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      Functional reconstitution of the Mycobacterium tuberculosis long-chain acyl-CoA carboxylase from multiple acyl-CoA subunits

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          Abstract

          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 (ACCase) whose subunit composition and physiological roles have not yet been clearly established. A rather controversial data in the literature refers to the exact protein composition and substrate specificity of the enzyme complex that produces the long-chain α-carboxy-acyl-CoAs; one of the substrates involved in the last step of condensation mediated by the polyketide synthase Pks13 to synthesize mature mycolic acids. Here we have successfully reconstituted the so called long-chain acyl-CoA carboxylase complex (LCC) from its purified components: the α-subunit AccA3, the ε-subunit AccE5 and the two β-subunits AccD4 and AccD5, and demonstrated that the four subunits are essential for its LCC activity. Furthermore, we also showed by substrate competition experiments and the use of a specific inhibitor of the AccD5 subunit, that its 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-C 24–26-CoA, used as condensing units for the biosynthesis of all the lipids present in this pathogen.

          Graphical abstract

          A new long-chain acyl-CoA carboxylase complex (LCC) formed by a biotinylated α subunit (AccA3), two different carboxyltransferase subunits β(AccD4) and β′ (AccD5) and an ε subunit (AccE5), was characterized and showed to be able to carboxylate short- as well as long-chain acyl-CoAs. This supercomplex could provide the substrates needed for fatty acids and mycolic acid biosynthesis, as well as for methyl-branched lipid biosynthesis of M. tuberculosis.

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          Author and article information

          Journal
          101229646
          32231
          FEBS J
          FEBS J.
          The FEBS journal
          1742-464X
          1742-4658
          18 March 2017
          19 March 2017
          April 2017
          01 April 2018
          : 284
          : 7
          : 1110-1125
          Affiliations
          [1 ]Laboratory of Physiology and Genetics of Actinomycetes, Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina, France
          [2 ]CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Département Tuberculose et Biologie des Infections, 205 route de Narbonne BP64182, F-31077 Toulouse, France
          [3 ]Université de Toulouse, UPS, IPBS, F-31077 Toulouse, France
          Author notes
          [* ]Corresponding author: Hugo Gramajo ( gramajo@ 123456ibr-conicet.gov.ar ), Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET). Ocampo y Esmeralda. Rosario (2000), Argentina., Tel.: +54 341 4237070 ext. 642, Fax: +54 341 4237070 ext. 607
          Article
          PMC5393044 PMC5393044 5393044 nihpa854182
          10.1111/febs.14046
          5393044
          28222482
          Categories
          Article

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