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

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.异柠檬酸脱氢酶（isocitrate dehydrogenases, IDHs）通过可逆磷酸化调控三羧酸循环（tricarboxylic acid cycle, TCA cycle）与乙醛酸分流（glyoxylate shunt）之间的碳流分布，该过程可影响病原菌毒力。现有研究仅证实NADP+特异性异柠檬酸脱氢酶（NADP+-specific IDHs, NADP-IDHs）可发生磷酸化，而NAD+特异性异柠檬酸脱氢酶（NAD+-specific IDHs, NAD-IDHs）是否易发生磷酸化仍不明确。本研究首次对源自嗜麦芽窄食单胞菌（Stenotrophomonas maltophilia）与甘蔗黄单胞菌（Xanthomonas sacchari）的两种NAD-IDHs及其磷酸化调控机制进行了系统表征。研究人员通过质谱（mass spectrometry）技术鉴定出SmIDH中的Ser80为磷酸化位点，并通过定点突变（site-directed mutagenesis）实验完成了功能验证。乙酸盐诱导可使IDH/ICL比酶活比值降低约78%，这与磷酸化介导的调控规律相符。通过改造XsIDH与XsAceK的关键识别区域，本研究提升了XsIDH的磷酸化效率，为相关进化机制解析提供了新的见解。本研究可为后续针对嗜麦芽窄食单胞菌与甘蔗黄单胞菌的新型抗菌药物靶点研究提供助力。