Fig. 2: Development of highly efficient MsMDH^G11Q variant based on structural comparison between MsMDH and CgMDH.

a The OAA/malate binding site and b NADH/NAD⁺ binding site of the two crystal structures; MsMDH (left, green model) and CgMDH (right, magenta model). The conformation of OAA is obtained from a superimposed structure of Methylobacterium extorquens MDH (PDB code 4ROS). The mobile loop is distinguished by different color schemes of light blue (MsMDH) and gray (CgMDH). The observed residual differences are indicated by red color. c Site-directed mutagenesis and the relative activities of the MsMDH and CgMDH variants in comparison with the activity of MsMDH (n = 3 independent experiments). Data are presented as mean values ± standard deviation. Each of the corresponding variants are indicated by the same color scheme and arrow. The MsMDH^A224S variant, which is labeled as ‘Inclusion body’, was expressed insoluble. The MsMDH^G11Q variant is indicated by a green star. d Optimal pH of the MsMDH^G11Q variant (n = 3 independent experiments). Data are presented as mean values ± standard deviation. The specific activity at pH 10.0 was determined from a single data. e Catalytic performance of the MsMDH^G11Q variant at different pH (n = 3 independent experiments). Data are presented as mean values ± standard deviation. f kcat, g km, h kcat/km, and i ki values of the MsMDH^G11Q variant at different pH. The ki value of the MsMDH^G11Q variant at pH 9.0 is shown in number because its value is significantly higher than the rest of the ki values. Data f–i are presented as parameters ± standard error. The standard errors from determining the kinetic parameters using OriginPro 2019 software (n = the number of mean velocity data at specific pH) are shown as bars.