Analysis of coenzyme A activated compounds in actinomycetes

Cabruja, Matías; Lyonnet, Bernardo Bazet; Millan, Gustavo Enrique; Gramajo, Hugo; Gago, Gabriela

doi:10.1007/s00253-016-7635-0

Cited by 9 publications

(10 citation statements)

References 43 publications

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“…Acyl-CoAs extracted from a fixed number of cells were analysed by LC-MS as previously described [31]. As shown in figure 4 a , an increase in the content of acetyl-CoA and malonyl-CoA was observed in the cultures grown in the presence of ATc.…”

Section: Resultsmentioning

confidence: 99%

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A conditional mutant of the fatty acid synthase unveils unexpected cross talks in mycobacterial lipid metabolism

et al. 2017

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Unlike most bacteria, mycobacteria rely on the multi-domain enzyme eukaryote-like fatty acid synthase I (FAS I) to make fatty acids de novo. These metabolites are precursors of the biosynthesis of most of the lipids present both in the complex mycobacteria cell wall and in the storage lipids inside the cell. In order to study the role of the type I FAS system in Mycobacterium lipid metabolism in vivo, we constructed a conditional mutant in the fas-acpS operon of Mycobacterium smegmatis and analysed in detail the impact of reduced de novo fatty acid biosynthesis on the global architecture of the cell envelope. As expected, the mutant exhibited growth defect in the non-permissive condition that correlated well with the lower expression of fas-acpS and the concomitant reduction of FAS I, confirming that FAS I is essential for survival. The reduction observed in FAS I provoked an accumulation of its substrates, acetyl-CoA and malonyl-CoA, and a strong reduction of C12 to C18 acyl-CoAs, but not of long-chain acyl-CoAs (C19 to C24). The most intriguing result was the ability of the mutant to keep synthesizing mycolic acids when fatty acid biosynthesis was impaired. A detailed comparative lipidomic analysis showed that although reduced FAS I levels had a strong impact on fatty acid and phospholipid biosynthesis, mycolic acids were still being synthesized in the mutant, although with a different relative species distribution. However, when triacylglycerol degradation was inhibited, mycolic acid biosynthesis was significantly reduced, suggesting that storage lipids could be an intracellular reservoir of fatty acids for the biosynthesis of complex lipids in mycobacteria. Understanding the interaction between FAS I and the metabolic pathways that rely on FAS I products is a key step to better understand how lipid homeostasis is regulated in this microorganism and how this regulation could play a role during infection in pathogenic mycobacteria.

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Section: Resultsmentioning

confidence: 99%

“…Acyl-CoAs were extracted following the protocol described by Cabruja et al [ 31 ]. Bacterial cultures were harvested by centrifugation at 5800 g for 15 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

A conditional mutant of the fatty acid synthase unveils unexpected cross talks in mycobacterial lipid metabolism

et al. 2017

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“…Recently, the mycobacterial EchA6 protein was shown to be a shuttle of long-chain acyl-CoAs, possibly for the FAS-II system [27]; one can imagine that this mechanism might also serve for other metabolic complexes that also use very long-chain fatty acyl-CoAs as substrates, such as the LCC complex. The low K m determined for the C 20 -CoA, 10 and 5 fold lower than the values obtained for acetyl-CoA and propionyl-CoA, respectively, is coherent with the low concentration of the very long-chain acyl-CoAs found within mycobacteria cells [28]. Yet, the catalytic efficiency of the LCC complex is between 10 and 50 fold lower, compared with the ACC and PCC activities of the same complex, respectively; probably reflecting the lower need for very long-chain carboxy-acyl-CoAs with respect to malonyl-CoA and methylmalonyl-CoA which are required for an array of fatty acid elongation pathways in mycobacteria.…”

Section: Discussionmentioning

confidence: 68%

“…In the reactions containing C 16 -CoA as substrate, C 24 -CoA was used as standard. Separation of the different acyl-CoAs was made by ion pairing-reverse phase high performance liquid chromatography (IP-RP-HPLC) as described in Cabruja et al [28]. UV 260nm chromatogram peaks were integrated using Bruker Daltonics´s Compass™ DataAnalysis (Bruker Corporation, Billerica, MA, United States) software; peak areas were copied to spreadsheets and used for the quantification of the substrates and products.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2017

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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-C24–26-CoA, used as condensing units for the biosynthesis of all the lipids present in this pathogen.

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A review of the synthesis, bioanalysis, and chemical reactivity of xenobiotic acyl-coenzyme a thioesters

Skonberg¹,

Olsen²

2023

Med Chem Res

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Analysis of coenzyme A activated compounds in actinomycetes

Cited by 9 publications

References 43 publications

A conditional mutant of the fatty acid synthase unveils unexpected cross talks in mycobacterial lipid metabolism

A conditional mutant of the fatty acid synthase unveils unexpected cross talks in mycobacterial lipid metabolism

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

A review of the synthesis, bioanalysis, and chemical reactivity of xenobiotic acyl-coenzyme a thioesters

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