Characterization of short-chain dehydrogenase/reductase homologues of Escherichia coli (YdfG) and Saccharomyces cerevisiae (YMR226C)

Short-chain dehydrogenase/reductase homologues from Escherichia coli (YdfG) and Saccharomyces cerevisiae (YMR226C) show high sequence similarity to serine dehydrogenase from Agrobacterium tumefaciens. We cloned each gene encoding YdfG and YMR226C into E. coli JM109 and purified them to homogeneity from the E. coli clones. YdfG and YMR226C consist of four identical subunits with a molecular mass of 27 and 29 kDa, respectively. Both enzymes require NADP(+) as a coenzyme and use L-serine as a substrate. Both enzymes show maximum activity at about pH 8.5 for the oxidation of L-serine. They also catalyze the oxidation of D-serine, L-allo-threonine, D-threonine, 3-hydroxyisobutyrate, and 3-hydroxybutyrate. The k(cat)/K(m) values of YdfG for L-serine, D-serine, L-allo-threonine, D-threonine, L-3-hydroxyisobutyrate, and D-3-hydroxyisobutyrate are 105, 29, 199, 109, 67, and 62 M(-1) s(-1), and those of YMR226C are 116, 110, 14600, 7540, 558, and 151 M(-1) s(-1), respectively. Thus, YdfG and YMR226C are NADP(+)-dependent dehydrogenases acting on 3-hydroxy acids with a three- or four-carbon chain, and L-allo-threonine is the best substrate for both enzymes.     

Enantioselective synthesis of (S)-phenylephrine by recombinant Escherichia coli cells expressing the short-chain dehydrogenase/reductase gene from Serratia quinivorans BCRC 14811

BackgroundAn amino alcohol dehydrogenase gene (RE_AADH) from Rhodococcus erythropolis BCRC 10909 has been used for the conversion of 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) to (S)-phenylephrine [(S)-PE]. However RE_AADH uses NADPH as cofactor, and only limited production of (S)-PE from HPMAE is achieved.MethodsA short-chain dehydrogenase/reductase gene (SQ_SDR) from Serratia quinivorans BCRC 14811 was expressed in Escherichia coli BL21 (DE3) for the conversion of HPMAE to (S)-PE.ResultsThe SQ_SDR enzyme was capable of converting 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 E. coli 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 RE_AADH as the biocatalyst.ConclusionThe SQ_SDR enzyme with its high catalytic activity and strong preference for NADH as a cofactor provided a significant advantage in bioreduction.     

Characterization of human DHRS4: an inducible short-chain dehydrogenase/reductase enzyme with 3beta-hydroxysteroid dehydrogenase activity

Human DHRS4 is a peroxisomal member of the short-chain dehydrogenase/reductase superfamily, but its enzymatic properties, except for displaying NADP(H)-dependent retinol dehydrogenase/reductase activity, are unknown. We show that the human enzyme, a tetramer composed of 27 kDa subunits, is inactivated at low temperature without dissociation into subunits. The cold inactivation was prevented by a mutation of Thr177 with the corresponding residue, Asn, in cold-stable pig DHRS4, where this residue is hydrogen-bonded to Asn165 in a substrate-binding loop of other subunit. Human DHRS4 reduced various aromatic ketones and α-dicarbonyl compounds including cytotoxic 9,10-phenanthrenequinone. The overexpression of the peroxisomal enzyme in cultured cells did not increase the cytotoxicity of 9,10-phenanthrenequinone. While its activity towards all-trans-retinal was low, human DHRS4 efficiently reduced 3-keto-C₁₉/C₂₁-steroids into 3β-hydroxysteroids. The stereospecific conversion to 3β-hydroxysteroids was observed in endothelial cells transfected with vectors expressing the enzyme. The mRNA for the enzyme was ubiquitously expressed in human tissues and several cancer cells, and the enzyme in HepG2 cells was induced by peroxisome-proliferator-activated receptor α ligands. The results suggest a novel mechanism of cold inactivation and role of the inducible human DHRS4 in 3β-hydroxysteroid synthesis and xenobiotic carbonyl metabolism.     

Molecular determinants for the stereospecific reduction of 3-ketosteroids and reactivity towards all-trans-retinal of a short-chain dehydrogenase/reductase (DHRS4)

DHRS4, a member of the short-chain dehydrogenase/reductase superfamily, reduces all-trans-retinal and xenobiotic carbonyl compounds. Human DHRS4 differs from other animal enzymes in kinetic constants for the substrates, particularly in its low reactivity to retinoids. We have found that pig, rabbit and dog DHRS4s reduce benzil and 3-ketosteroids into S-benzoin and 3α-hydroxysteroids, respectively, in contrast to the stereoselectivity of human DHRS4 which produces R-benzoin and 3β-hydroxysteroids. Among substrate-binding residues predicted from the crystal structure of pig DHRS4, F158 and L161 in the animal DHRS4 are serine and phenylalanine, respectively, in the human enzyme. Double mutation (F158S/L161F) of pig DHRS4 led to an effective switch of its substrate affinity and stereochemistry into those similar to human DHRS4. The roles of the two residues in determining the stereospecificity in 3-ketosteroid reduction were confirmed by reverse mutation (S158F/F161L) in the human enzyme. The stereochemical control was evaluated by comparison of the 3D models of pig wild-type and mutant DHRS4s with the modeled substrates. Additional mutation of T177N into the human S158F/F161L mutant resulted in almost complete kinetic conversion into a pig DHRS4-type form, suggesting a role of N177 in forming the substrate-binding cavity through an intersubunit interaction in pig and other animal DHRS4s, and explaining why the human enzyme shows low reactivity towards retinoids.     

Semi-quantitative colony immunoassay for determining and optimizing protein expression in Saccharomyces cerevisiae and Escherichia coli

This work describes a quick semi-quantitative colony immunoassay (QSCI) method for immunoblot detection of intracellularly expressed proteins in both yeast and bacterial cells. After induction of protein expression, only 4.5 h is required for cell breakage, protein detection, and data analysis. This protocol was used to screen and unambiguously identify Saccharomyces cerevisiae cells efficiently overexpressing glutathione S-transferase (GST)-tagged Yih1 in addition to cells expressing the myc-tagged large 297-kDa Gcn1 protein. In addition, the method was used to identify Escherichia coli cells efficiently expressing His6-tagged Yih1 and a GST-tagged Gcn1 fragment, respectively. The protocol allows the use of both epitope-specific and protein-specific antibodies. The same colony immunoassay can also be used to determine the minimal concentration of inducing agent sufficient for induction of optimal protein expression (e.g., galactose for yeast, isopropyl β-d-1-thiogalactopyranoside [IPTG] for E. coli). To our knowledge, this is the first report on a rapid low-cost procedure that allows the calibration of inducing agent on solid medium.     

At5g50600 encodes a member of the short-chain dehydrogenase reductase superfamily with 11beta- and 17beta-hydroxysteroid dehydrogenase activities associated with Arabidopsis thaliana seed oil bodies

In a previous work, we presented evidence for the presence of a protein encoded by At5g50600 in oil bodies (OBs) from Arabidopsis thaliana [P. Jolivet, E. Roux, S. D'Andrea, M. Davanture, L. Negroni, M. Zivy, T. Chardot, Protein composition of oil bodies in Arabidopsis thaliana ecotype WS, Plant Physiol. Biochem. 42 (2004) 501-509]. Using specific antibodies and proteomic techniques, we presently confirm the existence of this protein, which is a member of the short-chain steroid dehydrogenase reductase superfamily. We have measured its activity toward various steroids (cholesterol, dehydroepiandrosterone, cortisol, corticosterone, estradiol, estrone) and NAD(P)(H), either within purified OBs or as a purified bacterially expressed chimera. Both enzymatic systems (OBs purified from A. thaliana seeds as well as the chimeric enzyme) exhibited hydroxysteroid dehydrogenase (HSD) activity toward estradiol (17beta-hydroxysteroid) with NAD+ or NADP+, NADP+ being the preferred cofactor. Low levels of activity were observed with cortisol or corticosterone (11beta-hydroxysteroids), but neither cholesterol nor DHEA (3beta-hydroxysteroids) were substrates, whatever the cofactor used. Similar activity profiles were found for both enzyme sources. Purified OBs were found to be also able to catalyze estrone reduction (17beta-ketosteroid reductase activity) with NADPH. The enzyme occurring in A. thaliana OBs can be classified as a NADP+-dependent 11beta-,17beta-hydroxysteroid dehydrogenase/17beta-ketosteroid reductase. This enzyme probably corresponds to AtHSD1, which is encoded by At5g50600. However, its physiological role and substrates still remain to be determined.     

Identification and characterization of retinoid-active short-chain dehydrogenases/reductases in Drosophila melanogaster

Background

In chordates, retinoid metabolism is an important target of short-chain dehydrogenases/reductases (SDRs). It is not known whether SDRs play a role in retinoid metabolism of protostomes, such as Drosophila melanogaster.

Methods

Drosophila genome was searched for genes encoding proteins with ∼ 50% identity to human retinol dehydrogenase 12 (RDH12). The corresponding proteins were expressed in Sf9 cells and biochemically characterized. Their phylogenetic relationships were analyzed using PHYLIP software.

Results

A total of six Drosophila SDR genes were identified. Five of these genes are clustered on chromosome 2 and one is located on chromosome X. The deduced proteins are 300 to 406 amino acids long and are associated with microsomal membranes. They recognize all-trans-retinaldehyde and all-trans-3-hydroxyretinaldehyde as substrates and prefer NADPH as a cofactor. Phylogenetically, Drosophila SDRs belong to the same branch of the SDR superfamily as human RDH12, indicating a common ancestry early in bilaterian evolution, before a protostome–deuterostome split.

Conclusions

Similarities in the substrate and cofactor specificities of Drosophila versus human SDRs suggest conservation of their function in retinoid metabolism throughout protostome and deuterostome phyla.

General significance

The discovery of Drosophila retinaldehyde reductases sheds new light on the conversion of β-carotene and zeaxantine to visual pigment and provides a better understanding of the evolutionary roots of retinoid-active SDRs.     

Cysteine-125 is the catalytic residue of Saccharomyces cerevisiae free methionine-R-sulfoxide reductase

Free methionine-R-sulfoxide reductase (fRMsr) is a new type of methionine sulfoxide reductase that catalyzes the reduction of free methionine-R-sulfoxide to methionine. This enzyme cannot reduce oxidized methionine residues in proteins. While three Cys residues, Cys-91, Cys-101 and Cys-125, have been demonstrated to be involved in the catalysis by Saccharomyces cerevisiae fRMsr, their specific functions have not been fully established. In this work, we performed in vivo growth complementation experiments using S. cerevisiae cells lacking all three known methionine sulfoxide reductases. Cells containing a C125S construct, in which Cys-125 in fRMsr was replaced with Ser, did not grow in methionine sulfoxide medium, whereas cells containing C91S, C101S, or C91/101S constructs could grow in this medium. In addition, when assayed with thioredoxin and glutaredoxin reduction systems, the C125S form was inactive, whereas C91S and C101S had 1-2% and 9-10%, respectively, of the activity of the wild-type fRMsr. These data show that Cys-125 is the catalytic residue in fRMsr.     

The Saccharomyces cerevisiae succinate dehydrogenase does not require heme for ubiquinone reduction

The coupling of succinate oxidation to the reduction of ubiquinone by succinate dehydrogenase (SDH) constitutes a pivotal reaction in the aerobic generation of energy. In Saccharomyces cerevisiae, SDH is a tetramer composed of a catalytic dimer comprising a flavoprotein subunit, Sdh1p and an iron-sulfur protein, Sdh2p and a heme b-containing membrane-anchoring dimer comprising the Sdh3p and Sdh4p subunits. In order to investigate the role of heme in SDH catalysis, we constructed an S. cerevisiae strain expressing a mutant enzyme lacking the two heme axial ligands, Sdh3p His-106 and Sdh4p Cys-78. The mutant enzyme was characterized for growth on a non-fermentable carbon source, for enzyme assembly, for succinate-dependent quinone reduction and for its heme b content. Replacement of both Sdh3p His-106 and Sdh4p Cys-78 with alanine residues leads to an undetectable level of cytochrome b₅₆₂. Although enzyme assembly is slightly impaired, the apocytochrome SDH retains a significant ability to reduce quinone. The enzyme has a reduced affinity for quinone and its catalytic efficiency is reduced by an order of magnitude. To better understand the effects of the mutations, we employed atomistic molecular dynamic simulations to investigate the enzyme's structure and stability in the absence of heme. Our results strongly suggest that heme is not required for electron transport from succinate to quinone nor is it necessary for assembly of the S. cerevisiae SDH.     

Computational analysis of phenotypic space in heterologous polyketide biosynthesis—Applications to Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae

Polyketides represent a class of natural product small molecules with an impressive range of medicinal activities. In order to improve access to therapeutic polyketide compounds, heterologous metabolic engineering has been applied to transfer polyketide genetic pathways from often fastidious native hosts to more industrially-amenable heterologous hosts such as Escherichia coli, Saccharomyces cerevisiae, or Streptomyces coelicolor. Efforts thus far have resulted in titers either inferior to the native host and significantly below the theoretical yield, emphasizing the need to computationally investigate and engineer the interaction between native and heterologous metabolism for the improved production of heterologous polyketide compounds. In this work, we applied flux balance analysis on genome-scale models to simulate cellular metabolism and 6-deoxyerythronolide B (the cyclized polyketide precursor to erythromycin) production in three common heterologous hosts (E. coli, Bacillus subtilis, and S. cerevisiae) under a variety of carbon-source and medium compositions. We then undertook minimization of metabolic adjustment optimization to identify single and double gene-knockouts that resulted in increased polyketide production while maintaining cellular growth. For the production of 6-deoxyerythronolide B, the results suggest B. subtilis and E. coli are better heterologous hosts when compared to S. cerevisiae and that several single and multiple gene-knockout mutants are computationally predicted to improve specific production, in some cases, over 25-fold.     

Purification and characterization of Put1p from Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the PUT1 and PUT2 genes are required for the conversion of proline to glutamate. The PUT1 gene encodes Put1p, a proline dehydrogenase (PRODH) enzyme localized in the mitochondrion. Put1p was expressed and purified from Escherichia coli and shown to have a UV-visible absorption spectrum that is typical of a bound flavin cofactor. A K_m value of 36 mM proline and a k_cat = 27 s⁻¹ were determined for Put1p using an artificial electron acceptor. Put1p also exhibited high activity using ubiquinone-1 (CoQ₁) as an electron acceptor with a k_cat = 9.6 s⁻¹ and a K_m of 33 μM for CoQ₁. In addition, knockout strains of the electron transfer flavoprotein (ETF) homolog in S. cerevisiae were able to grow on proline as the sole nitrogen source demonstrating that ETF is not required for proline utilization in yeast.     

Enzymatic synthesis of an orlistat intermediate using a mutant short-chain dehydrogenase from Novosphingobium aromaticivorans

Methyl (R)-3-hydroxytetradeconoate ((R)-MHOT) is a crucial chiral intermediate for the chemical synthesis of the anti-obesity drug, orlistat. Here, (R)-MHOT was prepared from methyl 3-oxotetradecanoate (MOT) using a mutant of the short-chain dehydrogenase/reductase (SDR) from Novosphingobium aromaticivorans (NaSDR). Mutant NaSDR-G145A/I199L had a 3.23 times greater k_cat value than that of wild type toward MOT. The conditions for the expression of recombinant NaSDR-G145A/I199L were further investigated and obtained cells were used for gram-scale preparation of (R)-MHOT with 50 g/L of MOT. The target product was extracted and confirmed by gas chromatography; the enantiomeric excess value of (R)-MHOT was 99.0 %. Molecular docking analysis was used to reveal the molecular basis of the enhanced catalytic activity of NaSDR-G145A/I199L; NaSDR-G145A/I199L presented a more effective docking posture than NaSDR. This is the first reported use of SDR for preparing (R)-MHOT via the reduction of MOT. Our study provides a foundation for greener preparation of (R)-MHOT.     

Comparison of the genetic activity of aflatoxins B1 and G1 in Escherichia coli and Saccharomyces cerevisiae

The ability of aflatoxins B₁ and G₁ to induce back mutations to arg⁺ in Escherichia coli K-12/343/113 was compared with induction of mitotic gene conversion to ade⁺ in the diploid yeast strain Saccharomyces cerevisiae D4, ade₂^?. In analogy to previous results with other microorganisms, the compounds were not genetically active per se, indicating that under the experimental conditions employed none of the tester strains were able to activate the compounds to mutagenic products.In experiments using liver homegenates (S-9 fraction) of male Golden Syrian hamsters previously treated with phenobarbital, aflatoxin B₁ exhibited strong genetic activity both in E. coli and in S. cerevisiae, whereas the mutagenic activity of aflatoxin G₁ was markedly lower and could be detected only in the E. coli tester strain. These results correlate the findings that aflatoxin G₁ is a less potent carcinogen and mutagen than aflatoxin B₁.     

Exploring the active site of yeast xylose reductase by site-directed mutagenesis of sequence motifs characteristic of two dehydrogenase/reductase family types

Starting from a common tyrosine, yeast xylose reductases (XRs) contain two conserved sequence motifs corresponding to the catalytic signatures of single-domain reductases/epimerases/dehydrogenases (Tyrⁿ-(X)₃-Lysⁿ⁺⁴) and aldo/keto reductases (AKRs) (Tyrⁿ-(X)₂₈-Lysⁿ⁺²⁹). Tyr⁵¹, Lys⁵⁵ and Lys⁸⁰ of XR from Candida tenuis were replaced by site-directed mutagenesis. The purified Tyr⁵¹→ Phe and Lys⁸⁰→Ala mutants showed turnover numbers and catalytic efficiencies for NADH-dependent reduction of -xylose between 2500- and 5000-fold below wild-type levels, suggesting a catalytic role of both residues. Replacing Lys⁵⁵ by Asn, a substitution found in other AKRs, did not detectably affect binding of coenzymes, and enzymatic catalysis to carbonyl/alcohol interconversion. The contribution of Tyr⁵¹ to rate enhancement of aldehyde reduction conforms with expectations for the general acid catalyst of the enzymatic reaction.     

Excision of the oxidatively formed 5-hydroxyhydantoin and 5-hydroxy-5-methylhydantoin pyrimidine lesions by Escherichia coli and Saccharomyces cerevisiae DNA N-glycosylases

Background

(5R?) and (5S?) diastereomers of 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd) are major oxidation products of 2′-deoxycytidine and thymidine respectively. If not repaired, when present in cellular DNA, these base lesions may be processed by DNA polymerases that induce mutagenic and cell lethality processes.

Methods

Synthetic oligonucleotides that contained a unique 5-hydroxyhydantoin (5-OH-Hyd) or 5-hydroxy-5-methylhydantoin (5-OH-5-Me-Hyd) nucleobase were used as probes for repair studies involving several E. coli, yeast and human purified DNA N-glycosylases. Enzymatic reaction mixtures were analyzed by denaturing polyacrylamide gel electrophoresis after radiolabeling of DNA oligomers or by MALDI-TOF mass spectrometry measurements.

Results

In vitro DNA excision experiments carried out with endo III, endo VIII, Fpg, Ntg1 and Ntg2, show that both base lesions are substrates for these DNA N-glycosylases. The yeast and human Ogg1 proteins (yOgg1 and hOgg1 respectively) and E. coli AlkA were unable to cleave the N-glycosidic bond of the 5-OH-Hyd and 5-OH-5-Me-Hyd lesions. Comparison of the k_cat/K_m ratio reveals that 8-oxo-7,8-dihydroguanine is only a slightly better substrate than 5-OH-Hyd and 5-OH-5-Me-Hyd. The kinetic results obtained with endo III indicate that 5-OH-Hyd and 5-OH-5-Me-Hyd are much better substrates than 5-hydroxycytosine, a well known oxidized pyrimidine substrate for this DNA N-glycosylase.

Conclusions

The present study supports a biological relevance of the base excision repair processes toward the hydantoin lesions, while the removal by the Fpg and endo III proteins are effected at better or comparable rates to that of the removal of 8-oxoGua and 5-OH-Cyt, two established cellular substrates.

General significance

The study provides new insights into the substrate specificity of DNA N-glycosylases involved in the base excision repair of oxidized bases, together with complementary information on the biological role of hydantoin type lesions.     

Activation of translation via reduction by thioredoxin-thioredoxin reductase in Saccharomyces cerevisiae

Previously we reported that in vitro translation activity in extracts of Saccharomyces cerevisiae was stimulated by dithiothreitol (DTT) and further increased by the addition of thioredoxin (TRX1) [Choi, S.K. (2007) Thioredoxin-mediated regulation of protein synthesis by redox in Saccharomyces cerevisiae. Kor. J. Microbiol. Biotechnol. 35, 36-40]. To identify the pathway affecting translation, we cloned and purified thioredoxin reductase 1 (TRR1), thioredoxin reductase 2 (TRR2), glutaredoxin 1 (GRX1) and glutaredoxin reductase 1 (GLR1) as fusion proteins. Thioredoxin-mediated activation of translation was more effectively stimulated by NADPH or NADH than by DTT. Moreover, addition of TRR1 led to a further increase of translation in the presence of thioredoxin plus NADPH. These findings indicate that redox control via the thioredoxin-thioredoxin reductase system plays an important role in the regulation of translation.     

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.     

Expression of Aeromonas caviae ST pyruvate dehydrogenase complex components mediate tellurite resistance in Escherichia coli

Potassium tellurite (K₂TeO₃) is harmful to most organisms and specific mechanisms explaining its toxicity are not well known to date. We previously reported that the lpdA gene product of the tellurite-resistant environmental isolate Aeromonas caviae ST is involved in the reduction of tellurite to elemental tellurium. In this work, we show that expression of A. caviae ST aceE, aceF, and lpdA genes, encoding pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase, respectively, results in tellurite resistance and decreased levels of tellurite-induced superoxide in Escherichia coli. In addition to oxidative damage resulting from tellurite exposure, a metabolic disorder would be simultaneously established in which the pyruvate dehydrogenase complex would represent an intracellular tellurite target. These results allow us to widen our vision regarding the molecular mechanisms involved in bacterial tellurite resistance by correlating tellurite toxicity and key enzymes of aerobic metabolism.