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

• Published in: The FEBS journal Vol. 284; no. 7; pp. 1110 - 1125
• Main Authors: Bazet Lyonnet, Bernardo, Diacovich, Lautaro, Gago, Gabriela, Spina, Lucie, Bardou, Fabienne, Lemassu, Anne, Quémard, Annaïk, Gramajo, Hugo
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.04.2017; Wiley
• Subjects: Acetyl Coenzyme A - metabolism; Acyl Coenzyme A - metabolism; acyl‐CoA carboxylase; Antibiotic resistance; Antibiotics; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Biochemistry, Molecular Biology; Biosynthesis; Biotechnology; Carbon-Carbon Ligases - genetics; Carbon-Carbon Ligases - metabolism; Carboxylation; Catalysis; Chains; Cloning, Molecular; Composition; Condensation; Enzyme Assays; Enzymes; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Kinetics; Life Sciences; lipid biosynthesis; Lipids; Malonyl Coenzyme A - metabolism; Microbiology and Parasitology; Mycobacterium tuberculosis; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - metabolism; Mycolic acids; Mycolic Acids - metabolism; Pathogenicity; Pathogens; Polyketide synthase; Polyketide Synthases - genetics; Polyketide Synthases - metabolism; Protein composition; Protein Engineering; Protein Subunits - genetics; Protein Subunits - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Substrate Specificity; Substrates; Subunit structure; Tuberculosis; acyl-CoA carboxylase; lipid biosynthesis; mycolic acids; Mycobacterium tuberculosis
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.14046

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 β 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. A new long‐chain acyl‐CoA carboxylase complex, formed by a biotinylated α subunit, two different carboxyltransferase subunits β and β′ and an ε subunit, was characterized and shown to be able to carboxylate short‐ as well as long‐chain acyl‐CoAs. This supercomplex could provide the substrates needed for fatty acid and mycolic acid biosynthesis as well as for methyl‐branched lipid biosynthesis of Mycobacterium tuberculosis.