Semi-Rational Design of Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum Improved Its Activity toward Hydroxypyruvate for D-serine Synthesis

Abstract

D-serine plays an essential role in the field of medicine and cosmetics. Diaminopimelate dehydrogenase (DAPDH) is a kind of oxidoreductase that can reduce keto acid into the corresponding D-amino acid. Because of its high stereoselectivity and lack of by-product production, DAPDH has become the preferred enzyme for the efficient one-step synthesis of D-amino acids. However, the types of DAPDH with a reductive amination function reported so far are limited. Although the DAPDH from Symbiobacterium thermophilum (StDAPDH) demonstrates reductive amination activity toward a series of macromolecular keto acids, activity toward hydroxypyruvate (HPPA) for D-serine synthesis has not been reported. In this study, we investigated the activity of the available StDAPDH/H227V toward HPPA by measuring the desired product D-serine. After homologous structure modeling and docking analysis concerning the substrate-binding pocket, four residues, D92, D122, M152, and N253, in the active pocket were predicted for catalyzing HPPA. Through single-point saturation mutation and iterative mutation, a mutant D92E/D122W/M152S was obtained with an 8.64-fold increase in enzyme activity, exhibiting a specific activity of 0.19 U/mg and kcat value of 3.96 s−1 toward HPPA. Using molecular dynamics simulation, it was speculated that the increase in enzyme activity might be related to the change in substrate pocket size and the enhancement of the interactions between the substrate and key residues.