Dissertations / Theses on the topic 'Short-chain dehydrogenase/reductase (SDR)'

The classification of protein sequences is a subfield in the area of Bioinformatics that attracts a substantial interest today. Machine Learning algorithms are here believed to be able to improve the performance of the classification phase. This thesis considers the application of different Machine Learning algorithms to the classification problem of a data set of short-chain dehydrogenases/reductases (SDR) proteins. The classification concerns both the division of the proteins into the two main families, Classic and Extended, and into their different subfamilies. The results of the different algorithms are compared to select the most appropriate algorithm for this particular classification problem.
Klassificeringen av proteinsekvenser är ett område inom Bioinformatik, vilket idag drar till sig ett stort intresse. Maskininlärningsalgoritmer anses här kunna förbättra utförandet av klassificeringsfasen. Denna uppsats rör tillämpandet av olika maskininlärningsalgoritmer för klassificering av ett dataset med short-chain dehydrogenases/reductases (SDR) proteiner. Klassificeringen rör både indelningen av proteinerna i två huvudklasser, Classic och Extended, och deras olika subklasser. Resultaten av de olika algoritmerna jämförs för att välja ut den mest lämpliga algoritmen för detta specifika klassificeringsproblem.
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The main effect of lipid peroxidation, which often occurs in response to oxidative stress, is the production of different toxic aldehydes. In particular, over the years, the lipid peroxidation-derived aldehyde 4-hydroxy-trans-2-nonenal (HNE) has received much attention for its dual role in the pathogenesis of several diseases and as signaling molecule. HNE metabolism is reported to mainly occur through its conjugation with glutathione (GSH) and the subsequent formation of 3-glutathionyl-4-hydroxynonanal (GSHNE) [1, 2]. This molecule is susceptible to both oxidative and reductive transformations, which occur through the action of either the NADPH-dependent activity of aldose reductase (AKR1B1) [1] or through the NAD(P)+ -dependent activity of aldehyde dehydrogenase, respectively [3, 4]. Recently, we have demonstrated the implication of a new NADP+-dependent enzymatic activity able to oxidize GSHNE to its corresponding acid 3-glutathionyl-nonanoic-γ-lactone (GSHNA-γ-lactone) [5]. The enzyme was purified from a human astrocytoma cells line (ADF) to electrophoretic homogeneity as protein doublet in SDS-PAGE, with an apparent molecular weight of 31-32 kDa. Proteomic analysis identified both proteins as human CBR1, also known as NADP+ 15-hydroxyprostaglandine dehydrogenase with 74% of homology and proved their migration differences due to the occurrence of a carboxyethyl moiety at Lys239 [5]. This modification has been already described for the human enzyme and has been demonstrated to have no effect on the protein activity and specificity [6, 7]. The enzyme efficiently catalyzes the oxidation of GSHNE, while it is practically inactive towards 4-hydroxy trans-2-nonenal and other HNE-S-thiolated adducts containing an incomplete glutathionyl moiety [5]. Nucleotide sequence analysis of hCBR1 cDNA from ADF cells completely matched with the human wild type counterpart [5], excluding any gain-of-function mutations in the cDNA-derived protein sequence of hCBR1 [8, 9]. Highly purified human recombinant carbonyl reductase 1 (E.C. 1.1.1.184, hCBR1), which preserves its ability to oxidize specifically GSHNE, is also shown to efficiently act as aldehyde reductase on glutathionylated alkanals, namely 3-glutathionyl-4-hydroxynonanal (GSHNE), 3-glutathionyl-nonanal, 3-glutathionyl-hexanal and 3-glutathionyl-propanal [10]. The presence of the glutathionyl moiety appears as a necessary requirement for the susceptibility of these compounds to the NADPH-dependent reduction by hCBR1. In fact the corresponding alkanals and alkenals, and the cysteinyl and γ-glutamyl-cysteinyl alkanals adducts were either ineffective or very poorly active as CBR1 substrates [10]. Mass spectrometry analysis reveals the ability of hCBR1 to reduce GSHNE to the corresponding 3-glutathionyl-1,4-dihydroxynonane (GSDHN) and at the same time to catalyze the oxidation of the hemiacetal form of GSHNE, generating the 3-glutathionylnonanoic-γ-lactone. These data are indicative of the ability of the enzyme to catalyze a disproportion reaction of the substrate through the redox recycle of the pyridine cofactor [10]. A rationale for the observed preferential activity of hCBR1 on different GSHNE diastereoisomers is given by molecular modelling. These results evidence the potential of hCBR1 acting on GSHNE to accomplish a dual role, both in terms of HNE detoxification and, through the production of GSDHN, in terms of involvement into the signalling cascade of the cellular inflammatory response.

The pea (Pisum sativum) tetrameric short-chain alcohol dehydrogenase-like protein (SAD) family consists of at least three highly similar members (SAD-A, -B, and -C). According to mRNA data, environmental stimuli induce SAD expression. The aim of this study was to characterize the SAD proteins by examining their catalytic function, distribution in pea, and induction in different tissues. In enzyme activity assays using a range of potential substrates, the SAD-C enzyme was shown to reduce one- or two-ring-membered quinones lacking long hydrophobic hydrocarbon tails. Immunological assays using a specific antiserum against the protein demonstrated that different tissues and cell types contain small amounts of SAD protein that was predominantly located within epidermal or subepidermal cells and around vascular tissue. Particularly high local concentrations were observed in the protoderm of the seed cotyledonary axis. Two bow-shaped rows of cells in the ovary and the placental surface facing the ovule also exhibited considerable SAD staining. Ultraviolet-B irradiation led to increased staining in epidermal and subepidermal cells of leaves and stems. The different localization patterns of SAD suggest functions both in development and in responses to environmental stimuli. Finally, the pea SAD-C promoter was shown to confer heterologous wound-induced expression in Arabidopsis (Arabidopsis thaliana), which confirmed that the inducibility of its expression is regulated at the transcriptional level.

Pteridine reductase 1 (PTR1) is an NADPH dependent reductase that catalyzes the reduction of several pterins and folates. The gene encoding this enzyme was originally identified in Leishmania based on its ability to provide resistance to the drug methotrexate (MTX). The DNA and amino acid sequences are known, and overproducing strains of Escherichia coli are available. PTR1 has been previously shown to be required for the salvage of oxidized pteridines (folate, biopterin, and others). Since Leishmaniaare folate and pterin auxotrophes, PTR1 is a possible target for novel anti-folate drugs for the treatment of leishmaniasis. PTR1 catalyzes the transfer of hydride from NADPH to the 2-amino-4-oxo-pteridine ring system yielding 7, 8-dihydropteridines, and to the pteridine ring system of 7, 8-dihydropteridines yielding 5,6, 7, 8-tetrahydropteridines. PTR1 shows a pH dependent substrate specificity. At pH 4.6 the specific activity of PTR1 is highest with pterins, while at pH 6.0 the specific activity of PTR1 was highest with folates. The sequence of PTR1 is only 20-30% homologous to the sequences of members of the short chain dehydrogenase/reductase enzyme family. Although this is typical for members of this enzyme family, it does not allow for unambiguous classification in this family. In fact, when the DNA sequence of PTR1was first determined, PTR1 was classified as an aldoketo reductase. To classify PTR1 definitively, further biochemical characterization was required. To provide this information, the work described here was undertaken: (i) the stereochemical and kinetic course of PTR1 was determined; (ii) residues important in catalysis and ligand binding were identified; and (iii) conditions for the crystallization of PTR1 were developed. The stereochemistry of hydride transfer The use of [3H]-folate, showed that the ultimate product of PTR1 was 5, 6, 7, 8-tetrahydrofolate. 4R-[3H]-NADPH and 4S-[3H]-NADPH were synthesized enzymatically and used as the cofactor for the reduction of folate. PTR1 was coupled to thymidylate synthase (TS), and tritium from 4S-[3H]-NADPH was transferred to thymidylate. Therefore, the pro-S hydride of NADPH was transferred to the si face of dihydrofolate (DHF; see figure I-1). The transfer of the pro-Shydride indicates that PTR1 is a B-side dehydrogenase which is consistent with its membership in the short chain dehydrogenase (SDR) family. The kinetic mechanism of PTR1 When NADPH was varied at several fixed concentrations of folate (and vice-versa) V/K (Vmax/KM) showed a dependence upon concentration of the fixed substrate. This is consistent with a ternary complex mechanism, in contrast to a substituted enzyme mechanism that exhibits no dependence of V/K on fixed substrate. Product inhibition patterns using NADP+ and 5-deazatetrahydrofolate (5dTHF, a stable product analog) were consistent with an ordered ternary complex mechanism in which NADPH binds first and NADP+ dissociates last. However, an enzyme-DHF binary complex was detected by fluorescence. Isotope partitioning experiments showed that the enzyme-DHF binary complex was not catalytically competent whereas the enzyme-NADPH complex was. Measurement of the tritium isotope effect on V/K (T(V/K)) at high and low dihydrofolate confirmed that PTR1 proceeds via a steady state ordered mechanism. Rapid quench analysis showed that dihydrofolate was a transient intermediate during the reduction of folate to tetrahydrofolate and that folate reduction is biphasic. Catalytic Residues of PTR1 The amino acid sequences of dihydropteridine reductase and 3-α, 20-β, hydroxy steroid dehydrogenase were aligned to that of PTR1. Based on the results of the alignment, site directed mutagenesis was used to investigate the role of specific residues in the catalytic cycle of PTR1. Variant enzymes were screened based on their ability to rescue a dihydrofolate reductase (DHFR) deficient strain of E. coli. Selected PTR1 variants (some complementing and some non-complementing) were purified and further characterized. Tyrosine 193 of the wild type enzyme was found to be involved in the reduction of pteridines, but not in the reduction of 7, 8-dihydropteridines, and eliminated the substrate inhibition of 7, 8-dihydropteridines observed with the wild type enzyme. Both PTR1(K197Q) and PTR1(Y193F/K197Q) had decreased activity for all substrates and low affinity for NADPH. In contrast to the wild type enzyme, NADPH displayed substrate inhibition towards PTR1(K197Q). All PTR1(D180) variants that were purified were inactive except for PTR1(D180C), which showed 2.5% of wild type activity with DHF. The binary complexes of PTR1(D180A) and PTR1(D180S) with NADPH showed a decrease in affinity for folate. Based on the kinetic properties of the PTR1 variants, roles for Y193, K197, and D180 are proposed. In conjunction with D180, Y193 acts as a proton donor to N8 of folate. K197 forms hydrogen bonds with NADPH in the active site and lowers the pKaof Y193. D180 participates in the protonation of N8 of folate and N5 of DHF. Crystallization of PTR1 and PTR1-ligand complexes The crystallization of PTR1 from L. major and L. tarentolea as unliganded and as binary and ternary complexes was attempted. Several crystal forms were obtained including L. major PTR1-NADPH-MTX crystals that diffracted to ~ 3.2 Å resolution. It was not possible to collect a full data set of any of the crystals. At their current stage, none of the crystal forms is suitable for structural work.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第19195号
農博第2134号
新制||農||1034(附属図書館)
学位論文||H27||N4941(農学部図書室)
32187
京都大学大学院農学研究科食品生物科学専攻
(主査)教授 谷 史人, 教授 保川 清, 准教授 橋本 渉
学位規則第4条第1項該当

Sinorhizobium meliloti maintains a complex lifestyle, including saprotrophy, rhizophere colonization and root hair infection leading to the formation of root nodules in which the plant provides sustenance in return for nitrogen fixation. S. meliloti cells use a variety of carbon substrates for growth; this omnivory probably contributes to competitive ability in the soil. Several candidates for contribution to the catabolic capacity are found within the family of short chain dehydrogenases /reductases (SDR), which catalyze NAD(P)(H) dependent oxidation / reduction reactions. The 6.7 Mb genome of S. meliloti contains 78 SDR-encoding genes distributed on all three replicons. In this work each of these genes were disrupted by single crossover mutagenesis. These mutants were screened for growth on 93 different compounds as carbon source, and phenotypes were found for 17 of the mutants, providing suggestions for potential substrates of the corresponding enzymes. Carbon sources for which phenotype was observed include sugar alcohols, leucine, lysine, ornithine, galactitol, rhamnose, arabinose, mono-methyl succinate and ribono-γ-lactone. In addition, one of the mutants was found to be a proline auxotroph. In several cases, the phenotypes were consistent with the phenotypes of deletion mutants in which large sections of pSymB were absent. Eight of the mutants exhibited symbiotic deficiency after inoculation of alfalfa, while viable cells of three of the mutants could not be isolated from the nodules even though nitrogen fixation occurred. The results suggest that the corresponding SDR enzymes are involved in a pathway that is required for maintenance of viability by cells throughout infection and nodule development. This work demonstrates that members of the SDR family contribute to both the catabolic capacity and the symbiotic interactions of S. meliloti. Further experiments will address the details of the biochemical pathways involved. Knowledge of the substrate specificities of these enzymes should also prove informative in the description and annotation of orthologs that are identified in other genome sequences.

碩士
國立中興大學
生命科學院碩士在職專班
98
In order to avoid involved in the chemical synthesis method, the present study was designed to use a biotransformation approach to produce L-PE from 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE). We found that S. quinivorans BCRC 14811 could convert HPMAE to L-PE with 15% of yield. Addition of 2-phenylethanol and acetophenone in the culture medium could increase conversion yield from 15% to 88% and 83%, respectively. A genomic library of S. quinivorans BCRC 14811 was constructed for the screening of clones capable of converting HPMAE to PE using pQE30 as cloning vector and HPMAE-sensitive Escherichia coli NovaBlue as host cell. Luria-Bertani plate containing 1 to 10 mM HPMAE were used as the selection medium. However no positive clone was obtained. Short-chain dehydrogenase / reductase (SDR) was cloned from S. quinivorans BCRC 14811 by PCR. When the sdr gene was expressed in E. coli BL21(DE3), the recombinant E. coli cell can convert 10 mM HPMAE to 8.9 mM D-PE with a yield of about 89% and 60 mM HPMAE to 56.2 mM D-PE with a conversion yield of 94%. The SDR was purified by immobilized metal affinity chromatography. Enzyme activity assay demonstrated that the SDR protein could uses NADPH and NADH as cofactors, which exhibit a specific activities of 257 U/mg and 285 U/mg, respectively.

All-trans retinoic acid (atRA) is an important morphogen in many developmental processes, including apoptosis, growth, organogenesis and differentiation. During the early embryonic development, atRA is synthesized in an irreversible reaction from all-trans retinal (atRAL), catalyzed mainly by retinal dehydrogenase 2 (RALDH2). The upstream metabolic pathway, including the redox reaction between all-trans retinol (atROL) and atRAL, mediated by short-chain dehydrogenase/reductase, however, is less understood during embryonic development.
Previously a Xenopus laevis short-chain dehydrogenase/reductase 3 (dhrs3) was identified as a gene differentially expressed in the Spemann-Mangold Organizer. In this study, dhrs3 was found to be expressed in the circumblastoporal ring, neuroectoderm and pronephros region, and was up-regulated by atRA signalling. By using loss-of-function and gain-of-function approaches, it was found that the phenotype induced by knockdown of dhrs3 mimicked those with an elevated level of atRA signalling, and overexpression of dhrs3 enhanced the phenotype of cyp26a1, which functions in degradation of atRA. In dhrs3 knock-down embryos (morphants), expression domain of the mesoderm markers brachyury was disrupted, and that of organizer marker lim1 were significantly expanded, suggesting altered mesoderm induction. Overexpression of dhrs3, on the other hand, exerted an opposite effect on lim1 by reducing its expression. dhrs3 also rescued the phenotype following raldh2 overexpression induced by exogenous atRAL, suggesting that dhrs3 competed with raldh2 for the same substrate, atRAL. In line with these findings, expression of the mid-brain, hindbrain and neural crest markers was posteriorized in dhrs3-overexpressing embryos, similar to the phenotype of atRA-deficient embryos induced by cyp26a1. These findings indicate that dhrs3 participates in the retinoid metabolism by reducing atRAL to atROL.
Xenopus dhrs3 morphants displayed a shortened anteroposterior axis, similar to that of atRA toxicity. Examination of convergent extension (CE) markers papc indicated a defect in the CE movement, which was also evidenced by the disrupted bra and not expression. Overall, the results of the present study suggest that dhrs3 regulates proper mesoderm patterning through regulating the CE movement.
Kam, Kin Ting.
Advisers: Yu Pang Eric Cho; Wood Yee Chan; Hui Zhao.
Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: .
Thesis (Ph.D.)--Chinese University of Hong Kong, 2011.
Includes bibliographical references (leaves [158]-184).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

博士
國立中興大學
分子生物學研究所
102
Chapter 1 (R)-phenylephrine [(R)-PE] is an adrenergic receptor agonist used to treat the common cold and is not associated with the side effects of ephedrine adrenergic drugs. We found a novel short-chain dehydrogenase/reductase (SQ_SDR) from Serratia quinivorans BCRC 14811 that strongly preferred NADH to NADPH as a cofactor and was capable of converting 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) to (S)-PE in the presence of NADH and NADPH, with specific activities of 26.5 ± 2.3 U/mg protein and 0.24 ± 0.01 U/mg protein, respectively, at 30°C and at a pH of 7.0. The Escherichia coli strain BL21 (DE3), expressing NADH-preferring SQ_SDR, converted HPMAE to (S)-PE with more than 99% enantiomeric excess, a conversion yield of 86.6% and a productivity of 20.2 mmol/l．h, which was much higher than our previous report using E. coli NovaBlue expressing NADPH-dependent amino alcohol dehydrogenase from Rhodococcus erythropolis BCRC 10909 as the biocatalyst. This new biocatalyst showed considerable stability in a process using 70 mM HPMAE, and a productivity of more than 17.9 mmol PE/l．h was obtained in the fourth cycle. Chapter 2 (R)-phenylephrine [(R)-PE] is an α1-adrenergic receptor agonist and is widely used as a nasal decongestant to treat common cold without the side effects of other epherdrine adrenergic drugs. We found a short-chain dehydrogenase/reductase (SM_SDR) from Serratia marcescens BCRC 10948 that was able to convert HPMAE to (R)-PE. The SM_SDR used NADPH and NADH as cofactors with specific activities of 17.35 ± 0.71 and 5.57 ± 0.07 mU/mg protein, respectively, at 30°C and at a pH of 7.0, indicating this enzyme could be categorized as NADPH-preferring short-chain dehydrogenase/reductase. The Escherichia coli strain BL21 (DE3) expressing SM_SDR could converted HPMAE to (R)-PE with more than 99% enantiomeric excess, the productivity and conversion yield were 0.57 mmol PE/l．h and 51.06 %, respectively, using 10 mM HPMAE. Fructose was the most effective carbon source for the conversion of HPMAE to (R)-PE. Key residues in the NAD(P)H-binding pocket of SM_SDR was modified by site-directed mutagenesis to improve the ability to use NADH as cofactor for (R)-PE production. The ratio of specific activity (NADH) / specific activity (NADPH) for SM_SDR variants with mutations of S18A/R19Q, A41D/S18A/R19Q and A41D/G143D/S18A/R19Q exhibited nearly 1-fold higher than that for wild-type SM_SDR. However, the specific activities of these mutated SM_SDRs using NADH as cofactor were not remarkably increase. Therefore, it’s necessary to mutate SM_SDR or screen other enzymes with high catalytic activity for industrial production of (R)-PE.

碩士
國立中興大學
分子生物學研究所
105
(R)-phenylephrine, (R)-PE, is often used as a medical treatment for blood pressure control and nasal decongestant. Conventional chemical synthesis is the current method being used for (R)-PE production. However, poor enantioselectivity and environmentally unfriendly are the major drawbacks and increase the production costs. Asymmetric bioreduction of prochiral ketone using purified enzymes or whole-cell system is more attractive method for production of optical pure (R)-PE. In our previous report, a novel NADPH-dependent short chain dehydrogenase/reductase (SM_SDR) from Serratia marcescens BCRC10948 was used for the reduction of 1-(3-hydroxyphenyl)-2-(methylamino)ethanone (HPME) to (R)-PE with more than 99% enantiomeric excess. However, a low conversion yield (51.06%) and productivity [0.57 (mmole/l．h)] limited the application of this method in industrial processes. In this study, the crystallographic structure of SM_SDR was used as a structural basis for site-directed mutagenesis to identify SM_SDR mutants that either enhance catalytic activities or change their cofactor preference from NADPH to NADH. A total of 35 mutants were constructed and then transformed into BL21(DE3) for whole cell conversion. Results showed that no significant change in conversion efficiency was observed for most of the mutants. However, mutational changes at Y40D, F98L, T193N, T193D, M195A, N196S, L206Y and N185Q resulted in completely lost of their enzymatic activities. Mutations at position F202 were designed to enhance the binding affinity with HPMAE or expand the binding pocket. Results revealed that F202A, F202K, F202R, F202D, F202E displayed at least 1.3-fold increase in (R)-PE production when using 10 mM HPMAE as substrate. The effect of different carbon sources and HPMAE concentrations on whole-cell bioconversion were evaluated. Results showed that 2% glycerol and 40 mM HPMAE, respectively, are the best conditions for (R)-PE production. However, the variants F202A, F202K, F202R, F202D and F202E have 1.84, 2.02, 1.53, 1.64 and 1.87 fold increase in (R)-PE production than wild type SM_SDR at 50 mM HPMAE at 48 h. Homologous expression of the cloned wild type and F202A SM_SDR in S. marcescens BCRC10948 were also carried out to enhance the (R)-PE production. When 5% of the recombinant cells was used for bioconversion, the wild type and F202A produced 23.82±0.75 mM and 38.52±1.84 mM of (R)-PE, respectively, after 12 h reaction using 50 mM HPMAE as substrate. The (R)-PE production can increase to 43.79±2.57 mM when 10% of the F202A but not for wild type recombinant cells were used for reaction. Moreover, the F202A recombinant cells can be recycled for 6 times and remaining over 95% conversion efficiency when compared to the first cycle. The fed-batch culture was performed to further improve the (R)-PE production, results revealed that (R)-PE production was dramatically increased to 134.09±2.97 mM with conversion yield of 89.3% and productivity of 2.97 mmol/l．h.

碩士
國立清華大學
分子與細胞生物研究所
103
(R)-Phenylephrine [(R)-PE] is an α1-adrenergic receptor agonist widely used as a nasal decongestant and a cardiac agent without major side effects opposing to other adrenergic drugs such as ephedrine. In addition, the current mass-production procedure usually consists of (S) chiral form (50%). In an end to increase the specificity, a bio-catalytic transformation procedure using a novel short-chain dehydrogenase/reductase (SDR) from Serratia marcescens BCRC 10948 (Peng, G. J. et al.) to convert 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) into an enantioseletive (R)-PE (more than 99%) has been attempted. However, this method performs relatively low conversion yield and productivity. In this study, we aim to determine the crystallographic structure of SmSDR as a structural basis to engineer high-activity SmSDR variants. Here, we report the 1.47 Å atomic-resolution apo-form structure. A liganded complex was built using Discovery Studio. Several mutants were predicted and characterized based on a structure-guided approach. A double mutant SmSDR-F98YF202L was found to display the highest activity. Furthermore, this mutant demonstrated a much higher conversion yield and productivity in the whole-cell assay, suggesting a valuable engineered variant for pharmaceutical applications.