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

酵母新型醛还原酶基因的cDNA克隆及其在手性醇合成中的应用

• 批准号: 09660091
• 负责人: KITA Keiko
• 金额: $ 1.28万
• 依托单位: Tottori University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09660091/
• 关键词: Chiral alcohol; Chiral synton; Yeast; Aldehyde reductase; Glucose dehydrogenase; NADPH-binding; cDNA cloning; NADPH; 部位特異的変異

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

从Sporobolomyces salmonicolor AKU 4429中纯化了新的醛还原酶（AR），ARII和ARIII，其将4-氯-3-氧代丁酸乙酯（4-COBE）还原为4-氯-3-羟基丁酸乙酯（CHBE），其中NADPH作为辅因子。ARII催化4-COBE立体特异性还原为（S）-CHBE（92.7%对映体过量（e.e.）），相反，ARIII将4-COBE还原为R-CHBE（38. 4% e.e.）。ARII减少脂肪族和芳香族醛，和羰基化合物，如蒽醌，但不接受醛糖作为底物。该酶是相对分子质量为34，000的单体蛋白质。其等电点为5.0。ARII的NH 2-末端氨基酸序列与ARI不同，ARI催化4-COBB立体特异性还原为R-CHBE（100% e.e.）。我们克隆并测序了S.鲑鱼AKU 4429。ARII基因包含1，032 bp，被4个内含子中断，并编码37，315 Da的多肽。 ...更多信息 序列与哺乳动物3β-羟基类固醇脱氢酶/植物二氢黄酮醇4-还原酶超家族成员的序列具有显著的相似性，但与醛酮还原酶超家族成员或先前从同一生物体分离的ARI的序列不相似。与原始酵母细胞相比，ARII蛋白在大肠杆菌中过量产生约2，000倍/g细胞。在E. coli中纯化得到的ARII与S.鲑鱼色的为了检测位于NH 2-末端区域的二核苷酸结合基序G β D219 β D2-X-X-G β D222 β D2-X-X-A β D225 β D2对ARII催化的贡献，我们交换了基序中的三个氨基酸残基，并纯化了相应的突变酶。4-COBE对该酶的底物抑制在G β D219突变体D2 → A和G β D222突变体D2 → A中不存在。当NADPH被等摩尔浓度的NADH替代时，A β D225突变体D2 → G酶可以还原4-COBE。少

项目成果

Keiko Kita: "Diversity of 4-chloroacetoacetate ethyl ester-reducing enzymes in yeasts and their application to chiral alcohol synthesis"J.Biosci.Bioeng.. 88(6). 591-598 (1999)

Keiko Kita：“酵母中 4-氯乙酰乙酸乙酯还原酶的多样性及其在手性醇合成中的应用”J.Biosci.Bioeng.. 88(6)。

Keiko Kita: "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"A

Keiko Kita：“鲑色孢子酵母 AKU4429 基因的克隆、过表达和诱变，该基因编码一种新的醛还原酶，该酶催化 4-氯-3-氧代丁酸乙酯立体选择性还原为-(S)-4-氯-3-羟基丁酸乙酯

K. Kita: "Purification and characterization of new aldehyde reductases from Sporobolomyces salmonicolor AKU4429"J. Mol. Catalysis B : Enzymatic. 6. 429-432 (1999)

K. Kita：“来自鲑色孢子酵母 AKU4429 的新型醛还原酶的纯化和表征”J。

清水 昌: "Chiral alcohol synthesis with microbial carbonyl reductases in a water-organic solvent two-phase system" Annals New York Academy of Sciences. 864. 87-95 (1998)

Masaru Shimizu：“在水-有机溶剂两相系统中用微生物羰基还原酶合成手性醇”纽约科学院年鉴 864. 87-95 (1998)。

K. Kita: "Cloning, overexpression, and mutagenesis of the Sporobo-lomyces 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"

K. Kita：“鲑色孢子酵母 AKU4429 基因的克隆、过表达和诱变，该基因编码一种新的醛还原酶，该酶催化 4-氯-3-氧代丁酸乙酯立体选择性还原为乙基-(S)-4-氯-