Abstract
Mycobacterium smegmatis G (MbsG) is a flavin-dependent monooxygenase that catalyzes the NAD(P)H- and oxygen-dependent hydroxylation of the terminal amino group on the side chain of l-lysine in the biosynthetic pathway of the siderophore mycobactin. Mycobactins are essential for mycobacterium growth under iron-limiting conditions encountered during infection in mammals. Thus, enzymes involved in the biosynthesis of mycobactin represent potential drug targets. MbsG was expressed in Escherichia coli and purified using metal affinity and ionic exchange chromatographies. Recombinant MbsG represents the first member of this class of enzymes isolated in the active form, with a tightly bound FAD cofactor. The k cat value for formation of hydroxylated l-lysine under steady-state conditions was 5.0 min -1, and K m values of 0.21 mM for l-lysine, 1.1 mM for NADH, and 2.4 mM for NADPH were calculated. The enzyme functioned as an oxidase when the activity of MbsG was measured by monitoring oxygen consumption in the absence of l-lysine, oxidizing NADH and NADPH with k cat values of 59 and 49 min -1, respectively. Under these conditions, MbsG produced both hydrogen peroxide and superoxide. In contrast, when l-lysine was present, the reaction became more coupled, producing hydroxylated l-lysine and decreasing the oxidase activity. These results suggest that substrate binding modulates the function of MbsG from an oxidase to a monooxygenase. © 2011 American Chemical Society.
Recommended Citation
R. Robinson and P. Sobrado, "Substrate Binding Modulates the Activity of Mycobacterium Smegmatis G, a Flavin-dependent Monooxygenase Involved in the Biosynthesis of Hydroxamate-containing Siderophores," Biochemistry, vol. 50, no. 39, pp. 8489 - 8496, American Chemical Society, Oct 2011.
The definitive version is available at https://doi.org/10.1021/bi200933h
Department(s)
Chemistry
International Standard Serial Number (ISSN)
1520-4995; 0006-2960
Document Type
Article - Journal
Document Version
Citation
File Type
text
Language(s)
English
Rights
© 2024 American Chemical Society, All rights reserved.
Publication Date
04 Oct 2011
PubMed ID
21870809
