Maruyama Reiji, Nishizawa Mikio, Itoi Yasushi, Ito Seiji, Inoue Masami
Department of Cell Engineering, Faculty of Engineering, Toyama University, 3190 Gofuku, 930-8555, Toyama, Japan.
J Biotechnol. 2002 Mar 28;94(2):157-69. doi: 10.1016/s0168-1656(01)00426-6.
The diketone compound, benzil is reduced to (S)-benzoin with living Bacillus cereus cells. Recently, we isolated a gene responsible for benzil reduction, and Escherichia coli cells in which this gene was overexpressed transformed benzil to (S)-benzoin. Although this benzil reductase showed high identity to the short-chain dehydrogenase/reductase (SDR) family, enzymological features were unknown. Here, we demonstrated that many B. cereus strains had benzil reductase activity in vivo, and that the benzil reductases shared 94-100% amino acid identities. Recombinant B. cereus benzil reductase produced optically pure (S)-benzoin with NADPH in vitro, and the ketone group distal to a benzene ring was asymmetrically reduced. B. cereus benzil reductase showed 31% amino acid identity to the yeast open reading frame YIR036C protein and 28-30% to mammalian sepiapterin reductases, sharing the seven residues consensus for the SDR family. We isolated the genes encoding yeast YIR036C protein and gerbil sepiapterin reductase, and both recombinant proteins also reduced benzil to (S)-benzoin in vitro. Green fluorescent protein-tagged B. cereus benzil reductase distributed in the bipolar cytoplasm in B. cereus cells. Asymmetric reduction with B. cereus benzil reductase, yeast YIR036C protein and gerbil sepiapterin reductase will be utilized to produce important chiral compounds.
二酮化合物苯偶酰可被活的蜡样芽孢杆菌细胞还原为(S)-苯偶姻。最近，我们分离出了一个负责苯偶酰还原的基因，该基因过表达的大肠杆菌细胞可将苯偶酰转化为(S)-苯偶姻。尽管这种苯偶酰还原酶与短链脱氢酶/还原酶（SDR）家族具有高度同源性，但其酶学特性尚不清楚。在此，我们证明许多蜡样芽孢杆菌菌株在体内具有苯偶酰还原酶活性，且这些苯偶酰还原酶的氨基酸同源性为94%-100%。重组蜡样芽孢杆菌苯偶酰还原酶在体外可利用NADPH产生光学纯的(S)-苯偶姻，且苯环远端的酮基被不对称还原。蜡样芽孢杆菌苯偶酰还原酶与酵母开放阅读框YIR036C蛋白的氨基酸同源性为31%，与哺乳动物蝶呤还原酶的同源性为28%-30%，共享SDR家族的七个共有残基。我们分离出了编码酵母YIR036C蛋白和沙鼠蝶呤还原酶的基因，这两种重组蛋白在体外也可将苯偶酰还原为(S)-苯偶姻。绿色荧光蛋白标记的蜡样芽孢杆菌苯偶酰还原酶分布于蜡样芽孢杆菌细胞的双极细胞质中。蜡样芽孢杆菌苯偶酰还原酶、酵母YIR036C蛋白和沙鼠蝶呤还原酶的不对称还原将被用于生产重要的手性化合物。
