木薯MeGRXC3 通过其关键功能位点介导对MeCAT1 酶活性的特异性调控OA

Glutaredoxins play a critical role in plant stress responses by modulating the redox state of cysteine residues in target proteins.Previous studies have shown that the cassava CC-type glutaredoxin MeGRXC3 interacts with catalase MeCAT1 and negatively regulates its enzyme activity,yet the underlying molecular mechanism remains unclear.To elucidate the regulatory effects of key functional sites of MeGRXC3 on MeCAT1 enzyme activity,this study constructed expression vectors carrying mu-tations in active domain(C21AC24A)and glutathione-binding domain(G75L)of MeGRXC3.Subcellular localization analysis indicated that mutations in these functional domains did not alter the intracellular localization of MeGRXC3.Yeast two-hybrid,bimolecular fluorescence complementation,and pull-down assays confirmed that MeGRXC3 interacts with MeCAT1 both in vivo and in vitro,and this interaction is independent of the aforementioned functional domains.However,transient expression in to-bacco and biochemical analysis revealed that the negative regulation of MeCAT1 enzyme activity by MeGRXC3 is closely related to these two functional domains:mutation of the active domain attenuated the inhibitory effect,while mutation of the glu-tathione-binding domain completely abolished the inhibition,leading to a significant enhancement of MeCAT1 enzyme activity.These results demonstrate that the catalytic active domain and the predicted glutathione-binding domain of MeGRXC3,while dispensable for its physical interaction with MeCAT1,are essential for inhibiting MeCAT1 enzymatic activity.This indicates that MeGRXC3 likely binds to MeCAT1 through other regions/domains,subsequently depending on its conserved functional sites to execute its inhibitory role.This finding provides a crucial foundation for further elucidating the precise molecular mechanism by which MeGRXC3 regulates MeCAT1 activity at the post-translational level.