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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Acyl-CoAs are crucial compounds involved in essential metabolic pathways such as the Krebs cycle and lipid, carbohydrate, and amino acid metabolisms, and they are also key signal molecules involved ...in the transcriptional regulation of lipid biosynthesis in many organisms. In this study, we took advantage of the high selectivity of mass spectrometry and developed an ion-pairing reverse-phase high-pressure liquid chromatography electrospray ionization high-resolution mass spectrometry (IP-RP-HPLC/ESI-HRMS) method to carry on a comprehensive analytical determination of the wide range of fatty acyl-CoAs present in actinomycetes. The advantage of using a QTOF spectrometer resides in the excellent mass accuracy over a wide dynamic range and measurements of the true isotope pattern that can be used for molecular formula elucidation of unknown analytes. As a proof of concept, we used this assay to determine the composition of the fatty acyl-CoA pools in
Mycobacterium
,
Streptomyces
, and
Corynebacterium
species, revealing an extraordinary difference in fatty acyl-CoA amounts and species distribution between the three genera and between the two species of mycobacteria analyzed, including the presence of different chain-length carboxy-acyl-CoAs, key substrates of mycolic acid biosynthesis. The method was also used to analyze the impact of two fatty acid synthase inhibitors on the acyl-CoA profile of
Mycobacterium smegmatis
, which showed some unexpected low levels of C
24
acyl-CoAs in the isoniazid-treated cells. This robust, sensitive, and reliable method should be broadly applicable in the studies of the wide range of bacteria metabolisms in which acyl-CoA molecules participate.
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.
1 Microbiology Division, Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
2 Department ...of Molecular Biology and Biochemistry and Department of Chemistry, University of California, Irvine, CA 92612, USA
3 Microbiology Division, Facultad de Ciencias Médicas, Universidad Nacional de Rosario, Argentina
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 µM. 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.
Correspondence Hugo Gramajo gramajo{at}ibr.gov.ar
Abbreviations: ACCase, acyl-CoA carboxylase; CT, carboxyltransferase; FAME, fatty acid methyl ester; FAS, fatty acid synthase; MAME, mycolic acid methyl ester; MDR, multi-drug resistant
Three supplementary figures, with associated methods, are available with the online version of this paper.
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