Functional reconstitution of the Mycobacterium tuberculosis long‐chain acyl‐CoA carboxylase from multiple acyl‐CoA subunits
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...
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Published in | The FEBS journal Vol. 284; no. 7; pp. 1110 - 1125 |
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Main Authors | , , , , , , , |
Format | Journal Article |
Language | English |
Published |
England
Blackwell Publishing Ltd
01.04.2017
Wiley |
Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 1742-464X 1742-4658 |
DOI: | 10.1111/febs.14046 |