Development of a Selective Activity-Based Probe for Adenylating Enzymes: Profiling MbtA Involved in Siderophore Biosynthesis from Mycobacterium tuberculosis

• Published in: ACS chemical biology 2012-10, Vol.7 (10), p.1653-1658
• Main Authors: Duckworth, Benjamin P, Wilson, Daniel J, Nelson, Kathryn M, Boshoff, Helena I, Barry, Clifton E, Aldrich, Courtney C
• Format: Article
• Language: English
• Subjects: Adenine - metabolism; Adenosine - analogs & derivatives; Adenosine - pharmacology; Antitubercular Agents - pharmacology; Ligases - antagonists & inhibitors; Ligases - isolation & purification; Microbial Sensitivity Tests; Models, Molecular; Mycobacterium tuberculosis - enzymology; Mycobacterium tuberculosis - metabolism; Siderophores - metabolism; Structure-Activity Relationship
• ISSN: 1554-8929; 1554-8937
• DOI: 10.1021/cb300112x

MbtA is an adenylating enzyme from Mycobacterium tuberculosis that catalyzes the first step in the biosynthesis of the mycobactins. A bisubstrate inhibitor of MbtA (Sal-AMS) was previously described that displays potent antitubercular activity under iron-replete as well as iron-deficient growth conditions. This finding is surprising since mycobactin biosynthesis is not required under iron-replete conditions and suggests off-target inhibition of additional biochemical pathways. As a first step toward a complete understanding of the mechanism of action of Sal-AMS, we have designed and validated an activity-based probe (ABP) for studying Sal-AMS inhibition in M. tuberculosis. This probe labels pure MbtA as well as MbtA in mycobacterial lysate, and labeling can be completely inhibited by preincubation with Sal-AMS. Furthermore, this probe provides a prototypical core scaffold for the creation of ABPs to profile any of the other 66 adenylating enzymes in Mtb or the multitude of adenylating enzymes in other pathogenic bacteria.