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Ser67Asp and His68Asp substitutions in candida parapsilosis carbonyl reductase alter the coenzyme specificity and enantioselectivity of ketone reduction.
Appl Environ Microbiol. 2009 Apr; 75(7):2176-83.AE

Abstract

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.

Authors+Show Affiliations

Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi 214122, People's Republic of China.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

19201968

Citation

Zhang, Rongzhen, et al. "Ser67Asp and His68Asp Substitutions in Candida Parapsilosis Carbonyl Reductase Alter the Coenzyme Specificity and Enantioselectivity of Ketone Reduction." Applied and Environmental Microbiology, vol. 75, no. 7, 2009, pp. 2176-83.
Zhang R, Xu Y, Sun Y, et al. Ser67Asp and His68Asp substitutions in candida parapsilosis carbonyl reductase alter the coenzyme specificity and enantioselectivity of ketone reduction. Appl Environ Microbiol. 2009;75(7):2176-83.
Zhang, R., Xu, Y., Sun, Y., Zhang, W., & Xiao, R. (2009). Ser67Asp and His68Asp substitutions in candida parapsilosis carbonyl reductase alter the coenzyme specificity and enantioselectivity of ketone reduction. Applied and Environmental Microbiology, 75(7), 2176-83. https://doi.org/10.1128/AEM.02519-08
Zhang R, et al. Ser67Asp and His68Asp Substitutions in Candida Parapsilosis Carbonyl Reductase Alter the Coenzyme Specificity and Enantioselectivity of Ketone Reduction. Appl Environ Microbiol. 2009;75(7):2176-83. PubMed PMID: 19201968.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Ser67Asp and His68Asp substitutions in candida parapsilosis carbonyl reductase alter the coenzyme specificity and enantioselectivity of ketone reduction. AU - Zhang,Rongzhen, AU - Xu,Yan, AU - Sun,Ying, AU - Zhang,Wenchi, AU - Xiao,Rong, Y1 - 2009/02/05/ PY - 2009/2/10/entrez PY - 2009/2/10/pubmed PY - 2009/5/5/medline SP - 2176 EP - 83 JF - Applied and environmental microbiology JO - Appl. Environ. Microbiol. VL - 75 IS - 7 N2 - 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. SN - 1098-5336 UR - https://www.unboundmedicine.com/medline/citation/19201968/Ser67Asp_and_His68Asp_substitutions_in_candida_parapsilosis_carbonyl_reductase_alter_the_coenzyme_specificity_and_enantioselectivity_of_ketone_reduction_ L2 - http://aem.asm.org/cgi/pmidlookup?view=long&pmid=19201968 DB - PRIME DP - Unbound Medicine ER -