Nonenzymatic Regeneration of Styrene Monooxygenase for Catalysis

Styrene monooxygenase (SMO) is a two-component flavoprotein catalyzing the selective epoxidation of various C═C double bonds. For cell-free catalysis, traditionally a cascade of NAD(P)H:flavin oxidoreductase, nicotinamide cofactor (NADH), and NADH regeneration enzyme is required to supply StyA with the reduced flavin adenine dinucleotide cofactor (FADH2) required for catalysis. Herein, we present a more direct and efficient FADH2 regeneration system using the nicotinamide cofactor mimic 1-benzyl-1,4-dihydronicotinamide (BNAH) as the sole reductant. Thus, BNAH replaces two enzymes and the nicotinamide cofactor, resulting in a significantly simplified reaction system.