Study on the phosphorylation of NAD+-specific isocitrate dehydrogenase in the pathogenic bacteria Stenotrophomonas maltophilia and Xanthomonas sacchari

Isocitrate dehydrogenases (IDHs) regulate the distribution of carbon flux between the TCA cycle and glyoxylate shunt through reversible phosphorylation that influences pathogen virulence. Current studies only indicate that NADP+-specific IDHs (NADP-IDHs) can be phosphorylated. Whether NAD+-specific IDHs (NAD-IDHs) are susceptible to phosphorylation remains unknown. In this study, two NAD-IDHs and their regulation by phosphorylation from Stenotrophomonas maltophilia and Xanthomonas sacchari were characterised for the first time. Ser80 was identified by mass spectrometry as the phosphorylation site in SmIDH, which was functionally validated through site-directed mutagenesis. Acetate induction led to an approximately 78% decrease in the ratio of IDH/ICL specific enzyme activity, consistent with phosphorylation-mediated regulation. By modifying key recognition regions in XsIDH and XsAceK, the phosphorylation efficiency of XsIDH was improved, revealing evolutionary insights. It may enable further investigations for the new antibacterial drug targets in S. maltophilia and X. sacchari.