Ser67Asp and His68Asp substitutions in candida parapsilosis carbonyl reductase alter the coenzyme specificity and enantioselectivity of ketone reduction.
A short-chain carbonyl reductase (SCR) from Candida parapsilosis catalyzes an anti-Prelog reduction of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol (PED) and exhibits coenzyme specificity for NADPH over NADH. By using site-directed mutagenesis, the mutants were designed with different combinations of Ser67Asp, His68Asp, and Pro69Asp substitutions inside or adjacent to the coenzyme binding pocket. All mutations caused a significant shift of enantioselectivity toward the (R)-configuration during 2-hydroxyacetophenone reduction. The S67D/H68D mutant produced (R)-PED with high optical purity and yield in the NADH-linked reaction. By kinetic analysis, the S67D/H68D mutant resulted in a nearly 10-fold increase and a 20-fold decrease in the k(cat)/K(m) value when NADH and NADPH were used as the cofactors, respectively, but maintaining a k(cat) value essentially the same with respect to wild-type SCR. The ratio of K(d) (dissociation constant) values between NADH and NADPH for the S67D/H68D mutant and SCR were 0.28 and 1.9 respectively, which indicates that the S67D/H68D mutant has a stronger preference for NADH and weaker binding for NADPH. Moreover, the S67D/H68D enzyme exhibited a secondary structure and melting temperature similar to the wild-type form. It was also found that NADH provided maximal protection against thermal and urea denaturation for S67D/H68D, in contrast to the effective protection by NADP(H) for the wild-type enzyme. Thus, the double point mutation S67D/H68D successfully converted the coenzyme specificity of SCR from NADP(H) to NAD(H) as well as the product enantioselectivity without disturbing enzyme stability. This work provides a protein engineering approach to modify the coenzyme specificity and enantioselectivity of ketone reduction for short-chain reductases.
Zhang
Rongzhen
R
Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi 214122, People's Republic of China.
Xu
Yan
Y
Sun
Ying
Y
Zhang
Wenchi
W
Xiao
Rong
R
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D003067:Coenzymes / Q000378:metabolism*; D004795:Enzyme Stability; D007659:Ketones / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D003067:Coenzymes; D007659:Ketones; D009243:NAD; D009249:NADP; D000429:Alcohol Oxidoreductases