RESUMEN
Mycobacterium tuberculosis produces a large number of structurally diverse lipids that have been implicated in the pathogenicity, persistence and antibiotic resistance of this organism. Most building blocks involved in the biosynthesis of all these lipids are generated by acyl-CoA carboxylases whose subunit composition and physiological roles have not yet been clearly established. Inconclusive data in the literature refer to the exact protein composition and substrate specificity of the enzyme complex that produces the long-chain α-carboxy-acyl-CoAs, which are substrates involved in the last step of condensation mediated by the polyketide synthase 13 to synthesize mature mycolic acids. Here we have successfully reconstituted the long-chain acyl-CoA carboxylase (LCC) complex from its purified components, the α subunit (AccA3), the ε subunit (AccE5) and the two ß subunits (AccD4 and AccD5), and demonstrated that the four subunits are essential for its activity. Furthermore, we also showed by substrate competition experiments and the use of a specific inhibitor that the AccD5 subunit's role in the carboxylation of the long acyl-CoAs, as part of the LCC complex, was structural rather than catalytic. Moreover, AccD5 was also able to carboxylate its natural substrates, acetyl-CoA and propionyl-CoA, in the context of the LCC enzyme complex. Thus, the supercomplex formed by these four subunits has the potential to generate the main substrates, malonyl-CoA, methylmalonyl-CoA and α-carboxy-C24-26 -CoA, used as condensing units for the biosynthesis of all the lipids present in this pathogen.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ligasas de Carbono-Carbono/metabolismo , Mycobacterium tuberculosis/metabolismo , Sintasas Poliquetidas/metabolismo , Subunidades de Proteína/metabolismo , Acetilcoenzima A/metabolismo , Acilcoenzima A/metabolismo , Proteínas Bacterianas/genética , Ligasas de Carbono-Carbono/genética , Clonación Molecular , Pruebas de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Cinética , Malonil Coenzima A/metabolismo , Mycobacterium tuberculosis/genética , Ácidos Micólicos/metabolismo , Sintasas Poliquetidas/genética , Ingeniería de Proteínas , Subunidades de Proteína/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Mycobacteria contain a large variety of fatty acids which are used for the biosynthesis of several complex cell wall lipids that have been implicated in the ability of the organism to resist host defenses. The building blocks for the biosynthesis of all these lipids are provided by a fairly complex set of acyl-CoA carboxylases (ACCases) whose subunit composition and roles within these organisms have not yet been clearly established. Previous biochemical and structural studies provided strong evidences that ACCase 5 from Mycobacterium tuberculosis is formed by the AccA3, AccD5 and AccE5 subunits and that this enzyme complex carboxylates acetyl-CoA and propionyl-CoA with a clear substrate preference for the latest. In this work we used a genetic approach to unambiguously demonstrate that the products of both accD5 and accE5 genes are essential for the viability of Mycobacterium smegmatis. By obtaining a conditional mutant on the accD5-accE5 operon, we also demonstrated that the main physiological role of this enzyme complex was to provide the substrates for fatty acid and mycolic acid biosynthesis. Furthermore, enzymatic and biochemical analysis of the conditional mutant provided strong evidences supporting the notion that AccD5 and/or AccE5 have an additional role in the carboxylation of long chain acyl-CoA prior to mycolic acid condensation. These studies represent a significant step towards a better understanding of the roles of ACCases in mycobacteria and confirm ACCase 5 as an interesting target for the development of new antimycobacterial drugs.
Asunto(s)
Ligasas de Carbono-Carbono/genética , Pared Celular/genética , Lípidos/biosíntesis , Mycobacterium smegmatis/genética , Acetilcoenzima A , Acilcoenzima A , Secuencia de Aminoácidos , Pared Celular/metabolismo , Ácidos Grasos/genética , Ácidos Grasos/metabolismo , Lipogénesis , Mycobacterium smegmatis/metabolismo , Ácidos Micólicos/metabolismoRESUMEN
Mycolic acids are essential for the survival, virulence and antibiotic resistance of the human pathogen Mycobacterium tuberculosis. Inhibitors of mycolic acid biosynthesis, such as isoniazid and ethionamide, have been used as efficient drugs for the treatment of tuberculosis. However, the increase in cases of multidrug-resistant tuberculosis has prompted a search for new targets and agents that could also affect synthesis of mycolic acids. In mycobacteria, the acyl-CoA carboxylases (ACCases) provide the building blocks for de novo fatty acid biosynthesis by fatty acid synthase (FAS) I and for the elongation of FAS I products by the FAS II complex to produce meromycolic acids. By generating a conditional mutant in the accD6 gene of Mycobacterium smegmatis, we demonstrated that AccD6 is the essential carboxyltransferase component of the ACCase 6 enzyme complex implicated in the biosynthesis of malonyl-CoA, the substrate of the two FAS enzymes of Mycobacterium species. Based on the conserved structure of the AccD5 and AccD6 active sites we screened several inhibitors of AccD5 as potential inhibitors of AccD6 and found that the ligand NCI-172033 was capable of inhibiting AccD6 with an IC(50) of 8 microM. The compound showed bactericidal activity against several pathogenic Mycobacterium species by producing a strong inhibition of both fatty acid and mycolic acid biosynthesis at minimal inhibitory concentrations. Overexpression of accD6 in M. smegmatis conferred resistance to NCI-172033, confirming AccD6 as the main target of the inhibitor. These results define the biological role of a key ACCase in the biosynthesis of membrane and cell envelope fatty acids, and provide a new target, AccD6, for rational development of novel anti-mycobacterial drugs.