Mutation of a key amino acid residue of meso-diaminopimelate dehydrogenase enhances the catalytic activity toward alkyl substituted 2-keto acids

Abstract:

[Background] Efficient biosynthesis of D-amino acids is highly desired. Meso-diaminopimelate dehydrogenase (DAPDH) synthesizes D-amino acids from 2-keto acids and ammonia. [Objective] To increase the catalytic activity against the alkyl substituted 2-keto acids. [Methods] Based on structural analysis and mutation results from previously selected sites, the saturation mutagenesis was carried out at the amino acid residue H227 of DAPDH from Symbiobacterium thermophilum (StDAPDH). The resulting mutant library was subjected to screening using D-alanine, D-2-aminobutyric acid, D-norvaline, and D-glutamic acid as substrates. [Results] The mutants H227Q and H227N were obtained. Mutant H227Q was found to have 10.9-, 11.5-, 8.6- and 7.6-folds improved enzyme activity toward pyruvic acid, 2-oxobutyric acid, 2-oxovaleric acid and 2-ketoglutaric acid, respectively, compared to that of wild-type enzyme. The kinetic parameters indicated that mutant H227Q increased the turnover number of the enzyme and the affinity of the enzyme for the substrate simultaneously, so that the catalytic efficiency (kcat/Km) of pyruvic acid was 9.4 folds higher than that of wild-type enzyme. Molecular modeling analysis of interaction between mutant H227Q and product amino acid, indicating that glutamine at position H227 forms a hydrogen bond with the carboxylic acid of the amino acid, so that the distance between the α-hydrogen atom of product amino acid and C4 of coenzyme nicotinamide ring was shortened. [Conclusion] Directed evolution technology has been successfully used to improve the catalytic activity of DADPH for alkyl-substituted 2-keto acids, which is helpful for the development of new high-efficiency biocatalysts. These efforts also provide guidance for our future engineering of this enzyme about more challenging D-amino acids.