School of Life Science, Shandong University of Technology, Zibo 255000, China.
Key Laboratory of Industrial Fermentation, Ministry of Education, Tianjin Key Lab of IndustrialMicrobiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457,China.
Int J Mol Sci. 2020 Mar 5;21(5):1788. doi: 10.3390/ijms21051788.
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 -diaminopimelate dehydrogenase (-DAPDH) from Symbiobacterium thermophilum, was the first -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.
通过还原胺化作用从 2-酮酸生产 D-氨基酸是一种很有吸引力的途径，因为它具有高产率和环境安全性。来自嗜热共生菌的 D-氨基庚二酸脱氢酶（StDAPDH）是第一个显示 2-酮酸胺化的 D-氨基庚二酸脱氢酶。此外，与其他内消旋二氨基庚二酸脱氢酶相比，StDAPDH 表现出出色的热稳定性。然而，StDAPDH 的辅因子是 NADP（H），在工业应用中比 NAD（H）少见。因此，需要对 StDAPDH 的辅因子进行工程改造。在这项研究中，靶向 NADPH 腺苷部分周围高度保守的辅因子结合位点，以确定辅因子特异性。发现环内的赖氨酸残基对于 StDAPDH 的辅因子特异性至关重要。用精氨酸取代赖氨酸导致丙酮酸与 NADH 作为辅因子的活性。K159R 与丙酮酸的亲和力与 NADH 或 NADPH 作为辅因子时相等，无论突变如何。分子动力学模拟表明，精氨酸的大位阻和 NADH 与精氨酸之间盐桥的相互作用可能限制了 NADH 的自由运动，促使突变体 K159R 形成稳定的活性构象。这些结果为进一步了解 StDAPDH 的催化机制以及 StDAPDH 的辅因子工程提供了指导。
