Insight into the Highly Conserved and Differentiated Cofactor-Binding Sites of meso-Diaminopimelate Dehydrogenase StDAPDH

Abstract

meso-Diaminopimelate dehydrogenase (meso-DAPDH) is a good candidate for one-step synthesis of d-amino acid from 2-keto acids. Our previous research revealed the classification of meso-DAPDH family and showed that type II meso-DAPDH, such as the meso-DAPDH from Symbiobacterium thermophilum (StDAPDH), could catalyze reductive amination. In this article, seven residues of StDAPDH, which are highly conserved in each subfamily but are different between two subfamilies, were targeted to explore the relationships between structure and function. Determination of kinetic parameters showed that the amino acid residues, including P69, K159, V68, S90, V14, and V156, played very important roles in the catalytic function of StDAPDH. Molecular dynamics simulation revealed that these point mutations reduced the productive conformations by the newly formed or eliminated interactions between the residues and ligands. These results strengthen our understanding of the catalytic mechanism and evolution of meso-DAPDH and can aid future endeavors in enzyme engineering.

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

“…Moreover, 15 hydrogen bond interactions were found between NADH and residues of the K159R mutant. As shown in our previous study, arginine was more easily able to form a salt bridge interaction than lysine [30]. In conclusion, the likely mechanism may be that, the larger steric hindrance of arginine compared to lysine and the formation of a salt bridge stabilized the active conformation of the enzyme when NADH was used as the cofactor.…”

“…Divergent evolution has been reported in the meso-DAPDH family [8]. Amino acid residues V14, V68, P69, T70, S90, V156, and K159 of StDAPDH have been reported in our previous study [30]. 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.…”

“…It has been reported that during catalysis by StDAPDH, H154 assists a water molecule to attract the amino acid intermediate, which is formed by hydride transfer from the Cα of meso-DAP to the C4N of the nicotinamide ring of NADP + [30]. This indicates that the orientation of the nicotinamide ring of NADP + plays a key role in catalysis by meso-DAPDH.…”

“…The K159R mutant was constructed in our previous study [30] following the protocol of whole-plasmid polymerase chain reaction in [8]. The bacterial strains producing wild-type and K159R mutant StDAPDH were maintained in our lab.…”

“…Cells were harvested by centrifugation and then disrupted by sonication (Scientz, Ningbo, China). Purified proteins were prepared by nickel chelate affinity chromatography using previously published protocols [8], which was newly cited by reference [30] and [39].…”