The mycobacterial acyltransferase PapA5 is required for biosynthesis of cell wall-associated phenolic glycolipids.

Phenolic glycolipids (PGLs) are non-covalently bound components of the outer membrane of many clinically relevant mycobacterial pathogens, and play important roles in pathogen biology. We report a mutational analysis that conclusively demonstrates that the conserved acyltransferase-encoding gene papA5 is essential for PGL production. In addition, we provide an in vitro acyltransferase activity analysis that establishes proof of principle for the competency of PapA5 to utilize diol-containing polyketide compounds of mycobacterial origin as acyl-acceptor substrates. Overall, the results reported herein are in line with a model in which PapA5 catalyses the acylation of diol-containing polyketides to form PGLs. These studies advance our understanding of the biosynthesis of an important group of mycobacterial glycolipids and suggest that PapA5 might be an attractive target for exploring the development of antivirulence drugs.

The dimycocerosate ester polyketide virulence factors of mycobacteria.

Recent advances in the study of mycobacterial lipids indicate that the class of outer membrane lipids known as dimycocerosate esters (DIMs) are major virulence factors of clinically relevant mycobacteria including Mycobacterium tuberculosis and Mycobacterium leprae. DIMs are a structurally intriguing class of polyketide synthase-derived wax esters discovered over seventy years ago, yet, little was known until recently about their biosynthesis. Availability of several mycobacterial genomes has accelerated progress toward clarifying steps in the DIM biosynthetic pathway and it is our belief that reviewing the bases of our current knowledge will clarify outstanding issues and help direct future endeavors.

Identification of phthiodiolone ketoreductase, an enzyme required for production of mycobacterial diacyl phthiocerol virulence factors.

Diacyl phthiocerol esters and their congeners are mycobacterial virulence factors. The biosynthesis of these complex lipids remains poorly understood. Insight into their biosynthesis will aid the development of rationally designed drugs that inhibit their production. In this study, we investigate a biosynthetic step required for diacyl (phenol)phthiocerol ester production, i.e., the reduction of the keto group of (phenol)phthiodiolones. We utilized comparative genomics to identify phthiodiolone ketoreductase gene candidates and provide a genetic analysis demonstrating gene function for two of these candidates. Moreover, we present data confirming the existence of a diacyl phthiotriol intermediate in diacyl phthiocerol biosynthesis. We also elucidate the mechanism underlying diacyl phthiocerol deficiency in some mycobacteria, such as Mycobacterium ulcerans and Mycobacterium kansasii. Overall, our findings shed additional light on the biosynthesis of an important group of mycobacterial lipids involved in virulence.

Crystal structure of PapA5, a phthiocerol dimycocerosyl transferase from Mycobacterium tuberculosis.

Polyketide-associated protein A5 (PapA5) is an acyltransferase that is involved in production of phthiocerol and phthiodiolone dimycocerosate esters, a class of virulence-enhancing lipids produced by Mycobacterium tuberculosis. Structural analysis of PapA5 at 2.75-A resolution reveals a two-domain structure that shares unexpected similarity to structures of chloramphenicol acetyltransferase, dihydrolipoyl transacetylase, carnitine acetyltransferase, and VibH, a non-ribosomal peptide synthesis condensation enzyme. The PapA5 active site includes conserved histidine and aspartic acid residues that are critical to PapA5 acyltransferase activity. PapA5 catalyzes acyl transfer reactions on model substrates that contain long aliphatic carbon chains, and two hydrophobic channels were observed linking the PapA5 surface to the active site with properties consistent with these biochemical activities and substrate preferences. An additional alpha helix not observed in other acyltransferase structures blocks the putative entrance into the PapA5 active site, indicating that conformational changes may be associated with PapA5 activity. PapA5 represents the first structure solved for a protein involved in polyketide synthesis in Mycobacteria.

Mycobacterial polyketide-associated proteins are acyltransferases: proof of principle with Mycobacterium tuberculosis PapA5.

Mycobacterium tuberculosis (Mt) produces complex virulence-enhancing lipids with scaffolds consisting of phthiocerol and phthiodiolone dimycocerosate esters (PDIMs). Sequence analysis suggested that PapA5, a so-called polyketide-associated protein (Pap) encoded in the PDIM synthesis gene cluster, as well as PapA5 homologs found in Mt and other species, are a subfamily of acyltransferases. Studies with recombinant protein confirmed that PapA5 is an acyltransferase [corrected]. Deletion analysis in Mt demonstrated that papA5 is required for PDIM synthesis. We propose that PapA5 catalyzes diesterification of phthiocerol and phthiodiolone with mycocerosate. These studies present the functional characterization of a Pap and permit inferences regarding roles of other Paps in the synthesis of complex lipids, including the antibiotic rifamycin.

Resistance to enediyne antitumor antibiotics by CalC self-sacrifice.

Antibiotic self-resistance mechanisms, which include drug elimination, drug modification, target modification, and drug sequestration, contribute substantially to the growing problem of antibiotic resistance among pathogenic bacteria. Enediynes are among the most potent naturally occurring antibiotics, yet the mechanism of resistance to these toxins has remained a mystery. We characterize an enediyne self-resistance protein that reveals a self-sacrificing paradigm for resistance to highly reactive antibiotics, and thus another opportunity for nonpathogenic or pathogenic bacteria to evade extremely potent small molecules.