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

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. (C) 2016 Elsevier Inc. All rights reserved.