The Acyl-AMP Ligase FadD32 and AccD4-containing Acyl-CoA Carboxylase Are Required for the Synthesis of Mycolic Acids and Essential for Mycobacterial Growth

Abstract

Mycolic acids are major and specific long-chain fatty acids of the cell envelope of several important human pathogens such as Mycobacterium tuberculosis, M. leprae, and Corynebacterium diphtheriae. Their biosynthesis is essential for mycobacterial growth and represents an attractive target for developing new antituberculous drugs. We have previously shown that the pks13 gene encodes condensase, the enzyme that performs the final condensation step of mycolic acid biosynthesis and is flanked by two genes, fadD32 and accD4. To determine the functions of the gene products we generated two mutants of C. glutamicum with an insertion/deletion within either fadD32 or accD4. The two mutant strains were deficient in mycolic acid production and exhibited the colony morphology that typifies the mycolate-less mutants of corynebacteria. Application of multiple analytical approaches to the analysis of the mutants demonstrated the accumulation of a tetradecylmalonic acid in the ⌬fadD32::km mutant and its absence from the ⌬accD4::km strain. The parental corynebacterial phenotype was restored upon the transfer of the wild-type fadD32 and accD4 genes in the mutants. These data demonstrated that both FadD32 and AccD4-containing acyl-CoA carboxylase are required for the production of mycolic acids. They also prove that the proteins catalyze, respectively, the activation of one fatty acid substrate and the carboxylation of the other substrate, solving the long-debated question of the mechanism involved in the condensation reaction. We used comparative genomics and applied a combination of molecular biology and proteomic technologies to the analysis of proteins that co-immunoprecipitated with AccD4. This resulted in the identification of AccA3 and AccD5 as subunits of the acyl-CoA carboxylase. Finally, we used conditionally replicative plasmids to show that both the fadD32 and accD4 genes are essential for the survival of M. smegmatis. Thus, in addition to Pks13, FadD32 and AccD4 are promising targets for the development of new antimicrobial drugs against pathogenic species of mycobacteria and related microorganisms.

Structural elucidation and genomic scrutiny of the C60–C100 mycolic acids of Segniliparus rotundus

“…As expected, the fas gene encoding de novo fatty acid synthase (FAS-I) displayed 59 % identity with that of M. tuberculosis H37Rv. The genes encoding the key enzymes of the mycolic acid condensation complex were present, including the condensing enzyme Pks13 (Portevin et al, 2004) and the Pks13-substrate activating proteins, the acyl-CoA carboxylase AccD4 and the acyl-AMP ligase FadD32 (Portevin et al, 2005); all of them were conserved in the same cluster and with an identity of 50-60 %. The genes encoding components of the elongation fatty acid synthase (FAS-II), producing the long meromycolic chains were also identified: genes encoding the acyl-CoA carboxylase subunit (AccD6) for formation of the FAS-II extender unit malonyl-CoA, the malonyl-CoA-ACP transacylase (FabD), the initiating b-ketoacyl-ACP synthase (FabH/Kas III) and the four genes encoding the successive steps of the FAS-II cycle.…”

AccD6, a Member of the Fas II Locus, Is a Functional Carboxyltransferase Subunit of the Acyl-Coenzyme A Carboxylase in Mycobacterium tuberculosis

“…In M. tuberculosis, the biotin carboxylation step is catalyzed by the ␣ subunit; there are three open reading frames (ORFs) that can encode the ␣ subunit (accA1 to -A3) in the genome. Carboxyl transfer is catalyzed by the ␤ subunit, and there are six ␤ subunits (accD1 to -D6) in the genome of the pathogen (6).Previously, the catalytic activities of the ␣ 3 , ␤ 4 , and ␤ 5 subunits were studied (10,11,22,24). However, the levels of expression of the various subunits have not been examined.…”

Crystal structure of the essential biotin‐dependent carboxylase AccA3 from Mycobacterium tuberculosis

“…Mycobacterium tuberculosis AccA3 thus provides the carboxylated biotin to all of the three essential AccD proteins 14, 15, 18, 19, 20. Moreover, the same interaction pattern has been found in MTb 11, 23.…”

“…MTb AccA3 has been shown to form functional Acetyl‐CoA Carboxylase complexes with AccD4, AccD5, and AccD6 14, 15, 18, 19, 20. The MTb AccA3‐AccD4 Acetyl‐CoA Carboxylase carboxylates long‐chain acyl‐CoA substrates.…”

“…The MTb AccA3‐AccD4 Acetyl‐CoA Carboxylase carboxylates long‐chain acyl‐CoA substrates. These are utilized in biosynthesis of mycobacterial specific very long‐chain fatty acids such as mycolic acids 11, 15, 18. AccA3–AccD5 have been shown to act mainly on propionyl‐CoA, producing methylmalonyl‐CoA, used for synthesis of methyl‐branched lipids, key components of the mycobacterial cell wall.…”