Genetic architecture of asthma in African American patients.

BACKGROUND: Asthma is a chronic inflammatory disorder with a strong genetic inheritance. Although more than 100 loci were reported through the genome-wide association study of European populations, the genetic underpinning of asthma in African American individuals remains largely elusive. OBJECTIVE: We aimed to identify genetic loci associated with asthma in African American individuals. METHODS: Three cohorts were genotyped at the Children's Hospital of Philadelphia by using the Illumina single-nucleotide polymorphism array platform. Genotype imputation was performed by using the Trans-Omics for Precision Medicine (TOPMed) reference panel, which includes whole genome sequencing data from more than 100,000 individuals. A meta-analysis of 3 Children's Hospital of Philadelphia cohorts and 10 Consortium on Asthma among African Ancestry Populations in the Americas cohorts, totaling 19,628 subjects, was conducted to identify genetic loci associated with asthma in African American individuals. RESULTS: Our study identified 12 loci surpassing the classical genome-wide significance threshold (5 × 10-8). Of those loci, 8 reached the stricter significance threshold (3 × 10-8). The 9p24.1 locus (rs10975467 [P = 1.63 × 10-8]) has previously been associated with asthma in European individuals. Six loci are associated with enhancer activities, 2 loci are in DNase I-hypersensitive regions, and all of them are associated with regulatory motifs. Moreover, the locus 11q13.4 (rs7480008) is an expression quantitative trait locus of XRRA1 in lung (P = 9.4 × 10-10), and the locus 13q14.3 (rs1543525) is a splicing quantitative trait locus of DHRS12 in lung (P = 1.1 × 10-13). CONCLUSIONS: Our findings provide candidate genetic loci for therapeutic target identification and prioritization for African populations.

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 in vitro assays or in vivo 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.

Genome-Wide Identification and Characterization of Short-Chain Dehydrogenase/Reductase (SDR) Gene Family in Medicago truncatula.

Short-chain dehydrogenase/reductase (SDR) belongs to the NAD(P)(H)-dependent oxidoreductase superfamily. Limited investigations reveal that SDRs participate in diverse metabolisms. A genome-wide identification of the SDR gene family in M. truncatula was conducted. A total of 213 MtSDR genes were identified, and they were distributed on all chromosomes unevenly. MtSDR proteins were categorized into seven subgroups based on phylogenetic analysis and three types including 'classic', 'extended', and 'atypical', depending on the cofactor-binding site and active site. Analysis of the data from M. truncatula Gene Expression Atlas (MtGEA) showed that above half of MtSDRs were expressed in at least one organ, and lots of MtSDRs had a preference in a tissue-specific expression. The cis-acting element responsive to plant hormones (salicylic acid, ABA, auxin, MeJA, and gibberellin) and stresses were found in the promoter of some MtSDRs. Many genes of MtSDR7C,MtSDR65C, MtSDR110C, MtSDR114C, and MtSDR108E families were responsive to drought, salt, and cold. The study provides useful information for further investigation on biological functions of MtSDRs, especially in abiotic stress adaptation, in the future.

A poplar short-chain dehydrogenase reductase plays a potential key role in biphenyl detoxification.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants with severe effects on human health and the biosphere. Plant-based remediation offers many benefits over conventional PCB remediation, but its development has been hampered by our poor understanding of biphenyl metabolism in eukaryotes, among other factors. We report here a major PCB-responsive protein in poplar, a plant model system capable of PCB uptake and translocation. We provide structural and functional evidence that this uncharacterized protein, termed SDR57C, belongs to the heterogeneous short-chain dehydrogenase reductase (SDR) superfamily. Despite sequence divergence, structural modeling hinted at structural and functional similarities between SDR57C and BphB, a central component of the Bph pathway for biphenyl/PCB degradation in aerobic bacteria. By combining gas chromatography/mass spectrometry (GC/MS) profiling with a functional complementation scheme, we found that poplar SDR57C can replace BphB activity in the upper Bph pathway of Pseudomonas furukawaii KF707 and therefore catalyze the oxidation of 2,3-dihydro-2,3-dihydroxybiphenyl (2,3-DHDB) to 2,3-dihydroxybiphenyl (2,3-DHB). Consistent with this biochemical activity, we propose a mechanism of action based on prior quantum studies, general properties of SDR enzymes, and the modeled docking of 2,3-DHDB to the SDR57C-NAD+ complex. The putative detoxifying capacity of SDR57C was substantiated through reverse genetics in Arabidopsis thaliana Phenotypic characterization of the SDR lines underscored an inducible plant pathway with the potential to catabolize toxic biphenyl derivatives. Partial similarities with aerobic bacterial degradation notwithstanding, real-time messenger RNA quantification indicates the occurrence of plant-specific enzymes and features. Our results may help explain differences in degradative abilities among plant genotypes and also provide elements to improve them.

Identification of the novel SDR42E1 gene that affects steroid biosynthesis associated with the oculocutaneous genital syndrome.

Hereditary connective tissue diseases form a heterogeneous group of disorders that affect collagen and extracellular matrix components. The cornea and the skin are among the major forms of connective tissues, and syndromes affecting both organs are often due to mutations in single genes. Brittle cornea syndrome is one of the pathologies that illustrates this association well. Furthermore, sex hormones are known to play a role in the maintenance of the structure and the integrity of the connective tissue including the skin and cornea, and may be involved in pathogenesis of oculocutaneous diseases. Herein, a double consanguineous family of Moroccan origin with two affected siblings, with suspected brittle cornea syndrome, was recruited. Ophthalmic examinations and genetic testing were performed in all the nuclear family individuals. Clinical examinations showed that the two affected boys presented with thinning of the cornea, blue sclera, keratoconus, hyperelasticity of the skin, joint hypermobility, muscle weakness, hearing loss and dental abnormalities that are compatible with the diagnosis of BCS disease. They showed however additional clinical signs including micropenis, hypospadias and cryptorchidism, suggesting abnormalities in endocrine pathways. Using a duo exome sequencing analysis performed in the mother and the propositus, we identified the novel homozygous missense mutation c.461G > A (p.Arg154Gln) in the short-chain dehydrogenase/reductase family 42E member 1 (SDR42E1) gene. This novel mutation, which co-segregated with the disease in the family, was predicted to be pathogenic by bioinformatics tools. SDR42E1 stability analysis using DynaMut web-server showed that the p.Arg154Gln mutations has a destabilizing effect with a ΔΔG value of -1.039 kcal/mol. As this novel gene belongs to the large family of short-chain dehydrogenases/reductases (SDR) thought to be involved in steroid biosynthesis, endocrinological investigations subsequently revealed that the two patients also had low levels of cholesterol. Karyotyping revealed a normal 46,XY karyotype for the two boys, excluding other causes of disorders of sex development due to chromosomal rearrangements. In conclusion, our study reveals that mutation in the novel SDR42E1 gene alters the steroid hormone synthesis and associated with a new syndrome we named oculocutaneous genital syndrome. In addition, this study highlights the role of SDR42E1 in the regulation of cholesterol metabolism in the maintenance of connective tissue and sexual maturation in humans.

Identification, Expression and Evolution of Short-Chain Dehydrogenases/Reductases in Nile Tilapia (Oreochromis niloticus).

The short-chain dehydrogenases/reductases (SDR) superfamily is involved in multiple physiological processes. In this study, genome-wide identification and comprehensive analysis of SDR superfamily were carried out in 29 animal species based on the latest genome databases. Overall, the number of SDR genes in animals increased with whole genome duplication (WGD), suggesting the expansion of SDRs during evolution, especially in 3R-WGD and polyploidization of teleosts. Phylogenetic analysis indicated that vertebrates SDRs were clustered into five categories: classical, extended, undefined, atypical, and complex. Moreover, tandem duplication of hpgd-a, rdh8b and dhrs13 was observed in teleosts analyzed. Additionally, tandem duplications of dhrs11-a, dhrs7a, hsd11b1b, and cbr1-a were observed in all cichlids analyzed, and tandem duplication of rdh10-b was observed in tilapiines. Transcriptome analysis of adult fish revealed that 93 SDRs were expressed in more than one tissue and 5 in one tissue only. Transcriptome analysis of gonads from different developmental stages showed that expression of 17 SDRs were sexually dimorphic with 11 higher in ovary and 6 higher in testis. The sexually dimorphic expressions of these SDRs were confirmed by in situ hybridization (ISH) and qPCR, indicating their possible roles in steroidogenesis and gonadal differentiation. Taken together, the identification and the expression data obtained in this study contribute to a better understanding of SDR superfamily evolution and functions in teleosts.

Quantitative analysis of mRNA expression levels of aldo-keto reductase and short-chain dehydrogenase/reductase isoforms in human livers.

The aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies are responsible for the reduction in compounds containing the aldehyde, ketone, and quinone groups. In humans, 12 AKR isoforms (AKR1A1, AKR1B1, AKR1B10, AKR1B15, AKR1C1, AKR1C2, AKR1C3, AKR1C4, AKR1D1, AKR1E2, AKR7A2, and AKR7A3) and 6 SDR isoforms (CBR1, CBR3, CBR4, HSD11B1, DHRS4, and DCXR) have been found to catalyze the reduction in xenobiotics, but their hepatic expression levels are unclear. The purpose of this study is to determine the absolute mRNA expression levels of these 18 isoforms in the human liver. In 22 human livers, all isoforms, except for AKR1B15, are expressed, and AKR1C2 (on average 1.6 × 106 copy/µg total RNA), AKR1C3 (1.3 × 106), AKR1C1 (1.3 × 106), CBR1 (9.7 × 105), and HSD11B1 (1.1 × 106) are abundant, representing 67% of the total expression of reductases in the liver. The expression levels of AKR1C2, AKR1C3, AKR1C1, CBR1, and HSD11B1 are significantly correlated with each other, except between AKR1C2 and CBR1, suggesting that they might be regulated by common factor(s). In conclusion, this study comprehensively determined the absolute expression of mRNA expression of each AKR and SDR isoform in the human liver.

Functional analysis of BPSS2242 reveals its detoxification role in Burkholderia pseudomallei under salt stress.

A bpss2242 gene, encoding a putative short-chain dehydrogenase/oxidoreductase (SDR) in Burkholderia pseudomallei, was identified and its expression was up-regulated by ten-fold when B. pseudomallei was cultured under high salt concentration. Previous study suggested that BPSS2242 plays important roles in adaptation to salt stress and pathogenesis; however, its biological functions are still unknown. Herein, we report the biochemical properties and functional characterization of BPSS2242 from B. pseudomallei. BPSS2242 exhibited NADPH-dependent reductase activity toward diacetyl and methylglyoxal, toxic electrophilic dicarbonyls. The conserved catalytic triad was identified and found to play critical roles in catalysis and cofactor binding. Tyr162 and Lys166 are involved in NADPH binding and mutation of Lys166 causes a conformational change, altering protein structure. Overexpression of BPSS2242 in Escherichia coli increased bacterial survival upon exposure to diacetyl and methylglyoxal. Importantly, the viability of B. pseudomallei encountered dicarbonyl toxicity was enhanced when cultured under high salt concentration as a result of BPSS2242 overexpression. This is the first study demonstrating that BPSS2242 is responsible for detoxification of toxic metabolites, constituting a protective system against reactive carbonyl compounds in B. pseudomallei..

DHRS12 inhibits the proliferation and metastasis of osteosarcoma via Wnt3a/ß-catenin pathway.

Aim: This experimental design was based on DHRS12 to explore its biological effects on osteosarcoma (OS). Materials & methods: The expression level of endogenous DHRS12 was analyzed by immunohistochemical analysis. DHRS12 was overexpressed in MG-63 and HOS cells by plasmid transfection. Cell proliferation, invasion, migration, apoptosis and western blot were used in the experiment. Results: The expression of DHRS12 was significantly reduced in OS. Overexpression of DHRS12 inhibited the proliferation, migration and invasion of MG-63 and HOS cells and induced apoptosis of OS cells. Overexpression of DHRS12 upregulated Bax, Caspase 9 and Caspase 3. Overexpression of DHRS12 resulted in inactivation of the Wnt3a/ß-catenin signaling pathway. Conclusion: Overexpression of DHRS12 inhibited the progression of OS via the Wnt3a/ß-catenin pathway.

Mice lacking the epidermal retinol dehydrogenases SDR16C5 and SDR16C6 display accelerated hair growth and enlarged meibomian glands.

Retinol dehydrogenases catalyze the rate-limiting step in the biosynthesis of retinoic acid, a bioactive lipid molecule that regulates the expression of hundreds of genes by binding to nuclear transcription factors, the retinoic acid receptors. Several enzymes exhibit retinol dehydrogenase activities in vitro; however, their physiological relevance for retinoic acid biosynthesis in vivo remains unclear. Here, we present evidence that two murine epidermal retinol dehydrogenases, short-chain dehydrogenase/reductase family 16C member 5 (SDR16C5) and SDR16C6, contribute to retinoic acid biosynthesis in living cells and are also essential for the oxidation of retinol to retinaldehyde in vivo Mice with targeted knockout of the more catalytically active SDR16C6 enzyme have no obvious phenotype, possibly due to functional redundancy, because Sdr16c5 and Sdr16c6 exhibit an overlapping expression pattern during later developmental stages and in adulthood. Mice that lack both enzymes are viable and fertile but display accelerated hair growth after shaving and also enlarged meibomian glands, consistent with a nearly 80% reduction in the retinol dehydrogenase activities of skin membrane fractions from the Sdr16c5/Sdr16c6 double-knockout mice. The up-regulation of hair-follicle stem cell genes is consistent with reduced retinoic acid signaling in the skin of the double-knockout mice. These results indicate that the retinol dehydrogenase activities of murine SDR16C5 and SDR16C6 enzymes are not critical for survival but are responsible for most of the retinol dehydrogenase activity in skin, essential for the regulation of the hair-follicle cycle, and required for the maintenance of both sebaceous and meibomian glands.

A Hedychium coronarium short chain alcohol dehydrogenase is a player in allo-ocimene biosynthesis.

KEY MESSAGE: An enzyme is crucial for the formation of Hedychium coronarium scent and defense responses, which may be responsible for the biosynthesis of allo-ocimene in H. coronarium. Hedychium coronarium can emit a strong scent as its main scent constituents are monoterpenes and their derivatives. Among these derivatives, allo-ocimene is not only a very important volatile substance in flower aroma, but is also crucial to plant defense. However, the molecular mechanism of allo-ocimene biosynthesis has not been characterized in plants. In this study, a new alcohol dehydrogenase gene, HcADH, was cloned. The amino acid sequences encoded by HcADH contained the most conserved motifs of short chain alcohol dehydrogenase/reductases (SDRs), which included NAD+ binding domain, TGxxx[AG]xG and active site YxxxK. Real-time PCR analyses showed that the HcADH was highly expressed in the outer labellum but was almost undetectable in vegetative organs. The change in its expression level in petals was positively correlated with the emission pattern of allo-ocimene during flower development. HcADH expression coincides also the release level of allo-ocimene among different Hedychium species. Although HcADH is not expressed in the leaves, HcADH expression and allo-ocimene release in leaves can be induced by mechanical wounding or methyl jasmonate (MeJA) treatment. In addition, the expression of HcADH induced by mechanical wounding can be prevented by acetylsalicylic acid, a jasmonic acid biosynthesis inhibitor, suggesting that jasmonic acid might participate in the transmission of wounding signals. Using the Barley stripe mosaic virus (BSMV)-VIGS method, it was found that BSMV:HcADH335 inoculation was able to down-regulate HcADH expression, decreasing only the release of allo-ocimene in flowers while the content of other volatile substances did not decrese. In vitro characterization showed that recombinant HcADH can catalyze geraniol into citral, and citral is an intermediate of allo-ocimene biosynthesis. HcADH may be responsible for the biosynthesis of allo-ocimene in H. coronarium, which is crucial for the formation of H. coronarium scent and defense function.

A novel short chain dehydrogenase from Bacillus megaterium for the conversion of the sesquiterpene nootkatol to (+)-nootkatone.

(+)-Nootkatone is a natural ingredient that occurs in grapefruit and certain other plants and is responsible for the characteristic smell of grapefruit. Due to its versatile applications in the flavor and fragrance industry as well as its application in some medical uses it recruits the interests of academic research along with industrial biotechnology. In the current work we present the application of a novel short chain dehydrogenase from Bacillus megaterium in an in vivo whole-cell biocatalyst system for the conversion of the intermediate nootkatol into the industrially valuable (+)-nootkatone. The newly identified dehydrogenase converted nootkatol selectively and efficiently into the final product. The conversion ratio of about 100% was achieved within 40 min yielding about 44 mg/L (+)-nootkatone. Furthermore, the herein identified dehydrogenase provides a new tool to overcome the limitation of the two-step enzymatic biotechnological process for the production of (+)-nootkatone.

Efficient synthesis of an antiviral drug intermediate using an enhanced short-chain dehydrogenase in an aqueous-organic solvent system.

(2R,3S)-N-tert-Butoxycarbonyl-3-amino-1-chloro-2-hydroxy-4-phenylbutane (1b) is key for the synthesis of the antiviral drug atazanavir. It can be obtained via the stereoselective bioreduction of (3S)-3-(N-Boc-amino)-1-chloro-4-phenyl-butanone (1a) with short-chain dehydrogenase/reductase (SDR). However, the stereoselective bioreduction of this hydrophobic and bulky substrate still remained a challenge because of the steric hindrance effect and low mass transfer rate. In this study, SDR isolated from Novosphingobium aromaticivorans (NaSDR) having low activity to 1a, which was engineered to enhance catalytic efficiency through active pocket iterative saturation mutagenesis (ISM). The obtained mutant (muSDR) (G141V/I195L) had 3.57 times higher kcat than the wild type (WT) towards 1a. Molecular docking analysis revealed considerable differences in the distance between the substrate and catalytic residues in WT and mutant SDR. Moreover, muSDR reduced 15 ketones with excellent enantioselectivity, indicating broad substrate acceptance. After optimization of expression and reaction conditions, the conversion was completed in a scale-up reaction (500 mL) using 50% toluene with 500 mM substrate without additional NADH. These results show that muSDR may be a valuable biocatalyst for future industrial applications.

Mapping key amino acid residues for the epimerase efficiency and stereospecificity of the sex pheromone biosynthetic short-chain dehydrogenases/reductases of Nasonia.

Males of the parasitic wasp genus Nasonia use blends of chiral hydroxylactones as sex pheromones to attract conspecific females. Whereas all Nasonia species use a mixture of (4R,5S)-5-hydroxy-4-decanolide (RS) and 4-methylquinazoline (MQ) as sex pheromones, Nasonia vitripennis evolved (4R,5R)-5-hydroxy-4-decanolide (RR) as an extra sex pheromone component. We recently identified and functionally characterized three short-chain dehydrogenases/reductases (SDRs) NV10127, NV10128, and NV10129 that are capable of catalyzing the epimerization of RS to RR via (4R)-5-oxo-4-decanolide (ODL) as intermediate. Despite their very high sequence identities of 88-98%, these proteins differ drastically in their ability to epimerize RS to RR and in their stereoselectivity when reducing ODL to RR/RS. Here, in order to unravel the sequence differences underlying these varying functional properties of NV1027, NV10128 and NV10129, we created chimeras of the three enzymes and monitored their catalytic activities in vitro. The results show that a few amino acid changes at the C-termini and active sites of Nasonia vitripennis SDRs lead to substantially altered RS to RR epimerization and ODL-reduction activities. Thus, our study adds to the understanding of pheromone evolution by showing that subtle mutations in key biosynthetic enzymes can result in drastic effects on the composition of chemical signals.

The short-chain dehydrogenases/reductases (SDR) gene: A new specific target for rapid detection of Mycobacterium tuberculosis complex by modified comparative genomic analysis.

BACKGROUND: Early detection of tuberculosis is one of the crucial steps for TB control. Although, the sensitivity of conventional methods like Lowenstein Jensen (LJ) culture and direct staining is quite low, molecular techniques like polymerase chain reaction (PCR) are more sensitive and be considered as useful tools for rapid detection of tuberculosis. Various genes like IS6110 and mpb64 have been used as target for detection of M. tuberculosis, but more research is needed to find the most specific targets. The short-chain dehydrogenases/reductases family (SDR) is one of a very large family of NAD- or NADP-dependent oxidoreductase enzymes which is present in all M. tuberculosis strains. The large part of SDR sequences in tuberculosis is completely conserved and different from non-tuberculosis mycobacterium. The aim of the study was to develop an in-house PCR assay using the SDR target for rapid detection of M. tuberculosis from clinical specimens. METHOD: M. tuberculosis-specific sequences were found using modified genome comparison method and the primers were designed by the Primer Premier 5.0 software. A PCR assay was developed targeting the nucleotide sequences within the SDR gene. A total of 50 cultivated specimens and 120 clinical specimens were evaluated by PCR. RESULTS: The clinical evaluation of SDR PCR assay showed high specificity (100%) and high sensitivity (88.5%). The analytical sensitivity was 10 fg of template DNA which is theoretically equivalent to 2 copy of genomic DNA per microliter. The SDR is a new specific target of M. tuberculosis and no cross-reactivity was observed to non-tuberculosis mycobacterium and other pathogenic bacteria. CONCLUSIONS: Based on our results, the SDR gene can be considered as a useful target for detection of M. tuberculosis complex from clinical specimens.

Stereoselective Bioreduction of Ethyl 3-Oxo-3-(2-Thienyl) Propanoate Using the Short-Chain Dehydrogenase/Reductase ChKRED12.

Ethyl (S)-3-hydroxy-3-(2-thienyl)propanoate((S)-HEES)acts as a key chiral intermediate for the blockbuster antidepressant drug duloxetine, which canbe achieved viathe stereoselective bioreduction ofethyl 3-oxo-3-(2-thienyl) propanoate (KEES) that containsa 3-oxoacyl structure.The sequences of the short-chain dehydrogenase/reductases from Chryseobacterium sp. CA49 were analyzed, and the putative3-oxoacyl-acyl-carrier-protein reductase, ChKRED12, was able to stereoselectivelycatalyze theNADPH-dependent reduction to produce (S)-HEES.The reductase activity of ChKRED12 towardsothersubstrates with 3-oxoacyl structure were confirmed with excellent stereoselectivity (>99% enantiomeric excess) in most cases. When coupled with a cofactor recycling system using glucose dehydrogenase, the ChKRED12 was able to catalyze the complete conversion of 100 g/l KEES within 12h, yielding the enantiopure product with >99% ee, showing a remarkable potential to produce (S)-HEES.

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.

Structure-guided design of Serratia marcescens short-chain dehydrogenase/reductase for stereoselective synthesis of (R)-phenylephrine.

Bioconversion is useful to produce optically pure enantiomers in the pharmaceutical industry, thereby avoiding problems with side reactions during organic synthesis processes. A short-chain dehydrogenase/reductase from Serratia marcescens BCRC 10948 (SmSDR) can stereoselectively convert 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) into (R)-phenylephrine [(R)-PE], which is marketed medically as a nasal decongestant agent. The whole-cell conversion process for the synthesis of (R)-PE using SmSDR was reported to have an unexpectedly low conversion rate. We reported the crystal structure of the SmSDR and designed profitable variants to improve the enzymatic activity by structure-guided approach. Several important residues in the structure were observed to form hydrophobic clusters that stabilize the mobile loops surrounding the pocket. Of these, Phe98 and Phe202 face toward each other and connect the upper curvature from the two arms (i.e., the α7 helix and loopß4-α4). The mutant structure of the double substitutions (F98YF202Y) exhibited a hydrogen bond between the curvatures that stabilizes the flexible arms. Site-directed mutagenesis characterization revealed that the mutations (F98Y, F98YF202Y, and F98YF202L) of the flexible loops that stabilize the region exhibited a higher transformation activity toward HPMAE. Together, our results suggest a robust structure-guided approach that can be used to generate a valuable engineered variant for pharmaceutical applications.

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.