Structural basis of the π-stacking network governing cofactor-substrate cooperativity of SbSOMT
SAM-dependent methyltransferases are ubiquitous enzyme catalyzing methylation of substrate by consuming S-adenosyl methionine (SAM) cofactor. In this study, we discovered the positive cofactor-substrate cooperativity of SbSOMT and uncovered the underlying mechanism at structural basis. The binding kinetics analyses show that SbSOMT exhibits bilateral positive cooperativity except the unproductive pairing between SAM and product due to the steric hindrance of an additional methyl group. Unprecedentedly, two cofactor-analogous inhibitors, S-adenosyl homocysteine (SAH) and sinefungin demonstrated opposite effects on substrate binding kinetics while attaining positive cooperativity. SAM-resembling sinefungin demonstrated higher foldchange in the acceleration of substrate association rate constant than deceleration of dissociation rate constant. Integrating SbSOMT structural insights at multiple states, we identified the dynamical W279-driven {pi}-stacking network governs the cofactor-substrate cooperativity. SbSOMT exhibits closed conformation at first-ligand bound states, where the C-terminal W279 serves as the central plane for {pi}-{pi} interaction between N-terminal H196, catalytic H282, and/or substrate. We propose that the binding of first ligand overcome the energy barrier represented by W279 {pi}-stacking network, leading to the conformational landscapes in favor to second binding event. Our study highlights the intrinsic design of SbSOMT in pertaining enzyme competency and serves as a foundation to expand the knowledge and applications of cofactor-substrate cooperativity of the structurally diverse methyltransferases.