Conformational transitions induced by NADH binding promote reduction half-reaction in...

2-Hydroxybiphenyl-3-monoxygenase from Pseudomonas azelaica is an effective catalyst of the regiospecific conversions of various aromatic compounds. A comprehensive understanding of the complete catalytic cycle, including the as yet unclear details of NADH binding, NADH/FAD interaction as well as related conformational changes could facilitate the rational design of improved enzyme variants for practical applications. Induced fit formation of a specific pocket for the nicotinamide ring at NADH binding has been revealed using advanced molecular simulation methods including metadynamics and QM/MM modeling. The resulting triple stacking interaction of the nicotinamide as well as isoalloxazine rings and evolutionarily correlated amino acid residues of the active site greatly contributes to the stabilization of the charge-transfer complex and determines the Pro-S stereospecificity of the hydride transfer and the low energy barrier 11 kcal/mol. Then the resulting FADH^- anion undergoes the consequent conformational transition of the FAD isoalloxazine ring from the open out to the closed in position which is followed by the binding of an oxygen molecule what is crucial for the next step of substrate oxidation and the completion of the catalytic cycle.