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Applied and Environmental Microbiology, April 2009, p. 2176-2183, Vol. 75, No. 7
0099-2240/09/$08.00+0 doi:10.1128/AEM.02519-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Ser67Asp and His68Asp Substitutions in Candida parapsilosis Carbonyl Reductase Alter the Coenzyme Specificity and Enantioselectivity of Ketone Reduction
,
Rongzhen Zhang,1
Yan Xu,1,4*
Ying Sun,1
Wenchi Zhang,2 and
Rong Xiao3
Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi 214122, People's Republic of China,1 National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, People's Republic of China,2 Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08854,3 State Key Laboratory for Food Science and Technology, Jiangnan University, Wuxi 214122, People's Republic of China4
Received 3 November 2008/ Accepted 30 January 2009
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 kcat/Km value when NADH and NADPH were used as the cofactors, respectively, but maintaining a kcat value essentially the same with respect to wild-type SCR. The ratio of Kd (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.
* Corresponding author. Mailing address: School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China. Phone: 86 510 85918201. Fax: 86 510 85864112. E-mail: biosean{at}yahoo.com.cn
Published ahead of print on 5 February 2009.
Supplemental material for this article may be found at http://aem.asm.org/.
Applied and Environmental Microbiology, April 2009, p. 2176-2183, Vol. 75, No. 7
0099-2240/09/$08.00+0 doi:10.1128/AEM.02519-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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