cDNA cloning of novel aldehyde reductase gene from yeast and its application to chiral alcohol synthesis

新的醛还原酶（AR）， ARII和ARIII，将4-氯-3-氧丁酸乙酯（4-COBE）减少到4-氯-3-羟基丁酸乙酯（CHBE），与NADPH作为一个辅因子，从孢子虫胺salmonicolor AKU 4429中净化。ARII催化了4-COBE到（S）-CHBE的立体空间减少（92.7%的显体优势（如E）），在对比下， ARIII将4-COBE减少到R-CHBE （38.4%等）。ARII减少了aliphatic和芳香醛，以及碳基化合物作为camphorquinone，但没有作为底物接受aldose。酶是一种单体蛋白质，其相对分子质量为34，000。其等电点为5.0。ARII的NH 2终氨基酸序列与ARII不同，将4-COBB的立体空间减少到R-CHBE （100% E.E.）。我们从S中克隆并测序了基因编码的NADPH依赖性ARII。salmonicolor AKU 4429 ARII基因含有1，032个基点，被4个intron打断，编码为37，315个多肽。 ... More 该序列显示出哺乳动物3β-羟基类固醇脱氢酶/植物二氢氟醚4-还原酶超家族成员的显著相似性，但不是aldo-keto还原酶超家族成员的那些成员，或使ARI先前从相同的组织中孤立。ARII蛋白质在大肠杆菌中过量产生，每g细胞折叠约2，000个，与原始年份细胞相比。在E.中表达的酶。大肠杆菌被净化为同源性，并显示出相同的催化性质，从S.中净化为ARII。salmonicolor。为了测试二核肽结合动机的贡献，Gy-D219 y-D2-X-X-Gy-D222 y-D2-X-X-A y-D225 y-D2，位于NH 2终端区域，用于ARII催化，我们更换了三种氨基酸溶液，并清除了对突变酶的反应。The substrate inhibition of the enzyme by 4-COBE was absent in the G-D219-D2 → A and G-D222-D2 → A mutant enzymes。The AイD225エD2 ￣ G mutant enzyme could reduce 4-COBE when NADPH was replaced by an equimolar concentration of NADH. Less（低）

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 … More 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