Structural dynamics of the π-stacking network governing cofactor-substrate cooperativity of SbSOMT methyltransferase

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 π-stacking network governs this cooperativity. Upon binding of the first ligand, H196, W279, and H282 rearrange to form a π-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.