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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