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Sakayu Shimizu - One of the best experts on this subject based on the ideXlab platform.
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cloning overexpression and mutagenesis of the sporobolomyces salmonicolor aku4429 gene encoding a new Aldehyde Reductase which catalyzes the stereoselective reduction of ethyl 4 chloro 3 oxobutanoate to ethyl s 4 chloro 3 hydroxybutanoate
Applied and Environmental Microbiology, 1999Co-Authors: Keiko Kita, Takanobu Fukura, Kohichi Nakase, Kenji Okamoto, Hideshi Yanase, Michihiko Kataoka, Sakayu ShimizuAbstract:Aldehyde Reductase (EC 1.1.1.2), aldose Reductase (EC 1.1.1.21), and carbonyl Reductase (EC 1.1.1.184) catalyze NADPH-dependent reduction of a variety of carbonyl compounds and are widely distributed in mammalian and plant tissues. These enzymes are members of the aldo-keto Reductase superfamily (4, 8); however, their physiological functions are not well understood. The amino acid sequences of aldose Reductases and Aldehyde Reductases exhibit significant levels of similarity, but the amino acid sequences of carbonyl Reductases do not (32). In previous papers, we described purification and characterization of three NADPH-dependent Aldehyde Reductases (ARI, ARII, and ARIII) of the red yeast Sporobolomyces salmonicolor AKU4429 (9, 14, 34). ARI is the most abundant Aldehyde Reductase in this yeast and catalyzes asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (4-COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate (4-CHBE) {enantiomeric excess for (R) = [(R − S)/(R + S] × 100 and vice versa}, a promising chiral building block for organic synthesis. In contrast, ARII is produced in considerably smaller amounts but reduces 4-COBE to the (S) enantiomer (92.7% enantiomeric excess), which is also a useful chiral building block for chemical synthesis of pharmaceuticals. In addition to the stereoselectivity of activity against 4-COBE, the N-terminal amino acid sequences of these two Aldehyde Reductases are quite different. Based on the amino acid sequence deduced from the cDNA sequence, ARI belongs to the aldo-keto Reductase superfamily (13). Recently, an NADPH-dependent Aldehyde Reductase (S1), which reduces 4-COBE to the (S) enantiomer (100% enantiomeric excess), was purified from Candida magnoliae AKU4643 (31). The substrate specificities, subunit structures, and N-terminal amino acid sequences of ARII and S1 are not similar. This indicates that the two enzymes belong to the different groups. In this study, we cloned and analyzed a cDNA clone of the Aldehyde Reductase gene (ARII) in order to compare the catalytic mechanisms of ARI and ARII and to understand the molecular basis of the stereospecific reduction of 4-COBE.
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stereoselective reduction of ethyl 4 chloro 3 oxobutanoate by escherichia coli transformant cells coexpressing the Aldehyde Reductase and glucose dehydrogenase genes
Applied Microbiology and Biotechnology, 1999Co-Authors: Michihiko Kataoka, Keiko Kita, Hideshi Yanase, Masaru Wada, Hiroshi Kawabata, Kazuhiko Yamamoto, Sakayu ShimizuAbstract:The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate [(R)-CHBE] using Escherichia coli cells, which coexpress both the Aldehyde Reductase gene from Sporobolomyces salmonicolor and the glucose dehydrogenase (GDH) gene from Bacillus megaterium as a catalyst was investigated. In an organic solvent-water two-phase system, (R)-CHBE formed in the organic phase amounted to 1610 mM (268 mg/ml), with a molar yield of 94.1% and an optical purity of 91.7% enantiomeric excess. The calculated turnover number of NADP+ to CHBE formed was 13 500 mol/mol. Since the use of E. coli JM109 cells harboring pKAR and pACGD as a catalyst is simple, and does not require the addition of GDH or the isolation of the enzymes, it is highly advantageous for the practical synthesis of (R)-CHBE.
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purification and characterization of an Aldehyde Reductase from candida magnoliae
Journal of Molecular Catalysis B-enzymatic, 1999Co-Authors: Masaru Wada, Michihiko Kataoka, Hiroshi Kawabata, Yoshihiko Yasohara, Noriyuki Kizaki, Junzo Hasegawa, Sakayu ShimizuAbstract:Abstract An NADPH-dependent Aldehyde Reductase was purified to homogeneity from Candida magnoliae AKU4643 through four steps, including Blue-Sepharose affinity chromatography. The relative molecular mass of the enzyme was estimated to be 33,000 on high performance gel-permeation chromatography and 35,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The substrate specificity of the enzyme was broad and resembled those of other aldo–keto Reductases. The partial amino acid sequences of the enzyme showed that it belongs to the aldo–keto Reductase superfamily. The enzyme catalyzed the stereoselective reduction of ethyl 4-chloro-3-oxobutanoate to the corresponding ( R )-alcohol, with a 100% enantiomeric excess. The enzyme was inhibited by 1 mM quercetin, CuSO 4 , ZnSO 4 and HgCl 2 . The thermostability of the enzyme was inferior to that of the ( S )-CHBE-producing enzyme from the same strain.
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enzymatic production of ethyl r 4 chloro 3 hydroxybutanoate asymmetric reduction of ethyl 4 chloro 3 oxobutanoate by an escherichia coli transformant expressing the Aldehyde Reductase gene from yeast
Applied Microbiology and Biotechnology, 1997Co-Authors: Michihiko Kataoka, Keiko Kita, Hideshi Yanase, Masaru Wada, Hiroshi Kawabata, Kazuhiko Yamamoto, L P S Rohani, Sakayu ShimizuAbstract:The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate (CHBE) using Escherichia coli JM109 (pKAR) cells expressing the Aldehyde Reductase gene from Sporobolomyces salmonicolor AKU4429 as a catalyst was studied. The reduction required NADP+, glucose and glucose dehydrogenase for NADPH regeneration. In an aqueous system, the substrate was unstable, and inhibition of the reaction by the substrate was also observed. Efficient conversion of COBE to (R)-CHBE with a satisfactory enantiomeric excess (ee) was attained on incubation with transformant cells in an n-butyl acetate/water two-phase system containing the above NADPH-regeneration system. Under the optimized conditions, with the periodical addition of COBE, glucose and glucose dehydrogenase, the (R)-CHBE yield reached 1530 mM (255 mg/ml) in the organic phase, with a molar conversion yield of 91.1% and an optical purity of 91% ee. The calculated turnover of NADP+, based on the amounts of NADP+ added and CHBE formed, was about 5100 mol/mol.
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cloning of the Aldehyde Reductase gene from a red yeast sporobolomyces salmonicolor and characterization of the gene and its product
Applied and Environmental Microbiology, 1996Co-Authors: Keiko Kita, Hideshi Yanase, Michihiko Kataoka, K Matsuzaki, T Hashimoto, Nobuo Kato, Maxey C M Chung, Sakayu ShimizuAbstract:An NADPH-dependent Aldehyde Reductase (ALR) isolated from a red yeast, Sporobolomyces salmonicolor, catalyzes the reduction of a variety of carbonyl compounds. To investigate its primary structure, we cloned and sequenced the cDNA coding for ALR. The Aldehyde Reductase gene (ALR) comprises 969 bp and encodes a polypeptide of 35,232 Da. The deduced amino acid sequence showed a high degree of similarity to other members of the aldo-keto Reductase superfamily. Analysis of the genomic DNA sequence indicated that the ALR gene was interrupted by six introns (two in the 5' noncoding region and four in the coding region). Southern hybridization analysis of the genomic DNA from S. salmonicolor indicated that there was one copy of the gene. The ALR gene was expressed in Escherichia coli under the control of the tac promoter. The enzyme expressed in E. coli was purified to homogeneity and showed the same catalytic properties as did the enzyme from S. salmonicolor.
John D Hayes - One of the best experts on this subject based on the ideXlab platform.
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characterization of the rat aflatoxin b1 Aldehyde Reductase gene akr7a1 structure and chromosomal localization of akr7a1 as well as identification of antioxidant response elements in the gene promoter
Carcinogenesis, 2003Co-Authors: Elizabeth M Ellis, Cara M Slattery, John D HayesAbstract:Rat aflatoxin B1 Aldehyde Reductase (called AFAR1 or AKR7A1) is a member of the aldo-keto Reductase 7 family, which metabolizes the environmental carcinogen aflatoxin B1. The expression of this enzyme is markedly increased in rat liver by cancer chemopreventive agents, many of which are believed to regulate gene expression through the antioxidant response element (ARE). In order to understand how this gene is regulated, two overlapping genomic clones have been isolated that contain most of the coding region for the enzyme; together they encompass 14.1 kb of DNA. Characterization of these clones has shown that rat AFAR1 is ~8 kb long and comprises seven exons and six introns. The seven exons are between 97 and 380 bp in size. The introns range in size from 194 bp to ~2.9 kb. Fluorescent in situ hybridization localized AFAR1 to rat chromosome 5q36.5, a region that is syntenic with human chromosome 1p35-1p36.1 where AKR7A2 resides. The transcriptional start site (TSS) was determined, using 5 0 -rapid amplification of cDNA ends, to be an A nucleotide 73 bp upstream from the ATG initiation codon. The 5 0 -flanking region of AFAR1 was isolated by polymerase chain reaction-based genome walking, and resulted in the isolation of ~900 bp of genomic DNA upstream from the TSS. Use of a gene expression reporter assay demonstrated that this cloned 5 0 -flanking region of AFAR1 could support transcription in the rat liver 34 (RL34) epithelial cell line. Within this upstream region of the promoter, a substantial number of sequences were found that are closely similar, but not identical, to the ‘core’ ARE consensus sequence. Between nucleotides ˇ810 and ˇ106 bp from the TSS 16 ARErelated sequences were identified. Four of these putative enhancers lay betweenˇ389 andˇ355 bp, and the motif 5 0 -GAGTGAG-3 0 was repeated three times within the 35 bp
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chemoprevention of aflatoxin b1 hepatocarcinogenesis by coumarin a natural benzopyrone that is a potent inducer of aflatoxin b1 Aldehyde Reductase the glutathione s transferase a5 and p1 subunits and nad p h quinone oxidoReductase in rat liver
Cancer Research, 2000Co-Authors: Vincent P Kelly, G E Neal, David J Judah, Elizabeth M Ellis, Margaret M Manson, Simon A Chanas, Graeme J Moffat, Ronald Mcleod, John D HayesAbstract:Structurally diverse compounds can confer resistance to aflatoxin B1 (AFB1) hepatocarcinogenesis in the rat. Treatment with either phytochemicals [benzyl isothiocyanate, coumarin (CMRN), or indole-3-carbinol] or synthetic antioxidants and other drugs (butylated hydroxyanisole, diethyl maleate, ethoxyquin, beta-naphthoflavone, oltipraz, phenobarbital, or trans-stilbene oxide) has been found to increase hepatic aldo-keto Reductase activity toward AFB1-diAldehyde and glutathione S-transferase (GST) activity toward AFB1-8,9-epoxide in both male and female rats. Under the conditions used, the natural benzopyrone CMRN was a major inducer of the AFB1 Aldehyde Reductase (AFAR) and the aflatoxin-conjugating class-alpha GST A5 subunit in rat liver, causing elevations of between 25- and 35-fold in hepatic levels of these proteins. Induction was not limited to AFAR and GSTA5: treatment with CMRN caused similar increases in the amount of the class-pi GST P1 subunit and NAD(P)H: quinone oxidoReductase in rat liver. Immunohistochemistry demonstrated that the overexpression of AFAR, GSTA5, GSTP1, and NAD(P)H:quinone oxidoReductase affected by CMRN is restricted to the centrilobular (periacinar) zone of the lobule, sometimes extending almost as far as the portal tract. This pattern of induction was also observed with ethoxyquin, oltipraz, and trans-stilbene oxide. By contrast, induction of these proteins by beta-naphthoflavone and diethyl maleate was predominantly periportal. Northern blotting showed that induction of these phase II drug-metabolizing enzymes by CMRN was accompanied by similar increases in the levels of their mRNAs. To assess the biological significance of enzyme induction by dietary CMRN, two intervention studies were performed in which the ability of the benzopyrone to inhibit either AFB1-initiated preneoplastic nodules (at 13 weeks) or AFB1-initiated liver tumors (at 50 weeks) was investigated. Animals pretreated with CMRN for 2 weeks prior to administration of AFB1, and with continued treatment during exposure to the carcinogen for a further 11 weeks, were protected completely from development of hepatic preneoplastic lesions by 13 weeks. In the longer-term dietary intervention, treatment with CMRN before and during exposure to AFB1 for a total of 24 weeks was found to significantly inhibit the number and size of tumors that subsequently developed by 50 weeks. These data suggest that consumption of a CMRN-containing diet provides substantial protection against the initiation of AFB1 hepatocarcinogenesis in the rat.
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chemoprevention of aflatoxin b1 hepatocarcinogenesis by coumarin a natural benzopyrone that is a potent inducer of aflatoxin b1 Aldehyde Reductase the glutathione s transferase a5 and p1 subunits and nad p h quinone oxidoReductase in rat liver
Cancer Research, 2000Co-Authors: Vincent P Kelly, G E Neal, David J Judah, Elizabeth M Ellis, Margaret M Manson, Simon A Chanas, Graeme J Moffat, Ronald Mcleod, John D HayesAbstract:Structurally diverse compounds can confer resistance to aflatoxin B 1 (AFB 1 ) hepatocarcinogenesis in the rat. Treatment with either phytochemicals [benzyl isothiocyanate, coumarin (CMRN), or indole-3-carbinol] or synthetic antioxidants and other drugs (butylated hydroxyanisole, diethyl maleate, ethoxyquin,β -naphthoflavone, oltipraz, phenobarbital, or trans -stilbene oxide) has been found to increase hepatic aldo-keto Reductase activity toward AFB 1 -diAldehyde and glutathione S -transferase (GST) activity toward AFB 1 -8,9-epoxide in both male and female rats. Under the conditions used, the natural benzopyrone CMRN was a major inducer of the AFB 1 Aldehyde Reductase (AFAR) and the aflatoxin-conjugating class-α GST A5 subunit in rat liver, causing elevations of between 25- and 35-fold in hepatic levels of these proteins. Induction was not limited to AFAR and GSTA5: treatment with CMRN caused similar increases in the amount of the class-π GST P1 subunit and NAD(P)H:quinone oxidoReductase in rat liver. Immunohistochemistry demonstrated that the overexpression of AFAR, GSTA5, GSTP1, and NAD(P)H:quinone oxidoReductase affected by CMRN is restricted to the centrilobular (periacinar) zone of the lobule, sometimes extending almost as far as the portal tract. This pattern of induction was also observed with ethoxyquin, oltipraz, and trans -stilbene oxide. By contrast, induction of these proteins by β-naphthoflavone and diethyl maleate was predominantly periportal. Northern blotting showed that induction of these phase II drug-metabolizing enzymes by CMRN was accompanied by similar increases in the levels of their mRNAs. To assess the biological significance of enzyme induction by dietary CMRN, two intervention studies were performed in which the ability of the benzopyrone to inhibit either AFB 1 -initiated preneoplastic nodules (at 13 weeks) or AFB 1 -initiated liver tumors (at 50 weeks) was investigated. Animals pretreated with CMRN for 2 weeks prior to administration of AFB 1 , and with continued treatment during exposure to the carcinogen for a further 11 weeks, were protected completely from development of hepatic preneoplastic lesions by 13 weeks. In the longer-term dietary intervention, treatment with CMRN before and during exposure to AFB 1 for a total of 24 weeks was found to significantly inhibit the number and size of tumors that subsequently developed by 50 weeks. These data suggest that consumption of a CMRN-containing diet provides substantial protection against the initiation of AFB 1 hepatocarcinogenesis in the rat.
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Major differences exist in the function and tissue-specific expression of human aflatoxin B1 Aldehyde Reductase and the principal human aldo-keto Reductase AKR1 family members.
Biochemical Journal, 1999Co-Authors: Tania O'connor, Linda S. Ireland, David J. Harrison, John D HayesAbstract:Complementary DNA clones encoding human aflatoxin B(1) Aldehyde Reductase (AKR7A2), Aldehyde Reductase (AKR1A1), aldose Reductase (AKR1B1), dihydrodiol dehydrogenase 1 (AKR1C1) and chlordecone Reductase (AKR1C4) have been expressed in Escherichia coli. These members of the aldo-keto Reductase (AKR) superfamily have been purified from E. coli as recombinant proteins. The recently identified AKR7A2 was shown to differ from the AKR1 isoenzymes in being able to catalyse the reduction of 2-carboxybenzAldehyde. Also, AKR7A2 was found to exhibit a narrow substrate specificity, with activity being restricted to succinic semiAldehyde (SSA), 2-nitrobenzAldehyde, pyridine-2-Aldehyde, isatin, 1,2-naphthoquinone (1,2-NQ) and 9,10-phenanthrenequinone. In contrast, AKR1A1 reduces a broad spectrum of carbonyl-containing compounds, displaying highest specific activity for SSA, 4-carboxybenzAldehyde, 4-nitrobenzAldehyde, pyridine-3-Aldehyde, pyridine-4-Aldehyde, 4-hydroxynonenal, phenylglyoxal, methylglyoxal, 2,3-hexanedione, 1, 2-NQ, 16-ketoestrone and d-glucuronic acid. Comparison between the kinetic properties of AKR7A2 and AKR1A1 showed that both recombinant enzymes exhibited roughly similar k(cat)/K(m) values for SSA, 1,2-NQ and 16-ketoestrone. Many of the compounds which are substrates for AKR1A1 also serve as substrates for AKR1B1, though the latter enzyme was shown to display a specific activity significantly less than that of AKR1A1 for most of the aromatic and aliphatic Aldehydes studied. Neither AKR1C1 nor AKR1C4 was found to possess high Reductase activity towards aliphatic Aldehydes, aromatic Aldehydes, aldoses or dicarbonyls. However, unlike AKR1A1 and AKR1B1, both AKR1C1 and AKR1C4 were able to catalyse the oxidation of 1-acenaphthenol and, in addition, AKR1C4 could oxidize di- and tri-hydroxylated bile acids. Specific antibodies raised against AKR7A2, AKR1A1, AKR1B1, AKR1C1 and AKR1C4 have been used to show the presence of all of the Reductases in human hepatic cytosol; the levels of AKR1B1 and AKR1C1 were markedly elevated in livers with alcohol-associated injury, and indeed AKR1B1 was only detectable in livers with evidence of alcoholic liver disease. Western blotting of extracts from brain, heart, kidney, liver, lung, prostate, skeletal muscle, small intestine, spleen and testis showed that AKR7A2 is present in all of the organs examined, and AKR1B1 is similarly widely distributed in human tissues. These experiments revealed however, that the expression of AKR1A1 is restricted primarily to brain, kidney, liver and small intestine. The AKR1C family members proved not to be as widely expressed as the other Reductases, with AKR1C1 being observed in only kidney, liver and testis, and AKR1C4 being found in liver alone. As human kidney is a rich source of AKR, the isoenzymes in this organ have been studied further. Anion-exchange chromatography of human renal cytosol on Q-Sepharose allowed resolution of AKR1A1, AKR1B1, AKR1C1 and AKR7A2, as identified by substrate specificity and Western blotting. Immunohistochemistry of human kidney demonstrated that AKR7A2 is expressed in a similar fashion to the AKR1 family members in proximal and distal convoluted renal tubules. Furthermore, both AKR7A2 and AKR1 members were expressed in renal carcinoma cells, suggesting that these groups of isoenzymes may be engaged in related physiological functions.
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molecular cloning expression and catalytic activity of a human akr7 member of the aldo keto Reductase superfamily evidence that the major 2 carboxybenzAldehyde Reductase from human liver is a homologue of rat aflatoxin b1 Aldehyde Reductase
Biochemical Journal, 1998Co-Authors: Linda S. Ireland, G E Neal, David J. Harrison, John D HayesAbstract:The masking of charged amino or carboxy groups by N-phthalidylation and O-phthalidylation has been used to improve the absorption of many drugs, including ampicillin and 5-fluorouracil. Following absorption of such prodrugs, the phthalidyl group is hydrolysed to release 2-carboxybenzAldehyde (2-CBA) and the pharmaceutically active compound; in humans, 2-CBA is further metabolized to 2-hydroxymethylbenzoic acid by reduction of the Aldehyde group. In the present work, the enzyme responsible for the reduction of 2-CBA in humans is identified as a homologue of rat aflatoxin B1-Aldehyde Reductase (rAFAR). This novel human aldo-keto Reductase (AKR) has been cloned from a liver cDNA library, and together with the rat protein, establishes the AKR7 family of the AKR superfamily. Unlike its rat homologue, human AFAR (hAFAR) appears to be constitutively expressed in human liver, and is widely expressed in extrahepatic tissues. The deduced human and rat protein sequences share 78% identity and 87% similarity. Although the two AKR7 proteins are predicted to possess distinct secondary structural features which distinguish them from the prototypic AKR1 family of AKRs, the catalytic- and NADPH-binding residues appear to be conserved in both families. Certain of the predicted structural features of the AKR7 family members are shared with the AKR6 beta-subunits of voltage-gated K+-channels. In addition to reducing the dialdehydic form of aflatoxin B1-8,9-dihydrodiol, hAFAR shows high affinity for the gamma-aminobutyric acid metabolite succinic semiAldehyde (SSA) which is structurally related to 2-CBA, suggesting that hAFAR could function as both a SSA Reductase and a 2-CBA Reductase in vivo. This hypothesis is supported in part by the finding that the major peak of 2-CBA Reductase activity in human liver co-purifies with hAFAR protein.
Michihiko Kataoka - One of the best experts on this subject based on the ideXlab platform.
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cloning overexpression and mutagenesis of the sporobolomyces salmonicolor aku4429 gene encoding a new Aldehyde Reductase which catalyzes the stereoselective reduction of ethyl 4 chloro 3 oxobutanoate to ethyl s 4 chloro 3 hydroxybutanoate
Applied and Environmental Microbiology, 1999Co-Authors: Keiko Kita, Takanobu Fukura, Kohichi Nakase, Kenji Okamoto, Hideshi Yanase, Michihiko Kataoka, Sakayu ShimizuAbstract:Aldehyde Reductase (EC 1.1.1.2), aldose Reductase (EC 1.1.1.21), and carbonyl Reductase (EC 1.1.1.184) catalyze NADPH-dependent reduction of a variety of carbonyl compounds and are widely distributed in mammalian and plant tissues. These enzymes are members of the aldo-keto Reductase superfamily (4, 8); however, their physiological functions are not well understood. The amino acid sequences of aldose Reductases and Aldehyde Reductases exhibit significant levels of similarity, but the amino acid sequences of carbonyl Reductases do not (32). In previous papers, we described purification and characterization of three NADPH-dependent Aldehyde Reductases (ARI, ARII, and ARIII) of the red yeast Sporobolomyces salmonicolor AKU4429 (9, 14, 34). ARI is the most abundant Aldehyde Reductase in this yeast and catalyzes asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (4-COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate (4-CHBE) {enantiomeric excess for (R) = [(R − S)/(R + S] × 100 and vice versa}, a promising chiral building block for organic synthesis. In contrast, ARII is produced in considerably smaller amounts but reduces 4-COBE to the (S) enantiomer (92.7% enantiomeric excess), which is also a useful chiral building block for chemical synthesis of pharmaceuticals. In addition to the stereoselectivity of activity against 4-COBE, the N-terminal amino acid sequences of these two Aldehyde Reductases are quite different. Based on the amino acid sequence deduced from the cDNA sequence, ARI belongs to the aldo-keto Reductase superfamily (13). Recently, an NADPH-dependent Aldehyde Reductase (S1), which reduces 4-COBE to the (S) enantiomer (100% enantiomeric excess), was purified from Candida magnoliae AKU4643 (31). The substrate specificities, subunit structures, and N-terminal amino acid sequences of ARII and S1 are not similar. This indicates that the two enzymes belong to the different groups. In this study, we cloned and analyzed a cDNA clone of the Aldehyde Reductase gene (ARII) in order to compare the catalytic mechanisms of ARI and ARII and to understand the molecular basis of the stereospecific reduction of 4-COBE.
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stereoselective reduction of ethyl 4 chloro 3 oxobutanoate by escherichia coli transformant cells coexpressing the Aldehyde Reductase and glucose dehydrogenase genes
Applied Microbiology and Biotechnology, 1999Co-Authors: Michihiko Kataoka, Keiko Kita, Hideshi Yanase, Masaru Wada, Hiroshi Kawabata, Kazuhiko Yamamoto, Sakayu ShimizuAbstract:The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate [(R)-CHBE] using Escherichia coli cells, which coexpress both the Aldehyde Reductase gene from Sporobolomyces salmonicolor and the glucose dehydrogenase (GDH) gene from Bacillus megaterium as a catalyst was investigated. In an organic solvent-water two-phase system, (R)-CHBE formed in the organic phase amounted to 1610 mM (268 mg/ml), with a molar yield of 94.1% and an optical purity of 91.7% enantiomeric excess. The calculated turnover number of NADP+ to CHBE formed was 13 500 mol/mol. Since the use of E. coli JM109 cells harboring pKAR and pACGD as a catalyst is simple, and does not require the addition of GDH or the isolation of the enzymes, it is highly advantageous for the practical synthesis of (R)-CHBE.
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purification and characterization of an Aldehyde Reductase from candida magnoliae
Journal of Molecular Catalysis B-enzymatic, 1999Co-Authors: Masaru Wada, Michihiko Kataoka, Hiroshi Kawabata, Yoshihiko Yasohara, Noriyuki Kizaki, Junzo Hasegawa, Sakayu ShimizuAbstract:Abstract An NADPH-dependent Aldehyde Reductase was purified to homogeneity from Candida magnoliae AKU4643 through four steps, including Blue-Sepharose affinity chromatography. The relative molecular mass of the enzyme was estimated to be 33,000 on high performance gel-permeation chromatography and 35,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The substrate specificity of the enzyme was broad and resembled those of other aldo–keto Reductases. The partial amino acid sequences of the enzyme showed that it belongs to the aldo–keto Reductase superfamily. The enzyme catalyzed the stereoselective reduction of ethyl 4-chloro-3-oxobutanoate to the corresponding ( R )-alcohol, with a 100% enantiomeric excess. The enzyme was inhibited by 1 mM quercetin, CuSO 4 , ZnSO 4 and HgCl 2 . The thermostability of the enzyme was inferior to that of the ( S )-CHBE-producing enzyme from the same strain.
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enzymatic production of ethyl r 4 chloro 3 hydroxybutanoate asymmetric reduction of ethyl 4 chloro 3 oxobutanoate by an escherichia coli transformant expressing the Aldehyde Reductase gene from yeast
Applied Microbiology and Biotechnology, 1997Co-Authors: Michihiko Kataoka, Keiko Kita, Hideshi Yanase, Masaru Wada, Hiroshi Kawabata, Kazuhiko Yamamoto, L P S Rohani, Sakayu ShimizuAbstract:The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate (CHBE) using Escherichia coli JM109 (pKAR) cells expressing the Aldehyde Reductase gene from Sporobolomyces salmonicolor AKU4429 as a catalyst was studied. The reduction required NADP+, glucose and glucose dehydrogenase for NADPH regeneration. In an aqueous system, the substrate was unstable, and inhibition of the reaction by the substrate was also observed. Efficient conversion of COBE to (R)-CHBE with a satisfactory enantiomeric excess (ee) was attained on incubation with transformant cells in an n-butyl acetate/water two-phase system containing the above NADPH-regeneration system. Under the optimized conditions, with the periodical addition of COBE, glucose and glucose dehydrogenase, the (R)-CHBE yield reached 1530 mM (255 mg/ml) in the organic phase, with a molar conversion yield of 91.1% and an optical purity of 91% ee. The calculated turnover of NADP+, based on the amounts of NADP+ added and CHBE formed, was about 5100 mol/mol.
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cloning of the Aldehyde Reductase gene from a red yeast sporobolomyces salmonicolor and characterization of the gene and its product
Applied and Environmental Microbiology, 1996Co-Authors: Keiko Kita, Hideshi Yanase, Michihiko Kataoka, K Matsuzaki, T Hashimoto, Nobuo Kato, Maxey C M Chung, Sakayu ShimizuAbstract:An NADPH-dependent Aldehyde Reductase (ALR) isolated from a red yeast, Sporobolomyces salmonicolor, catalyzes the reduction of a variety of carbonyl compounds. To investigate its primary structure, we cloned and sequenced the cDNA coding for ALR. The Aldehyde Reductase gene (ALR) comprises 969 bp and encodes a polypeptide of 35,232 Da. The deduced amino acid sequence showed a high degree of similarity to other members of the aldo-keto Reductase superfamily. Analysis of the genomic DNA sequence indicated that the ALR gene was interrupted by six introns (two in the 5' noncoding region and four in the coding region). Southern hybridization analysis of the genomic DNA from S. salmonicolor indicated that there was one copy of the gene. The ALR gene was expressed in Escherichia coli under the control of the tac promoter. The enzyme expressed in E. coli was purified to homogeneity and showed the same catalytic properties as did the enzyme from S. salmonicolor.
T M Penning - One of the best experts on this subject based on the ideXlab platform.
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competing roles of aldo keto Reductase 1a1 and cytochrome p4501b1 in benzo a pyrene 7 8 diol activation in human bronchoalveolar h358 cells role of akrs in p4501b1 induction
Chemical Research in Toxicology, 2006Co-Authors: Hao Jiang, Daljit Vudathala, Ian A Blair, T M PenningAbstract:Benzo[a]pyrene (BP) requires metabolic activation to electrophiles to exert its deleterious effects. We compared the respective roles of aldo-keto Reductase 1A1 (AKR1A1, Aldehyde Reductase) and P4501B1 in the formation of BP-7,8-dione and BP-tetrols, respectively, in intact bronchoalveolar cells manipulated to express either enzyme. Metabolite formation was confirmed by HPLC/MS and quantitatively measured by HPLC/UV/β-RAM. In TCDD-treated H358 cells (P4501B1 expression), the anti-BPDE hydrolysis product BP-tetrol-1 increased over 3−12 h to a constant level. In H358 AKR1A1 transfectants, formation of BP-7,8-dione was elevated for 3−12 h but significantly decreased after 24 h. Interestingly, BP-tetrols were also detected in AKR1A1 transfectants even though they do not constitutively express P4501A1/P4501B1 enzymes. Northern and Western blotting confirmed the induction of P4501B1 by BP-7,8-dione in parental cells and the induction of P4501B1 by BP-7,8-diol in AKR1A1-transfected cells. P4501B1 induction was b...
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the ubiquitous Aldehyde Reductase akr1a1 oxidizes proximate carcinogen trans dihydrodiols to o quinones potential role in polycyclic aromatic hydrocarbon activation
Biochemistry, 2001Co-Authors: Nisha T Palackal, Michael E Burczynski, Ronald G Harvey, T M PenningAbstract:Polycyclic aromatic hydrocarbons (PAHs) are metabolized to trans-dihydrodiol proximate carcinogens by human epoxide hydrolase (EH) and CYP1A1. Human dihydrodiol dehydrogenase isoforms (AKR1C1-AKR1C4), members of the aldo-keto Reductase (AKR) superfamily, activate trans-dihydrodiols by converting them to reactive and redox-active o-quinones. We now show that the constitutively and widely expressed human AKR, Aldehyde Reductase (AKR1A1), will oxidize potent proximate carcinogen trans-dihydrodiols to their corresponding o-quinones. cDNA encoding AKR1A1 was isolated from HepG2 cells, overexpressed in Escherichia coli, purified to homogeneity, and characterized. AKR1A1 oxidized the potent proximate carcinogen (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene with a higher utilization ratio (V(max)/K(m)) than any other human AKR. AKR1A1 also displayed a high V(max)/K(m) for the oxidation of 5-methylchrysene-7,8-diol, benz[a]anthracene-3,4-diol, 7-methylbenz[a]anthracene-3,4-diol, and 7,12-dimethylbenz[a]anthracene-3,4-diol. AKR1A1 displayed rigid regioselectivity by preferentially oxidizing non-K-region trans-dihydrodiols. The enzyme was stereoselective and oxidized 50% of each racemic PAH trans-dihydrodiol tested. The absolute stereochemistries of the reactions were assigned by circular dichroism spectrometry. AKR1A1 preferentially oxidized the metabolically relevant (-)-benzo[a]pyrene-7(R),8(R)-dihydrodiol. AKR1A1 also preferred (-)-benz[a]anthracene-3(R),4(R)-dihydrodiol, (+)-7-methylbenz[a]anthracene-3(S),4(S)-dihydrodiol, and (-)-7,12-dimethylbenz[a]anthracene-3(R),4(R)-dihydrodiol. The product of the AKR1A1-catalyzed oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene was trapped with 2-mercaptoethanol and characterized as a thioether conjugate of benzo[a]pyrene-7,8-dione by LC/MS. Multiple human tissue expression array analysis showed coexpression of AKR1A1, CYP1A1, and EH, indicating that trans-dihydrodiol substrates are formed in the same tissues in which AKR1A1 is expressed. The ability of this general metabolic enzyme to divert trans-dihydrodiols to o-quinones suggests that this pathway of PAH activation may be widespread in human tissues.
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metabolic activation of polycyclic aromatic hydrocarbon trans dihydrodiols by ubiquitously expressed Aldehyde Reductase akr1a1
Chemico-Biological Interactions, 2001Co-Authors: Nisha T Palackal, Michael E Burczynski, Ronald G Harvey, T M PenningAbstract:Polycyclic aromatic hydrocarbons (PAHs) are metabolized to trans-dihydrodiol proximate carcinogens by CYP1A1 and epoxide hydrolase (EH). CYP1A1 or aldo-keto Reductases (AKRs) from the 1C subfamily can further activate the trans-dihydrodiols by forming either anti-diol-epoxides or reactive and redox active o-quinones, respectively. To determine whether other AKR superfamily members can divert trans-dihydrodiols to o-quinones, the cDNA encoding human Aldehyde Reductase (AKR1A1) was isolated from hepatoma HepG2 cells using RT-PCR, subcloned into a prokaryotic expression vector, overexpressed in E. coli and purified to homogeneity in milligram amounts. Studies revealed that AKR1A1 preferentially oxidized the metabolically relevant (-)-[3R,4R]-dihydroxy-3,4-dihydrobenz[a]anthracene. AKR1A1 also displayed high utilization ratios (V(max)/K(m)) for the following PAH trans-dihydrodiols: (+/-)trans-3,4-dihydroxy-3,4-dihydro-7-methylbenz[a]anthracene, (+/-)trans-3,4-dihydroxy-3,4-dihydro-7,12-dimethylbenz[a]anthracene and (+/-)trans-7,8-dihydroxy-7,8-dihydro-5-methylchrysene. Multiple tissue expression (MTE) arrays were used to measure the co-expressed of CYP1A1, EH and AKR1A1. All the three enzymes co-expressed to sites of PAH activation. The high catalytic efficiency of AKR1A1 for potent proximate carcinogen trans-dihydrodiols and its presence in tissues that contain CYP1A1 and EH suggests that it plays an important role in this alternative pathway of PAH activation (supported by CA39504).
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fatty alcohols for wax esters in marinobacter aquaeolei vt8 two optional routes in the wax biosynthesis pathway
Applied and Environmental Microbiology, 2013Co-Authors: Eric M Lenneman, Janet M Ohlert, Nagendra P Palani, Brett M BarneyAbstract:The biosynthesis of wax esters in bacteria is accomplished by a unique pathway that combines a fatty alcohol and a fatty acyl coenzyme A substrate. Previous in vitro enzymatic studies indicated that two different enzymes could be involved in the synthesis of the required fatty alcohol in Marinobacter aquaeolei VT8. In this study, we demonstrate through a series of gene deletions and transcriptional analysis that either enzyme is capable of fulfilling the role of providing the fatty alcohol required for wax ester biosynthesis in vivo, but evolution has clearly selected one of these, a previously characterized fatty Aldehyde Reductase, as the preferred enzyme to perform this reaction under typical wax ester-accumulating conditions. These results complement previous in vitro studies and provide the first glimpse into the role of each enzyme in vivo in the native organism.
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characterization of a fatty acyl coa Reductase from marinobacter aquaeolei vt8 a bacterial enzyme catalyzing the reduction of fatty acyl coa to fatty alcohol
Biochemistry, 2011Co-Authors: Robert M Willis, Bradley D Wahlen, Lance C Seefeldt, Brett M BarneyAbstract:Fatty alcohols are of interest as a renewable feedstock to replace petroleum compounds used as fuels, in cosmetics, and in pharmaceuticals. One biological approach to the production of fatty alcohols involves the sequential action of two bacterial enzymes: (i) reduction of a fatty acyl-CoA to the corresponding fatty Aldehyde catalyzed by a fatty acyl-CoA Reductase, followed by (ii) reduction of the fatty Aldehyde to the corresponding fatty alcohol catalyzed by a fatty Aldehyde Reductase. Here, we identify, purify, and characterize a novel bacterial enzyme from Marinobacter aquaeolei VT8 that catalyzes the reduction of fatty acyl-CoA by four electrons to the corresponding fatty alcohol, eliminating the need for a separate fatty Aldehyde Reductase. The enzyme is shown to reduce fatty acyl-CoAs ranging from C8:0 to C20:4 to the corresponding fatty alcohols, with the highest rate found for palmitoyl-CoA (C16:0). The dependence of the rate of reduction of palmitoyl-CoA on substrate concentration was cooperativ...
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purification characterization and potential bacterial wax production role of an nadph dependent fatty Aldehyde Reductase from marinobacter aquaeolei vt8
Applied and Environmental Microbiology, 2009Co-Authors: Bradley D Wahlen, Lance C Seefeldt, Whitney S Oswald, Brett M BarneyAbstract:Wax esters, ester-linked fatty acids and long-chain alcohols, are important energy storage compounds in select bacteria. The synthesis of wax esters from fatty acids is proposed to require the action of a four-enzyme pathway. An essential step in the pathway is the reduction of a fatty Aldehyde to the corresponding fatty alcohol, although the enzyme responsible for catalyzing this reaction has yet to be identified in bacteria. We report here the purification and characterization of an enzyme from the wax ester-accumulating bacterium Marinobacter aquaeolei VT8, which is a proposed fatty Aldehyde Reductase in this pathway. The enzyme, a 57-kDa monomer, was expressed in Escherichia coli as a fusion protein with the maltose binding protein on the N terminus and was purified to near homogeneity by using amylose affinity chromatography. The purified enzyme was found to reduce a number of long-chain Aldehydes to the corresponding alcohols coupled to the oxidation of NADPH. The highest specific activity was observed for the reduction of decanal (85 nmol decanal reduced/min/mg). Short-chain and aromatic Aldehydes were not substrates. The enzyme showed no detectable catalysis of the reverse reaction, the oxidation of decanol by NADP+. The mechanism of the enzyme was probed with several site-specific chemical probes. The possible uses of this enzyme in the production of wax esters are discussed.