Characterization of Lipoyl Synthase from Mycobacterium tuberculosis

The prevalence of multiple and extensively drug-resistant strains of Mycobacterium tuberculosis (<i>Mtb</i>), the causative agent of tuberculosis, is on the rise, necessitating the identification of new targets to combat an organism that has infected one-third of the world’s population, according to the World Health Organization. The biosynthesis of the lipoyl cofactor is one possible target, given its critical importance in cellular metabolism and the apparent lack of functional salvage pathways in <i>Mtb</i> that are found in humans and many other organisms. The lipoyl cofactor is synthesized <i>de novo</i> in two committed steps, involving the LipB-catalyzed transfer of an octanoyl chain derived from fatty acid biosynthesis to a lipoyl carrier protein and the LipA-catalyzed insertion of sulfur atoms at C6 and C8 of the octanoyl chain. A number of <i>in vitro</i> studies of lipoyl synthases from Escherichia coli, Sulfolobus solfataricus, and Thermosynechococcus elongatus have been conducted, but the enzyme from <i>Mtb</i> has not been characterized. Herein, we show that LipA from <i>Mtb</i> contains two [4Fe–4S] clusters and converts an octanoyl peptide substrate to the corresponding lipoyl peptide product via the same C6-monothiolated intermediate as that observed in the E. coli LipA reaction. In addition, we show that LipA from <i>Mtb</i> forms a complex with the H protein of the glycine cleavage system and that the strength of association is dependent on the presence of <i>S</i>-adenosyl-l-methionine. We also show that LipA from <i>Mtb</i> can complement a <i>lipA</i> mutant of E. coli, demonstrating the commonalities of the two enzymes. Lastly, we show that the substrate for LipA, which normally acts on a post-translationally modified protein, can be reduced to carboxybenzyl-octanoyllysine