Identification of structural determinants of NAD(P)H selectivity and lysine binding in lysine N6-monooxygenase

•NbtG is flavin monooxygenase that catalyzes the hydroxylation of lysine.•We identified conserved residues that might be involved in NAD(P)H and lysine binding.•R301 is essential for NADPH selectivity.•E216 is important for Lys binding.

l-lysine (l-Lys) N6-monooxygenase (NbtG), from Nocardia farcinica, is a flavin-dependent enzyme that catalyzes the hydroxylation of l-Lys in the presence of oxygen and NAD(P)H in the biosynthetic pathway of the siderophore nocobactin. NbtG displays only a 3-fold preference for NADPH over NADH, different from well-characterized related enzymes, which are highly selective for NADPH. The structure of NbtG with bound NAD(P)+ or l-Lys is currently not available. Herein, we present a mutagenesis study targeting M239, R301, and E216. These amino acids are conserved and located in either the NAD(P)H binding domain or the l-Lys binding pocket. M239R resulted in high production of hydrogen peroxide and little hydroxylation with no change in coenzyme selectivity. R301A caused a 300-fold decrease on kcat/Km value with NADPH but no change with NADH. E216Q increased the Km value for l-Lys by 30-fold with very little change on the kcat value or in the binding of NAD(P)H. These results suggest that R301 plays a major role in NADPH selectivity by interacting with the 2′-phosphate of the adenine-ribose moiety of NADPH, while E216 plays a role in l-Lys binding.

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