Highly active mutants of carbonyl reductase S1 with inverted coenzyme specificity and production of optically active alcohols.
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 V(max) and K(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 dehydrogenase.
Morikawa
Souichi
S
Life Science Research Laboratories, Kaneka Corporation, Takasago, Hyogo, Japan. Souichi.Morikawa@kn.kaneka.co.jp
Nakai
Takahisa
T
Yasohara
Yoshihiko
Y
Nanba
Hirokazu
H
Kizaki
Noriyuki
N
Hasegawa
Junzo
J
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000438:Alcohols / Q000138:chemical synthesis* / Q000737:chemistry; D000595:Amino Acid Sequence; D003067:Coenzymes / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity; D013816:Thermodynamics
D000438:Alcohols; D003067:Coenzymes; D000429:Alcohol Oxidoreductases