New mechanistic frontiers in carbohydrate-active enzymes revealed by QM/MM simulations

Carbohydrate-active enzymes (CAZymes) catalyze reactions central to glycan degradation, biosynthesis, and remodeling. Although their mechanisms have traditionally been described using a limited set of canonical models, recent studies are revealing a broader and more dynamic mechanistic landscape. We discuss how QM/MM-based enhanced-sampling methods have expanded our understanding of CAZyme catalysis by uncovering substrate-assisted pathways, complex proton-transfer processes, unconventional catalytic residues, and the roles of substrate distortion and enzyme dynamics in shaping reactivity. Examples from glycoside hydrolases, polysaccharide lyases, and glycosyltransferases include mechanisms that challenge classical Koshland-type descriptions, syn and anti β-elimination pathways in alginate lyases, and inverting glycosyltransferases with unconventional catalytic bases or reaction pathways. We also highlight how these insights can guide inhibitor and activity-based probe development and the engineering of enzymes with altered catalytic properties, illustrating the growing contribution of computational chemistry to understanding and manipulating CAZyme catalysis.