Substrate binding modulates the activity of Mycobacterium smegmatis G, a flavin-dependent monooxygenase involved in the biosynthesis of hydroxamate-containing siderophores.

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.

• Authors: Reeder Robinson, Pablo Sobrado
• Journal: Biochemistry (Biochemistry) Vol. 50 Issue 39 Pg. 8489-96 (Oct 04 2011) ISSN: 1520-4995 [Electronic] United States
• PMID: 21870809 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• Hydroxamic Acids
• Oxazoles
• Siderophores
• mycobactins
• Flavin-Adenine Dinucleotide
• Mixed Function Oxygenases
• lysine N-epsilon hydroxylase

Topics

• Flavin-Adenine Dinucleotide (metabolism)
• Hydroxamic Acids (metabolism)
• Kinetics
• Mixed Function Oxygenases (isolation & purification, metabolism)
• Mycobacterium smegmatis (metabolism)
• Oxazoles (metabolism)
• Oxygen Consumption
• Siderophores (biosynthesis)
• Substrate Specificity