain dehydrogenases/reductases (SDRs) have found biocatalytic applications in C=O and C=C (enone) reduction. For NADPH-dependent C=N reduction, imine reductases (IREDs) have primarily been investigated for extension of the substrate range. Here, we show that SDRs are also suitable for a broad range of imine reductions. The SDR noroxomaritidine reductase (NR) is involved in Amaryllidaceae alkaloid biosynthesis, serving as an enone reductase. We have characterized NR by using a set of typical imine substrates and established that the enzyme is active with all four tested imine compounds (up to 99 % conversion, up to 92 % ee). Remarkably, NR reduced two keto compounds as well, thus highlighting this enzyme family's versatility. Using NR as a template, we have identified an as yet unexplored SDR from the Amaryllidacea Zephyranthes treatiae with imine-reducing activity (≤95 % ee). Our results encourage the future characterization of SDR family members as a means of discovering new imine-reducing enzymes.

YLL056C from Saccharomyces cerevisiae encodes a novel protein with aldehyde reductase activity.

The short-chain dehydrogenase/reductase (SDR) family, the largest family in dehydrogenase/reductase superfamily, is divided into "classical," "extended," "intermediate," "divergent," "complex," and "atypical" groups. Recently, several open reading frames (ORFs) were characterized as intermediate SDR aldehyde reductase genes in Saccharomyces cerevisiae. However, no functional protein in the atypical group has been characterized in S. cerevisiae till now. Herein, we report that an uncharacterized ORF YLL056C from S. cerevisiae was significantly upregulated under high furfural (2-furaldehyde) or 5-(hydroxymethyl)-2-furaldehyde concentrations, and transcription factors Yap1p, Hsf1p, Pdr1/3p, Yrr1p, and Stb5p likely controlled its upregulated transcription. This ORF indeed encoded a protein (Yll056cp), which was grouped into the atypical subgroup 7 in the SDR family and localized to the cytoplasm. Enzyme activity assays showed that Yll056cp is not a quinone or ketone reductase but an NADH-dependent aldehyde reductase, which can reduce at least seven aldehyde compounds. This enzyme showed the best Vmax, Kcat, and Kcat/Km to glycolaldehyde, but the highest affinity (Km) to formaldehyde. The optimum pH and temperature of this enzyme was pH 6.5 for reduction of glycolaldehyde, furfural, formaldehyde, butyraldehyde, and propylaldehyde, and 30°C for reduction of formaldehyde or 35°C for reduction of glycolaldehyde, furfural, butyraldehyde, and propylaldehyde. Temperature and pH affected stability of this enzyme and this influence varied with aldehyde substrate. Metal ions, salts, and chemical protective additives, especially at high concentrations, had different influence on enzyme activities for reduction of different aldehydes. This research provided guidelines for study of more uncharacterized atypical SDR enzymes from S. cerevisiae and other organisms.

OsHSD1, a hydroxysteroid dehydrogenase, is involved in cuticle formation and lipid homeostasis in rice

Cuticular wax, a hydrophobic layer on the surface of all aerial plant organs, has essential roles in plant growth and survival under various environments. Here we report a wax-deficient rice mutant oshsd1 with reduced epicuticular wax crystals and thicker cuticle membrane. Quantification of the wax components and fatty acids showed elevated levels of very-long-chain fatty acids (VLCFAs) and accumulation of soluble fatty acids in the leaves of the oshsd1 mutant. We determined the causative gene OsHSD1, a member of the short-chain dehydrogenase reductase family, through map-based cloning. It was ubiquitously expressed and responded to cold stress and exogenous treatments with NaCl or brassinosteroid analogs. Transient expression of OsHSD1-tagged green fluorescent protein revealed that OsHSD1 localized to both oil bodies and endoplasmic reticulum (ER). Dehydrogenase activity assays demonstrated that OsHSD1 was an NAD+/NADP+-dependent sterol dehydrogenase. Furthermore, OsHSD1 mutation resulted in faster protein degradation, but had no effect on the dehydrogenase activity. Together, our data indicated that OsHSD1 plays a specialized role in cuticle formation and lipid homeostasis, probably by mediating sterol signaling. This work provides new insights into oil-body associated proteins involved in wax and lipid metabolism.

Structural determinants in bacterial 2-keto-3-deoxy-D-gluconate dehydrogenase KduD for dual-coenzyme specificity.

Short-chain dehydrogenase/reductase (SDR) is distributed in many organisms, from bacteria to humans, and has significant roles in metabolism of carbohydrates, lipids, amino acids, and other biomolecules. An important intermediate in acidic polysaccharide metabolism is 2-keto-3-deoxy-d-gluconate (KDG). Recently, two short and long loops in Sphingomonas KDG-producing SDR enzymes (NADPH-dependent A1-R and NADH-dependent A1-R') involved in alginate metabolism were shown to be crucial for NADPH or NADH coenzyme specificity. Two SDR family enzymes-KduD from Pectobacterium carotovorum (PcaKduD) and DhuD from Streptococcus pyogenes (SpyDhuD)-prefer NADH as coenzyme, although only PcaKduD can utilize both NADPH and NADH. Both enzymes reduce 2,5-diketo-3-deoxy-d-gluconate to produce KDG. Tertiary and quaternary structures of SpyDhuD and PcaKduD and its complex with NADH were determined at high resolution (approximately 1.6 Å) by X-ray crystallography. Both PcaKduD and SpyDhuD consist of a three-layered structure, α/β/α, with a coenzyme-binding site in the Rossmann fold; similar to enzymes A1-R and A1-R', both arrange the two short and long loops close to the coenzyme-binding site. The primary structures of the two loops in PcaKduD and SpyDhuD were similar to those in A1-R' but not A1-R. Charge neutrality and moderate space at the binding site of the nucleoside ribose 2' coenzyme region were determined to be structurally crucial for dual-coenzyme specificity in PcaKduD by structural comparison of the NADH- and NADPH-specific SDR enzymes. The corresponding site in SpyDhuD was negatively charged and spatially shallow. This is the first reported study on structural determinants in SDR family KduD related to dual-coenzyme specificity. Proteins 2016; 84:934-947. © 2016 Wiley Periodicals, Inc.

Structural insights into the regulation of NADPH binding to reductase domains of nonribosomal peptide synthetases: A concerted loop movement model.

The termination module of nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) offloads the final product as an acid (occasionally also accompanied by cyclization) upon hydrolysis by employing thioesterase domains (TE-domains). Reductase domains (R-domains) of short-chain dehydrogenase/reductase (SDR) family offer an alternative offloading mechanism by reducing 4'-phosphopantetheine (4'-PPant) arm-tethered peptidyl chain, a thioester, to an aldehyde or an alcohol. Recent studies have highlighted their functional importance, for instance in the glycopeptidolipid (GPL) biosynthesis of Mycobacterium smegmatis, where the resulting alcoholic group is the site for subsequent modifications such as glycosylations. The mechanistic understanding of how these R-domains function in the context of multi-modular NRPS and PKS is poorly understood. In this study, conformational differences in functionally important loops, not reported previously, were identified in a new crystal form of R-domain which may be relevant to functioning in the context of assembly-line NRPS and PKS enzymology. Here, we propose a concerted loop movement model that allows gating of cofactor binding to these enzymes, enabling the release of the final product only after the substrate has reached the active site during biosynthesis, and therefore distinct from a canonical single domain SDR family of enzymes.