Short-chain Dehydrogenase Research Articles

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.

Crystal structure of a short-chain dehydrogenase/reductase from Burkholderia phymatum in complex with NAD.

Burkholderia phymatum is an important symbiotic nitrogen-fixing betaproteobacterium. B. phymatum is beneficial, unlike other Burkholderia species, which cause disease or are potential bioagents. Structural genomics studies at the SSGCID include characterization of the structures of short-chain dehydrogenases/reductases (SDRs) from multiple Burkholderia species. The crystal structure of a short-chain dehydrogenase from B. phymatum (BpSDR) was determined in space group C2221 at a resolution of 1.80 Å. BpSDR shares less than 38% sequence identity with any known structure. The monomer is a prototypical SDR with a well conserved cofactor-binding domain despite its low sequence identity. The substrate-binding cavity is unique and offers insights into possible functions and likely inhibitors of the enzymatic functions of BpSDR.

Efficient Biocatalytic Synthesis of (R)-2-Chloro-1-(3,4-difluorophenyl)ethanol by the Short-Chain Dehydrogenase PpKR8 from Paraburkholderia phymatum STM815

Bioreductions catalyzed by ketoreductases play an important role in the synthesis of chiral alcohols. However, the synthesis of (R)-2-chloro-1-(3,4-difluorophenyl)ethanol (24b), an important chiral intermediate for the synthesis of the anticoagulant ticagrelor, poses significant challenges in terms of high substrate concentration requirements that limit its production. In this study, a novel NADH-dependent, short-chain dehydrogenase, PpKR8, from Paraburkholderia phymatum STM815, exhibited excellent enantioselectivity and high activity for the production of (R)-CFPL (24b) through the reduction of 2-chloro-1-(3,4-difluorophenyl)ethanone (24a). The coexpression of PpKR8 and glucose dehydrogenase from Bacillus subtilis in Escherichia coli allowed up to 300 g/L (1.57 M) CFPO (24a) to be completely converted into 24b with 99.9% enantiomeric excess and a high space–time yield (728 g/(L day)). Substrate specificity assays demonstrated a broad substrate spectrum for PpKR8, which included 35 α/β-ketoesters, aromatic ketones, and heterocyclic ketones. Moreover, three additional optically pure chiral alcohols that are used as important drug intermediates were synthesized at high substrate concentrations (150–330 g/L), demonstrating the excellent industrial potential of PpKR8-mediated bioreductions.

GDP-Mannose 3,5-Epimerase: A View on Structure, Mechanism, and Industrial Potential.

GDP-mannose 3,5-epimerase (GM35E, GME) belongs to the short-chain dehydrogenase/reductase (SDR) protein superfamily and catalyses the conversion of GDP-d-mannose towards GDP-l-galactose. Although the overall reaction seems relatively simple (a double epimerization), the enzyme needs to orchestrate a complex set of chemical reactions, with no less than 6 catalysis steps (oxidation, 2x deprotonation, 2x protonation and reduction), to perform the double epimerization of GDP-mannose to GDP-l-galactose. The enzyme is involved in the biosynthesis of vitamin C in plants and lipopolysaccharide synthesis in bacteria. In this review, we provide a clear overview of these interesting epimerases, including the latest findings such as the recently characterized bacterial and thermostable GM35E representative and its mechanism revision but also focus on their industrial potential in rare sugar synthesis and glycorandomization.

Exploration of archaeal nucleotide sugar epimerases unveils a new and highly promiscuous GDP-Gal4E subgroup

• Promiscuity of phGal4E_1 on different NDP-sugars was explored. • First demonstration of C4 epimerization activity on GDP-L-galactose and GDP-L-fucose. • PhGal4E_1, afGal4E and pcGal4E could form a new subgroup within CEP1 C4-epimerases. • The heptagonal box model allows the identification of new specificities in CEP1 family enzymes. UDP-galactose 4-epimerase (Gal4E, EC 5.1.3.2) catalyses the interconversion of UDP-galactose and UDP-glucose by inverting the configuration of the 4’-hydroxyl group of the sugar moiety. This enzyme is one of the mechanistically best-characterized members of the Short-chain Dehydrogenase/Reductase (SDR) superfamily. Although Gal4E was widely studied throughout all domains of life, ranging from eukaryotes to archaea, its biochemical characterization was often limited to UDP-hexoses, neglecting the possibility that Gal4E might be promiscuous towards other NDP-sugars and derivatives thereof. In this study, we identified a novel Gal4E subgroup displaying an unprecedented specificity on guanosine diphosphate (GDP) sugars. As a proof of concept, a detailed biochemical investigation was performed on Gal4E from Pyrococcus horikoshii (phGal4E_1), which revealed that it has, in fact, a clear preference for GDP-sugars. In addition, we confirmed that it accepts a variety of other sugar moieties, including L-sugars like L-galactose and L-fucose.

A putative short-chain dehydrogenase Rv0148 of Mycobacterium tuberculosis affects bacterial survival and virulence.

During infection, Mycobacterium tuberculosis combats the stress generated by the host cells through the action of short-chain dehydrogenases/reductases (SDRs). Rv0148 belongs to the oxidoreductase family with the SDRs domain, which regulates the homeostasis of M. tuberculosis. In our earlier studyusing knockout mutant strain (∆0148), we reported that Rv0148 is involved in intermediary metabolism, drug resistance and cell homeostasis of M. tuberculosis. In the current study, we explored the functional role of Rv0148 using gene knockout mutant in-vitro and in-vivo models of infection. We report the ∆0148 is attenuated for virulence of M. tuberculosis. During human monocyte (THP-1) cell line infection, M. tuberculosis Δ0148 displayed reduced intracellular survival compared to the wild type at successive time points. Similarly, in a guinea pig animal model of aerosol infection, Δ0148 displayed a growth attenuation at 5- and 10-week post-infection in the lungs and spleen compared to the wild-type M. tuberculosis and Rv0148-complemented Δ0148 strains. Our study suggest that Rv0148 has a distinct role in the intracellular virulence of M. tuberculosis.

Predicted Functional Shifts Due to Type of Soil Microbiome and Watering of Two Wild Plants in Western Region of Saudi Arabia

The present study aimed to predict differential enrichment of pathways and compounds in the rhizosphere microbiomes of the two wild plants (Abutilon fruticosum and Nitrosalsola vermiculata) and to predict functional shifts in microbiomes due to water. Amplicon sequencing of 16S rRNA region V3–V4 was done and gene-based microbial compositions were enrolled in PICRUSt to predict enriched pathways and compounds. The results indicated that “ABC transporters” and “Quorum sensing” pathways are among the highest enriched pathways in rhizosphere microbiomes of the two wild plants compared with those of the bulk soil microbiomes. The highest enriched compounds in soil microbiomes of the two wild plants included five proteins and three enzymes participating in one or more KEGG pathways. Six of these eight compounds showed higher predicted enrichment in rhizosphere soil microbiomes, while only one, namely phosphate transport system substrate-binding protein, showed higher enrichment in the surrounding bulk soil microbiomes. In terms of differentially enriched compounds due to watering, only the dual-specific aspartyl-tRNA (Asn)/glutamyl-tRNA (Gln) amidotransferase subunit A showed higher enrichment in rhizosphere soil of the two wild plants after 24 h of watering. Two of the highly enriched compounds namely branched-chain amino acid transport system ATP-binding protein and branched-chain amino acid transport system substrate-binding protein, are encoded by genes stimulated by the plant’s GABA that participates in conferring biotic and abiotic stresses in plants and improves the plant’s growth performance. The 3-Oxoacyl-[ACP] reductase, a member of the short-chain alcohol dehydrogenase/ reductase (SDR) superfamily, participates in fatty acids elongation cycles and contributes to plant-microbe symbiotic relationships, while enoyl-CoA hydratase has a reverse action as it participates in “Fatty acid degradation” pathway. The methyl-accepting chemotaxis protein is an environmental signal that sense “Bacterial chemotaxis” pathway to help establishing symbiosis with plant roots by recruiting/colonizing of microbial partners (symbionts) to plant rhizosphere. This information justifies the high enrichment of compounds in plant rhizosphere. The dual-specific aspartyl-tRNA (Asn)/glutamyl-tRNA (Gln) amidotransferase subunit A contributes to the plant ability to respond to watering as it participates in attaching the correct amino acid during translation to its cognate tRNA species, while hydrolyzing incorrectly attached amino acid. These two actions reduce the influence of oxidative stress in generating misfolded proteins and in reducing fidelity of translation.

Characterization of a putative tropinone reductase from Tarenaya hassleriana with a broad substrate specificity.

A novel short-chain alcohol dehydrogenase from Tarenaya hassleriana labeled as putative tropinone reductase was heterologously expressed in Escherichia coli. Purified recombinant protein had molecular weight of approximately 30kDa on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. T. hassleriana tropinone reductase-like enzyme (ThTRL) had not detected oxidative activity. The optimum pH for enzyme activity of ThTRL was weakly acidic (pH 5.0). 50°C was the optimum temperature for ThTRL. The highest catalytic efficiency and substrate affinity for recombinant ThTRL were observed with (+)-camphorquinone (kcat /Km =814.3s-1 mM-1 , Km =44.25μM). ThTRL exhibited a broad substrate specificity and reduced various carbonyl compounds, including small lipophilic aldehydes and ketones, terpene ketones, and their structural analogs.

Dehydrogenase reductase 9 (SDR9C4) and related homologs recognize a broad spectrum of lipid mediator oxylipins as substrates

Bioactive oxylipins play multiple roles during inflammation and in the immune response, with termination of their actions partly dependent on the activity of yet-to-be characterized dehydrogenases. Here, we report that human microsomal dehydrogenase reductase 9 (DHRS9, also known as SDR9C4 of the short-chain dehydrogenase/reductase (SDR) superfamily) exhibits a robust oxidative activity toward oxylipins with hydroxyl groups located at carbons C9 and C13 of octadecanoids, C12 and C15 carbons of eicosanoids, and C14 carbon of docosanoids. DHRS9/SDR9C4 is also active toward lipid inflammatory mediator dihydroxylated Leukotriene B4 and proresolving mediators such as tri-hydroxylated Resolvin D1 and Lipoxin A4, although notably, with lack of activity on the 15-hydroxyl of prostaglandins. We also found that the SDR enzymes phylogenetically related to DHRS9, i.e., human SDR9C8 (or retinol dehydrogenase 16), the rat SDR9C family member known as retinol dehydrogenase 7, and the mouse ortholog of human DHRS9 display similar activity toward oxylipin substrates. Mice deficient in DHRS9 protein are viable, fertile, and display no apparent phenotype under normal conditions. However, the oxidative activity of microsomal membranes from the skin, lung, and trachea of Dhrs9−/− mice toward 1 μM Leukotriene B4 is 1.7- to 6-fold lower than that of microsomes from wild-type littermates. In addition, the oxidative activity toward 1 μM Resolvin D1 is reduced by about 2.5-fold with DHRS9-null microsomes from the skin and trachea. These results strongly suggest that DHRS9 might play an important role in the metabolism of a wide range of bioactive oxylipins in vivo.

The biosynthesis of thymol, carvacrol, and thymohydroquinone in Lamiaceae proceeds via cytochrome P450s and a short-chain dehydrogenase

Thymol and carvacrol are phenolic monoterpenes found in thyme, oregano, and several other species of the Lamiaceae. Long valued for their smell and taste, these substances also have antibacterial and anti-spasmolytic properties. They are also suggested to be precursors of thymohydroquinone and thymoquinone, monoterpenes with anti-inflammatory, antioxidant, and antitumor activities. Thymol and carvacrol biosynthesis has been proposed to proceed by the cyclization of geranyl diphosphate to γ-terpinene, followed by a series of oxidations via p-cymene. Here, we show that γ-terpinene is oxidized by cytochrome P450 monooxygenases (P450s) of the CYP71D subfamily to produce unstable cyclohexadienol intermediates, which are then dehydrogenated by a short-chain dehydrogenase/reductase (SDR) to the corresponding ketones. The subsequent formation of the aromatic compounds occurs via keto-enol tautomerisms. Combining these enzymes with γ-terpinene in invitro assays or invivo in Nicotiana benthamiana yielded thymol and carvacrol as products. In the absence of the SDRs, only p-cymene was formed by rearrangement of the cyclohexadienol intermediates. The nature of these unstable intermediates was inferred from reactions with the γ-terpinene isomer limonene and by analogy to reactions catalyzed by related enzymes. We also identified and characterized two P450s of the CYP76S and CYP736A subfamilies that catalyze the hydroxylation of thymol and carvacrol to thymohydroquinone when heterologously expressed in yeast and N. benthamiana Our findings alter previous views of thymol and carvacrol formation, identify the enzymes involved in the biosynthesis of these phenolic monoterpenes and thymohydroquinone in the Lamiaceae, and provide targets for metabolic engineering of high-value terpenes in plants.

Highly efficient synthesis of pharmaceutically relevant chiral 3-N-substituted-azacyclic alcohols using two enantiocomplementary short chain dehydrogenases

Bioreductions catalyzed by alcohol dehydrogenases (ADHs) play an important role in the synthesis of chiral alcohols. In this study, we reported two stereocomplementary alcohol dehydrogenases from Flavobacterium psychrophilum (FpADH) and Flavobacterium sp. (FsADH), which showed the potential industrial application in highly efficient synthesis of a series of enantiomerically pure 3-N-substituted-azacyclic alochols. Both the enzymes showed high catalytic activity toward the model substrate N-Boc-4-piperidone (NBPO) and presented a strict enantioselectivity for the corresponding alcohol products. In addition, both enzymes showed broad substrate scope, including ketoesters, acryl ketones, and heterocyclic ketones. Using glucose dehydrogenase coexpressed with each of the enzymes to realize the efficient coenzyme recycling, various pharmaceutically relevant chiral 3-N-Boc azacyclic alcohols were asymmetrically synthesized at high substrate concentrations (343.7–643.8 g/L) and low equilvelent of NADP+ (0.1 mM) with excellent enantioselectivity (> 99.5% e.e), which have met the requirements of biocatalytic processes in the industry, and demonstrated the feasibility of FpADH and FsADH for industrial application in the biotransformation of chiral 3-N-substituted-azacyclic alcohols. The molecular basis of the enantioselectivity and catalytic efficiency of both enzymes were revealed the by molecular docking and MD simulation analysis.

Reductive inactivation of the hemiaminal pharmacophore for resistance against tetrahydroisoquinoline antibiotics

Antibiotic resistance is becoming one of the major crises, among which hydrolysis reaction is widely employed by bacteria to destroy the reactive pharmacophore. Correspondingly, antibiotic producer has canonically co-evolved this approach with the biosynthetic capability for self-resistance. Here we discover a self-defense strategy featuring with reductive inactivation of hemiaminal pharmacophore by short-chain dehydrogenases/reductases (SDRs) NapW and homW, which are integrated with the naphthyridinomycin biosynthetic pathway. We determine the crystal structure of NapW·NADPH complex and propose a catalytic mechanism by molecular dynamics simulation analysis. Additionally, a similar detoxification strategy is identified in the biosynthesis of saframycin A, another member of tetrahydroisoquinoline (THIQ) antibiotics. Remarkably, similar SDRs are widely spread in bacteria and able to inactive other THIQ members including the clinical anticancer drug, ET-743. These findings not only fill in the missing intracellular events of temporal-spatial shielding mode for cryptic self-resistance during THIQs biosynthesis, but also exhibit a sophisticated damage-control in secondary metabolism and general immunity toward this family of antibiotics.

Phosphorylation of OsABA2 at Ser197 by OsMPK1 regulates abscisic acid biosynthesis in rice

The mitogen-activated protein kinase OsMPK1 is involved in abscisic acid (ABA) biosynthesis in rice (Oryza sativa L.). However, the underlying molecular mechanisms of OsMPK1 in regulating ABA biosynthesis are poorly understood. Here, by using yeast two-hybrid assay and firefly luciferase complementary imaging assay, we show that OsMPK1 physically interact with a short-chain dehydrogenase protein OsABA2. However, OsMPK5, a homolog of OsMPK1, does not interact with OsABA2. Further, OsMPK1 can phosphorylate OsABA2S197 in vitro. Phosphorylation at the position of OsABA2S197 does not affect its subcellular localization, but enhances the stability of OsABA2 protein. We also found that OsABA2 has feedback regulation on OsMPK1 kinase activity. Further research reveals that OsMPK1 and OsABA2 coordinately regulate the biosynthesis of ABA, and phosphorylation of OsABA2 at Ser197 by OsMPK1 plays a crucial role in regulating the biosynthesis of ABA. Finally, genetic analysis showed that OsABA2 can enhance the sensitivity of rice to ABA and the tolerance of rice to drought and salt stress.

Identification of an important function of CYP123: Role in the monooxygenase activity in a novel estradiol degradation pathway in bacteria

Nowadays, 17β-estradiol (E2) biodegradation pathway has still not been identified in bacteria. To bridge this gap, we have described a novel E2 degradation pathway in Rhodococcus sp. P14 in this study, which showed that estradiol could be first transferred to estrone (E1) and thereby further converted into 16-hydroxyestrone, and then transformed into opened estrogen D ring. In order to identify the genes, which may be responsible for the pathway, transcriptome analysis was performed during E2 degradation in strain P14. The results showed that the expression of a short-chain dehydrogenase (SDR) gene and a CYP123 gene in the same gene cluster could be induced significantly by E2. Based on gene analysis, this gene cluster was found to play an important role in transforming E2 to 16-hydroxyestrone. The function of CYP123 was unknown before this study, and was found to harbor the activity of 16-estrone hydratase. Moreover, the global response to E2 in strain P14 was also analyzed by transcriptome analysis. It was observed that various genes involved in the metabolism processes, like the TCA cycle, lipid and amino acid metabolism, as well as glycolysis showed a significant increase in mRNA levels in response to strain P14 that can use E2 as the single carbon source. Overall, this study provides us an in depth understanding of the E2 degradation mechanisms in bacteria and also sheds light about the ability of strain P14 to effectively use E2 as the major carbon source for promoting its growth.

Plastidial Expression of 3β-Hydroxysteroid Dehydrogenase and Progesterone 5β-Reductase Genes Confer Enhanced Salt Tolerance in Tobacco.

The short-chain dehydrogenase/reductase (SDR) gene family is widely distributed in all kingdoms of life. The SDR genes, 3β-hydroxysteroid dehydrogenase (3β-HSD) and progesterone 5-β-reductases (P5βR1, P5βR2) play a crucial role in cardenolide biosynthesis pathway in the Digitalis species. However, their role in plant stress, especially in salinity stress management, remains unexplored. In the present study, transplastomic tobacco plants were developed by inserting the 3β-HSD, P5βR1 and P5βR2 genes. The integration of transgenes in plastomes, copy number and transgene expression at transcript and protein level in transplastomic plants were confirmed by PCR, end-to-end PCR, qRT-PCR and Western blot analysis, respectively. Subcellular localization analysis showed that 3β-HSD and P5βR1 are cytoplasmic, and P5βR2 is tonoplast-localized. Transplastomic lines showed enhanced growth in terms of biomass and chlorophyll content compared to wild type (WT) under 300 mM salt stress. Under salt stress, transplastomic lines remained greener without negative impact on shoot or root growth compared to the WT. The salt-tolerant transplastomic lines exhibited enhanced levels of a series of metabolites (sucrose, glutamate, glutamine and proline) under control and NaCl stress. Furthermore, a lower Na+/K+ ratio in transplastomic lines was also observed. The salt tolerance, mediated by plastidial expression of the 3β-HSD, P5βR1 and P5βR2 genes, could be due to the involvement in the upregulation of nitrogen assimilation, osmolytes as well as lower Na+/K+ ratio. Taken together, the plastid-based expression of the SDR genes leading to enhanced salt tolerance, which opens a window for developing saline-tolerant plants via plastid genetic engineering.

Epinephrine affects gene expression levels and has a complex effect on biofilm formation in Micrococcus luteus strain C01 isolated from human skin

In this study, the effect of epinephrine on the biofilm formation of Micrococcus luteus C01 isolated from human skin was investigated in depth for the first time. This hormone has a complex effect on biofilms in various systems. In a system with polytetrafluoroethylene (PTFE) cubes, treatment with epinephrine at a physiological concentration of 4.9 × 10−9 M increased the total amount of 72-h biofilm biomass stained with crystal violet and increased the metabolic activity of biofilms, but at higher and lower concentrations, the treatment had no significant effect. On glass fiber filters, treatment with the hormone decreased the number of colony forming units (CFUs) and changed the aggregation but did not affect the metabolic activity of biofilm cells. In glass bottom plates examined by confocal microscopy, epinephrine notably inhibited the growth of biofilms. RNA-seq analysis and RT–PCR demonstrated reproducible upregulation of genes encoding Fe–S cluster assembly factors and cyanide detoxification sulfurtransferase, whereas genes encoding the co-chaperone GroES, the LysE superfamily of lysine exporters, short-chain alcohol dehydrogenase and the potential c-di-GMP phosphotransferase were downregulated. Our results suggest that epinephrine may stimulate matrix synthesis in M. luteus biofilms, thereby increasing the activity of NAD(H) oxidoreductases. Potential c-di-GMP pathway proteins are essential in these processes.

Genomic analysis of Gordonia polyisoprenivorans strain R9, a highly effective 17 beta-estradiol- and steroid-degrading bacterium

The increasing levels of estrogens and pollution by other steroids pose considerable challenges to the environment. In this study, the genome of Gordonia polyisoprenivorans strain R9, one of the most effective 17 beta-estradiol- and steroid-degrading bacteria, was sequenced and annotated. The circular chromosome of G. polyisoprenivorans R9 was 6,033,879 bp in size, with an average GC content of 66.91%. More so, 5213 putative protein-coding sequences, 9 rRNA, 49 tRNA, and 3 sRNA genes were predicted. The core-pan gene evolutionary tree for the genus Gordonia showed that G. polyisoprenivorans R9 is clustered with G. polyisoprenivorans VH2 and G. polyisoprenivorans C, with 93.75% and 93.8% similarity to these two strains, respectively. Altogether, the three G. polyisoprenivorans strains contained 3890 core gene clusters. Strain R9 contained 785 specific gene clusters, while 501 and 474 specific gene clusters were identified in strains VH2 and C, respectively. Furthermore, whole genome analysis revealed the existence of the steroids and estrogens degradation pathway in the core genome of all three G. polyisoprenivorans strains, although the G. polyisoprenivorans R9 genome contained more specific estrogen and steroid degradation genes. In strain R9, 207 ABC transporters, 95 short-chain dehydrogenases (SDRs), 26 monooxygenases, 21 dioxygenases, 7 aromatic ring-hydroxylating dioxygenases, and 3 CoA esters were identified, and these are very important for estrogen and steroid transport, and degradation. The results of this study could enhance our understanding of the role of G. polyisoprenivorans R9 in estradiol and steroid degradation as well as evolution within the G. polyisoprenivorans species.

SDR7-6, a short-chain alcohol dehydrogenase/reductase family protein, regulates light-dependent cell death and defence responses in rice.

Lesion mimic mutants resembling the hypersensitive response without pathogen attack are an ideal material to understand programmed cell death, the defence response, and the cross‐talk between defence response and development in plants. In this study, mic, a lesion mimic mutant from cultivar Yunyin treated with ethyl methanesulphonate (EMS), was screened. By map‐based cloning, a short‐chain alcohol dehydrogenase/reductase with an atypical active site HxxxK was isolated and designated as SDR7‐6. It functions as a homomultimer in rice and is localized at the endoplasmic reticulum. The lesion mimic phenotype of the mutant is light‐dependent. The mutant displayed an increased resistance response to bacterial blight, but reduced resistance to rice blast disease. The mutant and knockout lines showed increased reactive oxygen species, jasmonic acid content, antioxidant enzyme activity, and expression of pathogenicity‐related genes, while chlorophyll content was significantly reduced. The knockout lines showed significant reduction in grain size, seed setting rate, 1000‐grain weight, grain weight per plant, panicle length, and plant height. SDR7‐6 is a new lesion mimic gene that encodes a short‐chain alcohol dehydrogenase with atypical catalytic site. Disruption of SDR7‐6 led to cell death and had adverse effects on multiple agricultural characters. SDR7‐6 may act at the interface of the two defence pathways of bacterial blight and rice blast disease in rice.

Structural and Kinetic Insights Into the Molecular Basis of Salt Tolerance of the Short-Chain Glucose-6-Phosphate Dehydrogenase From Haloferax volcanii.

Halophilic enzymes need high salt concentrations for activity and stability and are considered a promising source for biotechnological applications. The model study for haloadaptation has been proteins from the Halobacteria class of Archaea, where common structural characteristics have been found. However, the effect of salt on enzyme function and conformational dynamics has been much less explored. Here we report the structural and kinetic characteristics of glucose-6-phosphate dehydrogenase from Haloferax volcanii (HvG6PDH) belonging to the short-chain dehydrogenases/reductases (SDR) superfamily. The enzyme was expressed in Escherichia coli and successfully solubilized and refolded from inclusion bodies. The enzyme is active in the presence of several salts, though the maximum activity is achieved in the presence of KCl, mainly by an increment in the kcat value, that correlates with a diminution of its flexibility according to molecular dynamics simulations. The high KM for glucose-6-phosphate and its promiscuous activity for glucose restrict the use of HvG6PDH as an auxiliary enzyme for the determination of halophilic glucokinase activity. Phylogenetic analysis indicates that SDR-G6PDH enzymes are exclusively present in Halobacteria, with HvG6PDH being the only enzyme characterized. Homology modeling and molecular dynamics simulations of HvG6PDH identified a conserved NLTX2H motif involved in glucose-6-phosphate interaction at high salt concentrations, whose residues could be crucial for substrate specificity. Structural differences in its conformational dynamics, potentially related to the haloadaptation strategy, were also determined.

A short-chain dehydrogenase/reductase (SDR) detection for the Isoflavone reductase gene in Bulbophyllum and other orchids

Bulbophyllum, the largest genus in Orchidaceae, is known for being rich in phenolic compounds. In this study, the polyphenol and antioxidant content of four Bulbophyllum species was compared to that of a Dendrobium sp. control. The transcript levels of eight genes involved in flavonoid biosynthesis and oxidative stress responses were then determined to identify correlations with flavonoid and antioxidant content. A positive correlation between flavonoid content and transcription level of isoflavone reductase (IFR), belonging to the short-chain dehydrogenase/reductase (SDR) superfamily, was observed in leaf and pseudobulb tissue of the orchids. IFR showed an extraordinarily high level of gene expression (max. 118-fold) in the pseudobulb tissue of Bulbophyllum species compared with that in the same tissue in several species of Dendrobium. A pair of primers derived from two conserved regions within the sequences of related SDRs (IFR, eugenol synthase, and phenylcoumaran benzylic ether reductase) from Dendrobium spp., Paphiopedilum sp., Phaseolus sp., Phoenix sp., Striga sp., and Vanda sp. were used to attain a full-length IFR from four Bulbophyllum spp., three Dendrobium spp. and one Vanda sp. The phylogenetic relationships of those SDRs reported in the NCBI database showed distinctive clusters between the closely related SDRs of each genus in the Orchidaceae, and the distantly related clusters of the other plant SDRs. This work describes an easy method for the quick detection and isolation of IFRs in Bulbophyllum and other orchid species.