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

Abstract l -lysine ( l -Lys) N 6 -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 k cat / K m value with NADPH but no change with NADH. E216Q increased the K m value for l -Lys by 30-fold with very little change on the k cat 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.