Family Report for: A85-Mycolyl-transferase

Mycobacterium and Corynebacterium are important genera of the Corynebacteriales order, the members of which are characterized by an atypical diderm cell envelope. Indeed the cytoplasmic membrane of these bacteria is surrounded by a thick mycolic acid-arabinogalactan-peptidoglycan (mAGP) covalent polymer. The mycolic acid-containing part of this complex associates with other lipids (mainly trehalose monomycolate (TMM) and trehalose dimycolate (TDM)) to form an outer membrane. The metabolism of mycolates in the cell envelope is governed by esterases called mycoloyltransferases that catalyze the transfer of mycoloyl chains from TMM to another TMM molecule or to other acceptors such as the terminal arabinoses of arabinogalactan or specific polypeptides. In this review we present an overview of this family of Corynebacteriales enzymes, starting with their expression, localization, structure and activity to finally discuss their putative functions in the cell. In addition, we show that Corynebacteriales possess multiple mycoloyltransferases encoding genes in their genome. The reason for this multiplicity is not known, as their function in mycolates biogenesis appear to be only partially redundant. It is thus possible that, in some species living in specific environments, some mycoloyltransferases have evolved to gain some new functions. In any case, the few characterized mycoloyltransferases are very important for the bacterial physiology and are also involved in adaptation in the host where they constitute major secreted antigens. Although not discussed in this review, all these functions make them interesting targets for the discovery of new antibiotics and promising vaccines candidates. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazu and Dr. Federica Migliardo.

The Mycobacterium tuberculosis 30 kDa major secretory protein (antigen 85B) is the most abundant protein exported by M. tuberculosis, as well as a potent immunoprotective antigen and a leading drug target. A mycolyl transferase of 285 residues, it is closely related to two other mycolyl transferases, each of molecular mass 32 kDa: antigen 85A and antigen 85C. All three catalyze transfer of the fatty acid mycolate from one trehalose monomycolate to another, resulting in trehalose dimycolate and free trehalose, thus helping to build the bacterial cell wall. We have determined two crystal structures of M. tuberculosis antigen 85B (ag85B), initially by molecular replacement using antigen 85C as a probe. The apo ag85B model is refined against 1.8 A data, to an R-factor of 0.196 (R(free) is 0.276), and includes all residues except the N-terminal Phe. The active site immobilizes a molecule of the cryoprotectant 2-methyl-2,4-pentanediol. Crystal growth with addition of trehalose resulted in a second ag85B crystal structure (1.9 A resolution; R-factor is 0.195; R(free) is 0.285). Trehalose binds in two sites at opposite ends of the active-site cleft. In our proposed mechanism model, the trehalose at the active site Ser126 represents the trehalose liberated by temporary esterification of Ser126, while the other trehalose represents the incoming trehalose monomycolate just prior to swinging over to the first trehalose site to displace the mycolate from its serine ester. Our proposed interfacial mechanism minimizes aqueous exposure of the apolar mycolates. Based on the trehalose-bound structure, we suggest a new class of antituberculous drugs, made by connecting two trehalose molecules by an amphipathic linker.

The antigen 85 (ag85) complex, composed of three proteins (ag85A, B and C), is a major protein component of the Mycobacterium tuberculosis cell wall. Each protein possesses a mycolyltransferase activity required for the biogenesis of trehalose dimycolate (cord factor), a dominant structure necessary for maintaining cell wall integrity. The crystal structure of recombinant ag85C from M. tuberculosis, refined to a resolution of 1.5 A, reveals an alpha/beta-hydrolase polypeptide fold, and a catalytic triad formed by Ser 124, Glu 228 and His 260. ag85C complexed with a covalent inhibitor implicates residues Leu 40 and Met 125 as components of the oxyanion hole. A hydrophobic pocket and tunnel extending 21 A into the core of the protein indicates the location of a probable trehalose monomycolate binding site. Also, a large region of conserved surface residues among ag85A, B and C is a probable site for the interaction of ag85 proteins with human fibronectin.

Mycobacterium and Corynebacterium are important genera of the Corynebacteriales order, the members of which are characterized by an atypical diderm cell envelope. Indeed the cytoplasmic membrane of these bacteria is surrounded by a thick mycolic acid-arabinogalactan-peptidoglycan (mAGP) covalent polymer. The mycolic acid-containing part of this complex associates with other lipids (mainly trehalose monomycolate (TMM) and trehalose dimycolate (TDM)) to form an outer membrane. The metabolism of mycolates in the cell envelope is governed by esterases called mycoloyltransferases that catalyze the transfer of mycoloyl chains from TMM to another TMM molecule or to other acceptors such as the terminal arabinoses of arabinogalactan or specific polypeptides. In this review we present an overview of this family of Corynebacteriales enzymes, starting with their expression, localization, structure and activity to finally discuss their putative functions in the cell. In addition, we show that Corynebacteriales possess multiple mycoloyltransferases encoding genes in their genome. The reason for this multiplicity is not known, as their function in mycolates biogenesis appear to be only partially redundant. It is thus possible that, in some species living in specific environments, some mycoloyltransferases have evolved to gain some new functions. In any case, the few characterized mycoloyltransferases are very important for the bacterial physiology and are also involved in adaptation in the host where they constitute major secreted antigens. Although not discussed in this review, all these functions make them interesting targets for the discovery of new antibiotics and promising vaccines candidates. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazu and Dr. Federica Migliardo.

The maintenance of the highly hydrophobic cell wall is central to the survival of Mycobacterium tuberculosis within its host environment. The antigen 85 proteins (85A, 85B, and 85C) of M. tuberculosis help maintain the integrity of the cell wall 1) by catalyzing the transfer of mycolic acids to the cell wall arabinogalactan and 2) through the synthesis of trehalose dimycolate (cord factor). Additionally, these secreted proteins allow for rapid invasion of alveolar macrophages via direct interactions between the host immune system and the invading bacillus. Here we describe two crystal structures: the structure of antigen 85C co-crystallized with octylthioglucoside as substrate, resolved to 2.0 A, and the crystal structure of antigen 85A, which was solved at a resolution of 2.7 A. The structure of 85C with the substrate analog identifies residues directly involved in substrate binding. Elucidation of the antigen 85A structure, the last of the three antigen 85 homologs to be solved, shows that the active sites of the three antigen 85 proteins are virtually identical, indicating that these share the same substrate. However, in contrast to the high level of conservation within the substrate-binding site and the active site, surface residues disparate from the active site are quite variable, indicating that three antigen 85 enzymes are needed to evade the host immune system.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is known to secrete a number of highly immunogenic proteins that are thought to confer pathogenicity, in part, by mediating binding to host tissues. Among these secreted proteins are the trimeric antigen 85 (Ag85) complex and the related MPT51 protein, also known as FbpC1. While the physiological function of Ag85, a mycolyltransferase required for the biosynthesis of the cell wall component alpha,alpha'-trehalose dimycolate (or cord factor), has been identified recently, the function of the closely related MPT51 (approximately 40% identity with the Ag85 components) remains to be established. The crystal structure of M.tuberculosis MPT51, determined to 1.7 A resolution, shows that MPT51, like the Ag85 components Ag85B and Ag85C2, folds as an alpha/beta hydrolase, but it does not contain any of the catalytic elements required for mycolyltransferase activity. Moreover, the absence of a recognizable alpha,alpha'-trehalose monomycolate-binding site and the failure to detect an active site suggest that the function of MPT51 is of a non-enzymatic nature and that MPT51 may in fact represent a new family of non-catalytic alpha/beta hydrolases. Previous experimental evidence and the structural similarity to some integrins and carbohydrate-binding proteins led to the hypothesis that MPT51 might have a role in host tissue attachment, whereby ligands may include the serum protein fibronectin and small sugars.

The Mycobacterium tuberculosis 30 kDa major secretory protein (antigen 85B) is the most abundant protein exported by M. tuberculosis, as well as a potent immunoprotective antigen and a leading drug target. A mycolyl transferase of 285 residues, it is closely related to two other mycolyl transferases, each of molecular mass 32 kDa: antigen 85A and antigen 85C. All three catalyze transfer of the fatty acid mycolate from one trehalose monomycolate to another, resulting in trehalose dimycolate and free trehalose, thus helping to build the bacterial cell wall. We have determined two crystal structures of M. tuberculosis antigen 85B (ag85B), initially by molecular replacement using antigen 85C as a probe. The apo ag85B model is refined against 1.8 A data, to an R-factor of 0.196 (R(free) is 0.276), and includes all residues except the N-terminal Phe. The active site immobilizes a molecule of the cryoprotectant 2-methyl-2,4-pentanediol. Crystal growth with addition of trehalose resulted in a second ag85B crystal structure (1.9 A resolution; R-factor is 0.195; R(free) is 0.285). Trehalose binds in two sites at opposite ends of the active-site cleft. In our proposed mechanism model, the trehalose at the active site Ser126 represents the trehalose liberated by temporary esterification of Ser126, while the other trehalose represents the incoming trehalose monomycolate just prior to swinging over to the first trehalose site to displace the mycolate from its serine ester. Our proposed interfacial mechanism minimizes aqueous exposure of the apolar mycolates. Based on the trehalose-bound structure, we suggest a new class of antituberculous drugs, made by connecting two trehalose molecules by an amphipathic linker.

The antigen 85 (ag85) complex, composed of three proteins (ag85A, B and C), is a major protein component of the Mycobacterium tuberculosis cell wall. Each protein possesses a mycolyltransferase activity required for the biogenesis of trehalose dimycolate (cord factor), a dominant structure necessary for maintaining cell wall integrity. The crystal structure of recombinant ag85C from M. tuberculosis, refined to a resolution of 1.5 A, reveals an alpha/beta-hydrolase polypeptide fold, and a catalytic triad formed by Ser 124, Glu 228 and His 260. ag85C complexed with a covalent inhibitor implicates residues Leu 40 and Met 125 as components of the oxyanion hole. A hydrophobic pocket and tunnel extending 21 A into the core of the protein indicates the location of a probable trehalose monomycolate binding site. Also, a large region of conserved surface residues among ag85A, B and C is a probable site for the interaction of ag85 proteins with human fibronectin.