Highly Active Mutants of Carbonyl Reductase S1 with Inverted Coenzyme Specificity and Production of Optically Active Alcohols
| Autor: | Hirokazu Nanba, Junzo Hasegawa, Takahisa Nakai, Yoshihiko Yasohara, Souichi Morikawa, Noriyuki Kizaki |
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| Rok vydání: | 2005 |
| Předmět: |
Models
Molecular Carbonyl Reductase Stereochemistry Molecular Sequence Data Mutant Coenzymes Reductase Formate dehydrogenase Applied Microbiology and Biotechnology Biochemistry Cofactor Substrate Specificity Analytical Chemistry Amino Acid Sequence Molecular Biology chemistry.chemical_classification Sequence Homology Amino Acid biology Organic Chemistry Mutagenesis Wild type Stereoisomerism General Medicine Alcohol Oxidoreductases Enzyme chemistry Alcohols Mutagenesis Site-Directed biology.protein Thermodynamics Biotechnology |
| Zdroj: | Bioscience, Biotechnology, and Biochemistry. 69:544-552 |
| ISSN: | 1347-6947 0916-8451 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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