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

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

Small RNA Mcr11 requires the transcription factor AbmR for stable expression and regulates genes involved in the central metabolism of Mycobacterium tuberculosis

“…Disruption of components in any of these complexes can cause growth defects in Mtb, some of which are dependent upon the nutrient mixture present in the growth media (Shi & Ehrt, ; Venugopal et al, ). The genes upstream of Rv3282 are accD5 and accE5 , which are needed for a long chain aceyl‐CoA carboxylase enzymatic complex that generates substrates for fatty acid and mycolic acid biosynthesis (Bazet Lyonnet et al, , ). fadA3 is expected to have a role in lipid degradation through beta‐oxidation, although its role in the metabolic processes of Mtb has not been well defined.…”

Small RNA Mcr11 regulates genes involved in the central metabolism ofMycobacterium tuberculosisand requires 3’ sequence along with the transcription factor AbmR for stable expression

“…Disruption of components in any of these complexes can cause growth defects in Mtb, some of which are dependent upon the nutrient mixture present in the growth media (18, 19). The genes upstream of Rv3282 are accD5 and accE5 , which are needed for a long chain aceyl-CoA carboxylase enzymatic complex that generates substrates for fatty acid and mycolic acid biosynthesis (53, 54).…”

Increasing the Ascomycin Yield by Relieving the Inhibition of Acetyl/Propionyl-CoA Carboxylase by the Signal Transduction Protein GlnB

“…Firstly, BC attaches a carbonate to the biotin prosthetic group of BCCP to form carboxybiotin, a reaction that depends on the hydrolysis of ATP. Secondly, CT transfers the carboxyl from biotin to acyl-CoA to produce the corresponding carboxylated acyl-CoA ( Oh et al, 2006 ; Lyonnet et al, 2017 ). In Escherichia coli , Azospirillum brasilense and Synechocystis sp.…”

“…In actinobacteria, both ACC and PCC are complex multi-subunit enzymes. They share the same α subunit composed of BC and BCCP functional domains, but possess a specific β subunit for substrate recognition and a specific ε subunit for protein-protein interactions ( Diacovich et al, 2002 ; Lyonnet et al, 2017 ). Furthermore, both ACC and PCC catalyze similar two-step carboxylation reactions ( Oh et al, 2006 ), and their catalytic substrates even overlap to a certain extent ( Diacovich et al, 2002 ).…”