Molecular basis for the diversification of lincosamide biosynthesis by pyridoxal phosphate-dependent enzymes
摘要
The biosynthesis of the lincosamide antibiotics lincomycin A and celesticetin involves the pyridoxal-5′-phosphate (PLP)-dependent enzymes LmbF and CcbF, which are responsible for bifurcation of the biosynthetic pathways. Despite recognizing the same S-glycosyl-l-cysteine structure of the substrates, LmbF catalyses thiol formation through β-elimination, whereas CcbF produces S-acetaldehyde through decarboxylation-coupled oxidative deamination. The structural basis for the diversification mechanism remains largely unexplored. Here we conduct structure–function analyses of LmbF and CcbF. X-ray crystal structures, docking and molecular dynamics simulations reveal that active-site aromatic residues play important roles in controlling the substrate binding mode and the reaction outcome. Furthermore, the reaction selectivity and oxygen-utilization of LmbF and CcbF were rationally engineered through structure- and calculation-based mutagenesis. Thus, the catalytic function of CcbF was switched to that of LmbF, and, remarkably, both LmbF and CcbF variants gained the oxidative-amidation activity to produce an unnatural S-acetamide derivative of lincosamide.