Engineering a d-lactate dehydrogenase that can super-efficiently utilize NADPH and NADH as cofactors.
Engineering the cofactor specificity of a natural enzyme often results in a significant decrease in its activity on original cofactor. Here we report that a NADH-dependent dehydrogenase (d-LDH) from Lactobacillus delbrueckii 11842 can be rationally engineered to efficiently use both NADH and NADPH as cofactors. Point mutations on three amino acids (D176S, I177R, F178T) predicted by computational analysis resulted in a modified enzyme designated as d-LDH*. The Kcat/Km of the purified d-LDH* on NADPH increased approximately 184-fold while the Kcat/Km on NADH also significantly increased, showing for the first time that a rationally engineered d-LDH could exhibit comparable activity on both NADPH and NADH. Further kinetic analysis revealed that the enhanced affinity with NADH or NADPH and the significant increased Kcat of d-LDH* resulted in the significant increase of d-LDH* activity on both NADPH and NADH. This study thus demonstrated that the cofactor specificity of dehydrogenase can be broadened by using targeted engineering approach, and the engineered enzyme can efficiently function in NADH-rich, or NADPH-rich, or NADH and NADPH-rich environment.
Department of Cellular Biology, University of Science and Technology of China, Hefei, China.|CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Hengkai
H
Meng
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
Pi
P
Liu
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.|Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
Hongbing
H
Sun
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Zhen
Z
Cai
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Jie
J
Zhou
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.|College of Pharmacy, Nankai University, Tianjin, China.
Jianping
J
Lin
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Yin
Y
Li
D003067:Coenzymes / Q000378:metabolism*; D003198:Computer Simulation; D007700:Kinetics; D045022:Lactate Dehydrogenases / Q000235:genetics* / Q000378:metabolism*; D048668:Lactobacillus delbrueckii / Q000201:enzymology*; D060847:Metabolic Engineering; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D017354:Point Mutation
D003067:Coenzymes; D009243:NAD; D009249:NADP; D045022:Lactate Dehydrogenases; C071195:D-lactate dehydrogenase