The mmaA2 gene of Mycobacterium tuberculosis encodes the distal cyclopropane synthase of the alpha-mycolic acid.

Infection with Mycobacterium tuberculosis (Mtb) remains a severe global health problem that has prompted an aggressive search for new antibiotic targets and vaccine strategies for this persistent pathogen. Recently, a wide variety of genetic determinants of Mtb pathogenicity have been identified, including several genes involved in the biogenesis of the complex Mtb cell envelope. Among these are the mycolic acid cyclopropane synthases, a family of proteins that modify the major cell envelope lipids of Mtb with a diversity of cyclopropane rings. Despite substantial sequence identity, these proteins catalyze highly specific cyclopropane modifications, including proximal modification of the alpha-mycolate (pcaA) and trans-cyclopropane modification (cmaA2). Here we report the mycolic acid modification function of a third cyclopropane synthase, mmaA2, through the creation and analysis of an M. tuberculosis mmaA2 null mutant. Unexpectedly, mmaA2 is essential for the distal cyclopropane modification of the alpha-mycolate, a function previously attributed to cmaA1. alpha-Mycolates of a cmaA1 null mutant were unaffected, demonstrating that cmaA1 is not required for alpha-mycolate modification. Although fully cyclopropanated methoxymycolates are produced in the mmaA2 mutant, cis-cyclopropanation is impaired, leading to accumulation of unsaturated methoxymycolate derivatives. This study establishes mmaA2 as a distal cyclopropane synthase of the alpha-mycolates of M. tuberculosis and the first cyclopropane synthase to modify both alpha- and oxygenated mycolates. These results expand our knowledge of the biosynthesis of the Mtb cell envelope and will allow further elucidation of the relationship between Mtb pathogenesis and the fine structure of mycolic acids.