Journal
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
Volume 75, Issue 7, Pages 2176-2183Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/AEM.02519-08
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Funding
- National Science Foundation of China [20776060]
- National Key Basic Research and Development Program of China [2003CB716008, 2009CB724706]
- Hi-Tech Research and Development Program of China [2007AA02Z200]
- Ministry of Education, People's Republic of China
- Changjiang Scholars and Innovative Research Team in University [IRT0532]
- Talents of Discipline to Universities [111-2-06]
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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.
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