Abstract
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.
Highlights
The meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum IAM14863 (StDAPDH) was the first meso-DAPDH reported to catalyze the asymmetric amination of 2-keto acids, with consequent D-amino acid production [1]
The combination of its substrate spectrum and thermostability indicate that StDAPDH has great potential in future enzyme engineering and industrial applications [1]
These residues were predicted as NADP+-binding residues and are highly conserved during the evolution of type II meso-DAPDHs, but they differ from the residues in type I meso-DAPDHs
Summary
The meso-diaminopimelate dehydrogenase (meso-DAPDH) from Symbiobacterium thermophilum IAM14863 (StDAPDH) was the first meso-DAPDH reported to catalyze the asymmetric amination of 2-keto acids, with consequent D-amino acid production [1]. Akita et al focused on the screening of more stable meso-DAPDHs for industrial production of D-amino acids. All members of the type II group, which is represented by StDAPDH, show obvious catalytic activity with 2-keto acids. The combination of its substrate spectrum and thermostability indicate that StDAPDH has great potential in future enzyme engineering and industrial applications [1]. Compared to NADP+/NADPH, NAD+/NADH has some advantages in industrial applications. There is little research reported on the cofactor engineering of meso-DAPDHs. In the present study, residue K159 was found to be the key residue for the cofactor specificity of StDAPDH. Molecular dynamics (MD) simulations were performed to investigate the underlying molecular mechanisms
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