Identification of Structural Determinants of NAD(P)H Selectivity and Lysine Binding in Lysine N⁶-monooxygenase

Abstract

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.

Department(s)

Chemistry

Comments

National Science Foundation, Grant 1021384

Keywords and Phrases

C4a-hydroperoxyflavin; Flavin-dependent monooxygenases; L-lysine hydroxylase; N-hydroxylating monooxygenases; Nocobactin; Siderophores; Virulence factor

International Standard Serial Number (ISSN)

1096-0384; 0003-9861

Document Type

Article - Journal

Document Version

Citation

File Type

text

Language(s)

English

Rights

© 2024 Elsevier, All rights reserved.

Publication Date

15 Sep 2016

PubMed ID

27503802

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