| Co-Investigator(Kenkyū-buntansha) |
KATAOKA Michihiko Kyoto University, Graduate School of Agriculture, Assistant Professor, 大学院・農学研究科, 助手 (90252494)
SHIMIZU Sakayu Kyoto University, Graduate School of Agriculture, Professor, 大学院・農学研究科, 教授 (70093250)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1998: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1997: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
New aldehyde reductases (AR), ARII and ARIII, which reduce ethyl 4-chloro-3-oxobutanoate (4-COBE) to ethyl 4-chloro-3-hydroxybutanoate (CHBE), with NADPH as a cofactor, were purified from Sporobolomyces salmonicolor AKU4429. ARII catalyzed the stereospecific reduction of 4-COBE to (S)-CHBE (92.7% enantiomeric excess (e.e.)), in contrast, ARIII reduced 4-COBE to R-CHBE (38.4% e.e.). ARII reduced aliphatic and aromatic aldehydes, and carbonyl compounds such as camphorquinone, but did not accept aldose as a substrate. The enzyme is a monomer protein with a relative molecular mass of 34,000. Its isoelectric point is 5.0. The NH2-terminal amino acid sequence of ARII is different from that of ARI, which catalyzes the stereospecific reduction of 4-COBB to R-CHBE (100% e.e.). We cloned and sequenced the gene encoding an NADPH-dependent ARII from S. salmonicolor AKU4429. The ARII gene comprises 1,032 bp, is interrupted by 4 introns, and encodes a polypeptide of 37,315 Da. The deduced amino acid
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sequence showed significant similarity to that of members of the mammalian 3β-hydroxysteroid dehydrogenase/plant dihydroflavonol 4-reductase superfamily, but not to those of members of the aldo-keto reductase superfamily or to that of ARI previously isolated from the same organism. The ARII protein was overproduced in Escherichia coli about 2,000-fold per g cells, compared to in the original yeast cells. The enzyme expressed in E. coli was purified to homogeneity, and showed the same catalytic properties as ARII purified from S. salmonicolor. To examine the contribution of the dinucleotide-binding motif, GィイD219ィエD2-X-X-GィイD222ィエD2-X-X-AィイD225ィエD2, located in the NH2-terminal region, for ARII catalysis, we exchanged three amino acid residues in the motif, and purified the respective mutant enzymes. The substrate inhibition of the enzyme by 4-COBE was absent in the GィイD219ィエD2 → A and GィイD222ィエD2 → A mutant enzymes. The AィイD225ィエD2 → G mutant enzyme could reduce 4-COBE when NADPH was replaced by an equimolar concentration of NADH. Less
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