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

Abstract

MbtA is an adenylating enzyme from <i>Mycobacterium tuberculosis</i> 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 <i>M. tuberculosis</i>. 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 <i>Mtb</i> or the multitude of adenylating enzymes in other pathogenic bacteria