Characterization of Enzymatic Properties of Single Mutants of Homoserine Dehydrogenase from Corynebacterium glutamicum

Site-directed mutagenesis was used to improve the catalytic activity of homoserine dehydrogenase (HSD) to reduce its feedback inhibition and repression by metabolites in the pathway. HSD was docked with the substrate homoserine, and its spatial structure was analyzed. Two key sites, Gly25 and Asp61, were selected for site-directed saturation mutation. The activity screening showed that the mutants A61L and G25G had significantly increased enzyme activity when compared to the wild type (WT). The kinetics and enzymatic properties of these two mutants were studied. It was found that compared to the WT enzyme, the K_m values of G25G and A61L decreased, the substrate affinity increased, and the enzyme activity increased by 1.21 and 1.35 times, respectively; the n value decreased, and the positive synergy increased. The optimum temperature for A61L and G25G was 40 ℃, the same as that for WT; the optimum pH for A61L and WT was 8.0, which was lower than that (8.5) for G25G. The half-lives of A61L and G25G were 1 h longer and 0.5 h shorter than that of WT, respectively. Low concentrations of K⁺, Mg²⁺, and Ca²⁺ could activate the mutants and WT, while different concentrations of methanol, ethanol, acetonitrile and dimethyl sulfoxide had significantly inhibitory effects on the mutants and WT. At inhibitor concentrations of 1–25 mmol/L, the inhibitory effect was significantly weaker on the mutants than on WT. The mutants G25G and A61L showed improved enzyme activity and weakened allosteric inhibition. This study provides a reference for optimizing the biosynthetic pathway of HSD and constructing strains capable of producing high yield of methionine, threonine and isoleucine.