ethyl-4-chloro-3-hydroxybutanoate and ethyl-4-chloro-3-oxobutanoate

Previously, we published cloning, overexpression, characterization and subsequent exploitation of a carbonyl reductase (cr) gene, belonging to general family aldo-keto reductase from Candida glabrata CBS138 to convert keto ester (COBE) to a chiral alcohol (ethyl-4-chloro-3-hydroxybutanoate or CHBE). Exploiting global transcription factor CRP, rDNA and transporter engineering, we have improved batch production of CHBE by trinomial bioengineering. Herein, we present the exploration of cr gene in Candida glabrata CBS138 through genome mining approach, in silico validation of its activity and selection of its biocatalytic phase. For exploration of the gene under investigation, 3 template genes were chosen namely Saccharomyces cerevisae YDR541c, YGL157w and YOL151w. The CR showed significant homology match, overlapping of substrate binding site and NADPH binding site with the template proteins. The binding affinity of COBE toward CR (-4.6 Kcal/ mol) was found higher than that of the template proteins (-3.5 to -4.5 Kcal/ mol). Biphasic biocatalysis with cofactor regeneration improved product titer 4∼5 times better than monophasic biotransformation. Currently we are working on DNA Shuffling as a next level of strain engineering and we demonstrate this approach herein as a future strategy of biochemical engineering.

Topics: Acetoacetates; Aldo-Keto Reductases; Amino Acid Sequence; Binding Sites; Biocatalysis; Biotransformation; Butyrates; Candida glabrata; Crystallography, X-Ray; Data Mining; Fungal Proteins; Gene Expression; Genome, Fungal; Kinetics; Metabolic Engineering; Models, Molecular; NADP; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity; Thermodynamics

To increase the biocatalytic activity of Escherichia coli CCZU-T15 whole cells, choline chloride/glycerol ([ChCl][Gly]) was firstly used as biocompatible solvent for the effective biotransformation of ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE]. Furthermore, L-glutamine (150 mM) was added into [ChCl][Gly]-water ([ChCl][Gly] 12.5 vol%, pH 6.5) media instead of NAD

Topics: Acetoacetates; Biotransformation; Butyrates; Choline; Culture Media; DNA, Recombinant; Dose-Response Relationship, Drug; Escherichia coli; Ethylene Glycol; Glutamine; Polysorbates; Water

Topics: Acetates; Acetoacetates; Alcohol Oxidoreductases; Biotransformation; Butyrates; Candida glabrata; DNA, Ribosomal; Dose-Response Relationship, Drug; Escherichia coli; Protein Engineering; Sesquiterpenes

To reduce dependence on the expensive cofactor and effectively biotransform ethyl 4-chloro-3-oxobutanoate, L-glutamine and glycine were found to enhance the content of intracellular NADH and the reductase activity. Adding the mixture of 200 mM of L-glutamine and 500 mM of glycine to the reaction media, a 1.67-fold of reductase activity was increased over the control without the addition of the two compounds. Moreover, β-cyclodextrin (0.4 mol β-cyclodextrin/mol ethyl 4-chloro-3-oxobutanoate) was also added into this reaction media, and the biocatalytic activity of the whole-cell biocatalyst of Escherichia coli CCZU-K14 was increased by 1.34-fold than that without β-cyclodextrin. In this β-cyclodextrin-water media containing L-glutamine (200 mM) plus glycine (500 mM), ethyl (S)-4-chloro-3-hydroxybutanoate (>99% ee) could be obtained from 3000 mM ethyl 4-chloro-3-oxobutanoate in the yield of 98.0% after 8h. All the positive features demonstrate the potential applicability of the bioprocess for the large-scale production of ethyl (S)-4-chloro-3-hydroxybutanoate.

Topics: Acetoacetates; beta-Cyclodextrins; Butyrates; Escherichia coli; Glutamine; Glycine; NAD; Water

An NADH-dependent reductase (ClCR) was discovered by genome data mining. After ClCR was overexpressed in E. coli BL21, recombinant E. coli CCZU-T15 with high reductase activity and excellent stereoselectivity for the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE] was screened using a modified high-throughput colorimetric screening strategy. After the reaction optimization, a highly stereoselective bioreduction of COBE into (S)-CHBE (>99% ee) with the resting cells of E. coli CCZU-T15 was successfully demonstrated in toluene-water (50:50, v/v) biphasic system. Biotransformation of 1000 mM COBE for 24 h in the biphasic system, (S)-CHBE (>99% ee) could be obtained in the high yield of 96.4%. Significantly, E. coli CCZU-T15 shows high potential in the industrial production of (S)-CHBE (>99% ee).

Topics: Acetoacetates; Bacterial Proteins; Butyrates; Data Mining; Escherichia coli; High-Throughput Screening Assays; Industrial Microbiology; NAD; Trans-Activators

To avoid adding NAD(+) and effectively transform ethyl 4-chloro-3-oxobutanoate, the mixture of l-glutamine (200mM) and d-xylose (250mM) was added into in n-butyl acetate-water (10:90, v/v) biphasic system instead of NAD(+) for increasing the biocatalytic efficiency. To further improve the synthesis of optically pure ethyl (R)-4-chloro-3-hydroxybutanoate (>99% ee), β-cyclodextrin was also added into this reaction media, and ethyl (R)-4-chloro-3-hydroxybutanoate (>99% ee) could be effectively synthesized from 800mM ethyl 4-chloro-3-oxobutanoate in the yield of 100% by whole-cells of recombinant E. coli CCZU-A13. Finally, the possible mechanism for improving the reductase activity by supplementation of l-glutamine, d-xylose and β-CD was proposed. In conclusion, this strategy has high potential for the effective biosynthesis of ethyl (R)-4-chloro-3-hydroxybutanoate (>99% ee).

Topics: Acetates; Acetoacetates; Bacterial Proteins; beta-Cyclodextrins; Butyrates; Culture Media; Escherichia coli; Glutamine; Oxidoreductases; Water; Xylose

A novel aldo-keto reductase (LEK) from Lodderomyces elongisporus NRRL YB-4239 (ATCC 11503) was discovered by genome database mining for carbonyl reduction. LEK was overexpressed in Escherichia coli BL21 (DE3), purified to homogeneity and the catalytic properties were studied. Among the substrates, ethyl 4-chloro-3-oxobutanoate was converted to ethyl (R)-4-chloro-3- hydroxybutanoate ((R)-CHBE), an important pharmaceutical intermediate, with an excellent enantiomeric excess (e.e.) (>99 %). The mutants W28A and S209G obtained by site-directed mutation were identified with much higher molar conversion yields and lower Km values. Further, the constructed coenzyme regeneration system with glucose as co-substrate resulted in a yield of 100 %, an enantioselectivity of >99 %, and the calculated production rate of 56.51 mmol/L/H. These results indicated the potential of LEK for the industrial production of (R)-CHBE and other valuable chiral alcohols.

Topics: Acetoacetates; Aldehyde Reductase; Aldo-Keto Reductases; Butyrates; Coenzymes; Mutagenesis, Site-Directed; Mutation; Saccharomycetales

Reductases convert some achiral ketone compounds into chiral alcohols, which are important materials for the synthesis of chiral drugs. The Saccharomyces cerevisiae reductase YOR120W converts ethyl-4-chloro-3-oxobutanoate (ECOB) enantioselectively into (R)-ethyl-4-chloro-3- hydroxybutanoate ((R)-ECHB), an intermediate of a pharmaceutical. As YOR120W requires NADPH as a cofactor for the reduction reaction, a cofactor recycling system using Bacillus subtilis glucose dehydrogenase was employed. Using this coupling reaction system, 100 mM ECOB was converted to (R)-ECHB. A homology modeling and site-directed mutagenesis experiment were performed to determine the NADPH-binding site of YOR120W. Four residues (Q29, K264, N267, and R270) were suggested by homology and docking modeling to interact directly with 2'-phosphate of NADPH. Among them, two positively charged residues (K264 and R270) were experimentally demonstrated to be necessary for NADPH 2'-phosphate binding. A mutant enzyme (Q29E) showed an enhanced enantiomeric excess value compared with that of the wild-type enzyme.

Topics: Acetoacetates; Bacillus subtilis; Binding Sites; Butyrates; Coenzymes; DNA Mutational Analysis; Glucose 1-Dehydrogenase; Models, Molecular; Mutagenesis, Site-Directed; NADP; Oxidoreductases; Protein Conformation; Saccharomyces cerevisiae

A novel NADPH-dependent reductase (CaCR) from Candida albicans was cloned for the first time. It catalyzed asymmetric reduction to produce ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE). It contained an open reading frame of 843 bp encoding 281 amino acids. When co-expressed with a glucose dehydrogenase in Escherichia coli, recombinant CaCR exhibited an activity of 5.7 U/mg with ethyl 4-chloro-3-oxobutanoate (COBE) as substrate. In the biocatalysis of COBE to (S)-CHBE, 1320 mM (S)-CHBE was obtained without extra NADP+/NADPH in a water/butyl acetate system, and the optical purity of the (S)-isomer was higher than 99% enantiomeric excess.

Topics: Acetates; Acetoacetates; Amino Acid Sequence; Biocatalysis; Butyrates; Candida albicans; Cloning, Molecular; Escherichia coli; Escherichia coli Proteins; Fungal Proteins; Gene Expression; Glucose Dehydrogenases; Molecular Sequence Data; NADP; Open Reading Frames; Oxidoreductases; Plasmids; Stereoisomerism; Water

A substrate-coupled biocatalytic process was developed based on the reactions catalyzed by an NADPH-dependent sorbose reductase (SOU1) from Candida albicans in which ethyl 4-chloro-3-oxobutanoate (COBE) was reduced to (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE], while NADPH was regenerated by the same enzyme via oxidation of sugar alcohols. (S)-CHBE yields of 1,140, 1,150, and 780 mM were obtained from 1,220 mM COBE when sorbitol, mannitol, and xylitol were used as co-substrates, respectively. Optimization of COBE and sorbitol proportions resulted in a maximum yield of (S)-CHBE (2,340 mM) from 2,500 mM COBE, and the enantiomeric excess was 99.6 %. The substrate-coupled system driven by SOU1 maintained a stable pH and a robust intracellular NADPH circulation; thus, pH adjustment and addition of extra coenzymes were unnecessary.

Topics: Acetoacetates; Butyrates; Candida albicans; Coenzymes; Hydrogen-Ion Concentration; NADP; Oxidation-Reduction; Sugar Alcohol Dehydrogenases; Sugar Alcohols

Asymmetric reduction of ethyl-4-chloro-3-oxobutanoate to (S)-ethyl-4-chloro-3-hydroxybutanoate in aqueous medium by resting cells of Candida parapsilosis ATCC 7330 was optimized. The influence of culture parameters (inoculum size, inoculum age and biocatalyst harvest time) and reaction parameters (co-substrate, resting cell, pH and substrate concentrations) on the asymmetric reduction were studied. It was found that these parameters significantly influenced the rate of the asymmetric reduction. Under the optimum conditions, the final concentration of (S)-ethyl-4-chloro-3-hydroxybutanoate, enantiomeric excess and the isolated yield of (S)-ethyl-4-chloro-3-hydroxybutanoate were 1.38 M (230 g/l), >99 and 95%, respectively. The space time yield was 115 mmol/lh, which is significantly higher than other whole cell biocatalysts reported so far.

Topics: Acetoacetates; Butyrates; Candida; Culture Media; Glucose; Hydrogen-Ion Concentration; Industrial Microbiology; Temperature

Ethyl (R, S)-4-chloro-3-hydroxybutanoate (ECHB) is a useful chiral building block for the synthesis of L-carnitine and hypercholesterolemia drugs. The yeast reductase, YOL151W (GenBank locus tag), exhibits an enantioselective reduction activity, converting ethyl-4-chlorooxobutanoate (ECOB) exclusively into (R)-ECHB. YOL151W was generated in Escherichia coli cells and purified via Ni- NTA and desalting column chromatography. It evidenced an optimum temperature of 45 degrees C and an optimum pH of 6.5-7.5. Bacillus subtilis glucose dehydrogenase (GDH) was also expressed in Escherichia coli, and was used for the recycling of NADPH, required for the reduction reaction. Thereafter, Escherichia coli cells co-expressing YOL151W and GDH were constructed. After permeablization treatment, the Escherichia coli whole cells were utilized for ECHB synthesis. Through the use of this system, the 30 mM ECOB substrate could be converted to (R)-ECHB.

Topics: Acetoacetates; Bacillus subtilis; Biotechnology; Butyrates; Escherichia coli; Gene Expression; Genetic Engineering; Glucose 1-Dehydrogenase; NADP; Oxidoreductases; Recombinant Proteins; Saccharomyces cerevisiae Proteins

An NADPH-dependent carbonyl reductase (PsCR) gene from Pichia stipitis was cloned. It contains an open reading frame of 849 bp encoding 283 amino acids whose sequence had less than 60% identity to known reductases that produce ethyl (S)-4-chloro-3-hydroxybutanoates (S-CHBE). When expressed in Escherichia coli, the recombinant PsCR exhibited an activity of 27 U/mg using ethyl 4-chloro-3-oxobutanoate (COBE) as a substrate. Reduction of COBE to (S)-CHBE by transformants in an aqueous mono-phase system for 18 h, gave a molar yield of 94% and an optical purity of the (S)-isomer of more than 99% enantiomeric excess.

Topics: Acetoacetates; Alcohol Oxidoreductases; Amino Acid Sequence; Butyrates; Cloning, Molecular; Escherichia coli; Fungal Proteins; Gene Expression; Kinetics; Molecular Sequence Data; Open Reading Frames; Oxidation-Reduction; Pichia; Sequence Alignment; Sequence Homology, Amino Acid

Escherichia coli M15 (pQE30-car0210) was constructed to express carbonyl reductase (CAR) by cloning the car gene from Candida magnoliae and inserting it into pQE30. By cultivating E. coli M15 (pQE30-car0210) and M15 (pQE30-gdh0310), 8.2-fold and 12.3-fold enhancements in specific enzymatic activity over the corresponding original strain were achieved, respectively. After separate cultivations, these two strains were then mixed together at appropriate ratio to construct a novel two-strain system, in which M15 (pQE30-car0210) expressed CAR for ethyl 4-chloro-3-oxobutanoate (COBE) bioreduction and M15 (pQE30-gdh0310) expressed glucose dehydrogenase (GDH) for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration. In this complex system, the effects of substrate concentration, the biomass ratio between two strains as well as reaction temperature were investigated for efficient bioreduction. The results showed that the bioreduction reaction could be completed effectively without any addition of GDH or NADPH/NADP(+). An optical purity of 99% (enantiometric efficiency) was obtained, and the yield of (S)-4-chloro-3-hydroxybutanoate ethyl ester reached 96.6% when initial concentration of COBE was 36.9 mM. The coupling reactions between two different strains were further explored by determining the profile of NADPH in the reaction broth.

Topics: Acetoacetates; Alcohol Oxidoreductases; Biomass; Bioreactors; Butyrates; Candida; Escherichia coli; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Genetic Engineering; Glucosephosphate Dehydrogenase; NADP; Oxidation-Reduction; Substrate Specificity; Temperature; Time Factors

Formate dehydrogenases (FDH) are useful for the regeneration of NADH, which is required for asymmetric reduction by several dehydrogenases and reductases. FDHs have relatively low activity and are labile, especially to alpha-haloketones, thus FDH cannot be applied to the industrial manufacture of optically active alpha-haloalcohols. To stabilize a FDH from Mycobacterium vaccae (McFDH) against the alpha-haloketone ethyl 4-chloroacetoacetate (ECAA), a set of cysteine-mutant enzymes was constructed. Sensitivity to ECAA of mutant C6S was similar to that of the wild-type enzyme, and mutants C249S and C355S showed little activity. In contrast, mutant C256S exhibited remarkable tolerance to ECAA. Surprisingly, mutant C146S was activated by several organic compounds such as ethyl acetate. An optimized mutant, C6A/C146S/C256V (McFDH-26), was obtained by combining several effective mutations. Ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-ECHB] was synthesized from ECAA to 49.9 g/l with an optical purity of more than 99% e.e. using recombinant Escherichia coli cells coexpressing McFDH-26 and a carbonyl reductase (KaCR1) from Kluyveromyces aestuarii.

Topics: Acetates; Acetoacetates; Alcohol Oxidoreductases; Amino Acid Sequence; Amino Acid Substitution; Butyrates; Cysteine; Enzyme Activators; Enzyme Stability; Escherichia coli; Formate Dehydrogenases; Ketones; Kluyveromyces; Mutagenesis, Site-Directed; Mutation, Missense; Mycobacterium; NAD; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid

Two recombinant strains, E. coli M15 (pQE30-alr0307) and E. coli M15 (pQE30-gdh0310), which were constructed to express, respectively, an NADPH-dependent aldehyde reductase gene and a glucose dehydrogenase gene, were mixed in an appropriate ratio and used for the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (R)-4-chloro-3-hydroxybutanoate. The former strain acted as catalyst and the latter functioned in NADPH regeneration. The biotransformation was completed effectively without any addition of glucose dehydrogenase or NADP+/NADPH. An optical purity of 99% (ee) was obtained and the product yield reached 90.5% from 28.5 mM: substrate.

Topics: Acetoacetates; Aldehyde Reductase; Butyrates; Escherichia coli; Glucose Dehydrogenases; Industrial Microbiology; NADP; Oxidation-Reduction; Recombinant Proteins

An NADPH-dependent aldehyde reductase (ALR, EC1.1.1.2) gene is cloned from Sporobolomyces salmonicolor ZJUB 105, and inserted into plasmid pQE30 to construct the expression plasmid (pQE30-ALR). A variety of E. coli strains were employed as hosts to obtain transformants with pQE30-ALR, respectively. Among these different types of transformants, the highest enzyme activity of ALR can be produced with E. coli M15 (pQE30-ALR). The bioactivity of ALR could be further improved significantly by the optimization of induction conditions. The results showed that the enzyme activity of ALR reached 6.48 U/mg protein, which is fifteen times higher than that of S. salmonicolor ZJUB 105. This recombinant strain was applied to the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3- hydroxybutanoate (CHBE). The results showed that the yield and optical purity of (R)-CHBE reached 98.5% and 99% e.e. (enantiomeric excess), respectively.

Topics: Acetoacetates; Aldehyde Reductase; Basidiomycota; Butyrates; Cloning, Molecular; Escherichia coli; Fungal Proteins; Oxidation-Reduction; Plasmids; Recombinant Proteins; Transformation, Bacterial

To compare NADH-regeneration systems for the synthesis of (S)-4-chloro-3-hydroxybutanoate (ECHB), a novel NADH-dependent carbonyl reductase (KaCR1), which reduced ethyl 4-chloroacetoacetate (ECAA) to form (S)-ECHB, was screened and purified from Kluyveromyces aestuarii and a gene encoding KaCR1 was cloned. Glucose dehydrogenase (GDH) and formate dehydrogenase (FDH) were compared as enzymes for NADH regeneration using Escherichia coli cells coexpressing each enzyme with KaCR1. E. coli cells coexpressing GDH produced 45.6 g/l of (S)-ECHB from 50 g/l of ECAA and E. coli cells coexpressing FDH, alternatively, produced only 19.0 g/l. The low productivity in the case of FDH was suggested to result from the low activity and instability of FDH.

Topics: Acetoacetates; Alcohol Oxidoreductases; Amino Acid Sequence; Base Sequence; Butyrates; Cloning, Molecular; Conserved Sequence; Escherichia coli; Fatty Acid Synthases; Formate Dehydrogenases; Glucose 1-Dehydrogenase; Kluyveromyces; Molecular Sequence Data; NAD; NADH, NADPH Oxidoreductases; Plasmids; Sequence Analysis, DNA; Substrate Specificity

Amberlite XAD 2 resin enhanced the asymmetric reduction of ethyl 4-chloroacetoacetate (ECA) to S-4-chloro-3-hydroxybutyric acid ethyl ester as catalyzed by Saccharomyces cerevisiae. The absorbed ECA was released slowly to the solution during the reaction so that the substrate inhibition and the spontaneous chemical hydrolysis of ECA were considerably lessened. With 75 g resin l(-1) and ECA at 74 mM, the reaction yield and the product's optical purity increased from 75% to 84% and from 88% to 93%, respectively.

Topics: Absorption; Acetoacetates; Butyrates; Catalysis; Hydrolysis; Oxidation-Reduction; Polystyrenes; Quality Control; Saccharomyces cerevisiae; Sensitivity and Specificity; Stereoisomerism

The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) was investigated. Escherichia coli cells expressing both the carbonyl reductase (S1) gene from Candida magnoliae and the glucose dehydrogenase (GDH) gene from Bacillus megaterium were used as the catalyst. In an organic-solvent-water two-phase system, (S)-CHBE formed in the organic phase amounted to 2.58 M (430 g/l), the molar yield being 85%. E. coli transformant cells coproducing S1 and GDH accumulated 1.25 M (208 g/l) (S)-CHBE in an aqueous monophase system by continuously feeding on COBE, which is unstable in an aqueous solution. In this case, the calculated turnover of NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) to CHBE was 21,600 mol/mol. The optical purity of the (S)-CHBE formed was 100% enantiomeric excess in both systems. The aqueous system used for the reduction reaction involving E. coli HB101 cells carrying a plasmid containing the S1 and GDH genes as a catalyst is simple. Furthermore, the system does not require the addition of commercially available GDH or an organic solvent. Therefore this system is highly advantageous for the practical synthesis of optically pure (S)-CHBE.

Topics: Acetoacetates; Alcohol Oxidoreductases; Aldehyde Reductase; Aldo-Keto Reductases; Base Sequence; Biotechnology; Butyrates; Enzyme Stability; Escherichia coli; Genes, Bacterial; Glucose 1-Dehydrogenase; Glucose Dehydrogenases; Oxidation-Reduction; Plasmids; Stereoisomerism; Transformation, Genetic

The enantioselectivity of ECAA to ECHB by eight fungi of four genus was evaluated. All strains showed (S)-selectivity, and Cylindrocarpon sclerotigenum IFO 31855 gave the highest yield and good optical purity (e.e.; >99%). Cell-free extract and acetone-dried cells of C. sclerotigenum IFO 31855 reduced ECAA to (S)-ECHB in the presence of NADPH (e.e.; >99%) and the e.e. was not decreased by heat treatment of the cell-free extract or the acetone-dried cells. The active fractions shown by two peaks on a DEAE-Toyopearl 650 M column gave preferentially (S)-ECHB (e.e.; >99%).

Topics: Acetoacetates; Biotransformation; Butyrates; Fungi; Hypocreales; Kinetics; Oxidation-Reduction; Stereoisomerism

We cloned and sequenced the gene encoding an NADPH-dependent aldehyde reductase (ARII) in Sporobolomyces salmonicolor AKU4429, which reduces ethyl 4-chloro-3-oxobutanoate (4-COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate. The ARII gene is 1,032 bp long, is interrupted by four introns, and encodes a 37,315-Da polypeptide. The deduced amino acid sequence exhibited significant levels of similarity to the amino acid sequences of members of the mammalian 3beta-hydroxysteroid dehydrogenase-plant dihydroflavonol 4-reductase superfamily but not to the amino acid sequences of members of the aldo-keto reductase superfamily or to the amino acid sequence of an aldehyde reductase previously isolated from the same organism (K. Kita, K. Matsuzaki, T. Hashimoto, H. Yanase, N. Kato, M. C.-M. Chung, M. Kataoka, and S. Shimizu, Appl. Environ. Microbiol. 62:2303-2310, 1996). The ARII protein was overproduced in Escherichia coli about 2, 000-fold compared to the production in the original yeast cells. The enzyme expressed in E. coli was purified to homogeneity and had the same catalytic properties as ARII purified from S. salmonicolor. To examine the contribution of the dinucleotide-binding motif G(19)-X-X-G(22)-X-X-A(25), which is located in the N-terminal region, during ARII catalysis, we replaced three amino acid residues in the motif and purified the resulting mutant enzymes. Substrate inhibition of the G(19)-->A and G(22)-->A mutant enzymes by 4-COBE did not occur. The A(25)-->G mutant enzyme could reduce 4-COBE when NADPH was replaced by an equimolar concentration of NADH.

Topics: Acetoacetates; Aldehyde Reductase; Amino Acid Sequence; Animals; Butyrates; Cloning, Molecular; Introns; Kinetics; Mammals; Mitosporic Fungi; Molecular Sequence Data; Molecular Weight; Mutagenesis, Site-Directed; Phylogeny; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity