Abstact

SAM-dependent methyltransferases are enzymes that catalyze the transfer of a methyl group from a cofactor to a substrate through an ordered or random sequential bi-bi mechanism. However, the structural dynamics governing binding cooperativity between the cofactor and substrate remain understudied. In this study, we demonstrate that SbSOMT, a plant O-methyltransferase, exhibits bilateral positive cooperativity between the cofactor and substrate, except the unproductive SbSOMT-SAM-pterostilbene complex. Furthermore, SbSOMT displayed substrate-binding kinetics that shift in response to the nature of bound-cofactor. Sinefungin-bound SbSOMT exhibited positive cooperativity primarily attributed to an increased substrate association rate constant (k(on)), whereas SAH-bound SbSOMT displayed positive cooperativity driven primarily by a decreased dissociation rate constant (k(off)). Structural analysis implies that these cooperativity switch and divergent binding kinetics stem from the interactions between the cofactor and substrate at the methyl binding site. Integrating structural insights reveals that a dynamic W279 pi-stacking network governs this cooperativity. Upon binding of the first ligand, H196, W279, and H282 rearrange to form a pi-stacking network, in which W279 serves as the essential central plane that also stacks with the substrate. Accordingly, W279A mutagenesis substantially impaired the substrate affinity, cooperativity and enzymatic activity.