Altered Cofactor Preference of Thermostable StDAPDH by a Single Mutation at K159

International Journal of Molecular Sciences

Huihui Song¹  Qinyuan Ma²  Zhiyun Li³  Mingfei Liu³  Xiuzhen Gao³  Xinming Sun³ 

[1] Tianjin Key Lab of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China;Key Laboratory of Industrial Fermentation, Ministry of Education;School of Life Science, Shandong University of Technology, Zibo 255000, China;

关键词: meso-diaminopimelate dehydrogenase;    d-amino acid;    thermostable enzyme;    cofactor engineering;    molecular dynamics simulations;   

DOI  :  10.3390/ijms21051788

来源: DOAJ

【 摘 要 】

D-amino acid production from 2-keto acid by reductive amination is an attractive pathway because of its high yield and environmental safety. StDAPDH, a meso-diaminopimelate dehydrogenase (meso-DAPDH) from Symbiobacterium thermophilum, was the first meso-DAPDH to show amination of 2-keto acids. Furthermore, StDAPDH shows excellent thermostability compared to other meso-DAPDHs. However, the cofactor of StDAPDH is NADP(H), which is less common than NAD(H) in industrial applications. Therefore, cofactor engineering for StDAPDH is needed. In this study, the highly conserved cofactor binding sites around the adenosine moiety of NADPH were targeted to determine cofactor specificity. Lysine residues within a loop were found to be critical for the cofactor specificity of StDAPDH. Replacement of lysine with arginine resulted in the activity of pyruvic acid with NADH as the cofactor. The affinity of K159R to pyruvic acid was equal with NADH or NADPH as the cofactor, regardless of the mutation. Molecular dynamics simulations revealed that the large steric hindrance of arginine and the interaction of the salt bridge between NADH and arginine may have restricted the free movement of NADH, which prompted the formation of a stable active conformation of mutant K159R. These results provide further understanding of the catalytic mechanism of StDAPDH and guidance for the cofactor engineering of StDAPDH.

【 授权许可】

Unknown