Highly active mutants of carbonyl reductase S1 with inverted coenzyme specificity and production of optically active alcohols
2005
Morikawa, S. (Kaneka Corp., Takasago, Hyogo (Japan)) | Nakai, T. | Yasohara, Y. | Nanba, H. | Kizaki, N. | Hasegawa, J.
A wild type NADPH-dependent carbonyl reductase from Candida magnoliae (reductase S1) has been found not to utilize NADH as a coenzyme. A mutation to exchange the coenzyme specificity in reductase S1 has been designed by computer-aided methods, including three-dimensional structure modeling and in silico screening of enzyme mutants. Site-directed mutagenesis has been used to introduce systematic substitutions of seven or eight amino acid residues onto the adenosine-binding pocket of the enzyme according to rational computational design. The resulting S1 mutants show NADH-dependency and have lost their ability to utilize NADPH as a coenzyme, but retain those catalytic activities. Kinetic parameter Vsub(max) and Ksub(m) values of those mutants for NADH are 1/3- to 1/10-fold those of the wild type enzyme for NADPH. As a model system for industrial production of optically active alcohols, the S1 mutants can be applied to an asymmetric reduction of ketones, cooperating with a coenzyme-regeneration system that uses an NAD-dependent formate dehydro-genase.
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