The two carboxylases of Corynebacterium glutamicum essential for fatty acid and mycolic acid synthesis

Abstract

The suborder Corynebacterianeae comprises bacteria like Mycobacterium tuberculosis and Corynebacterium glutamicum, and these bacteria contain in addition to the linear fatty acids, unique alpha-branched beta-hydroxy fatty acids, called mycolic acids. Whereas acetyl-coenzyme A (CoA) carboxylase activity is required to provide malonyl-CoA for fatty acid synthesis, a new type of carboxylase is apparently additionally present in these bacteria. It activates the alpha-carbon of a linear fatty acid by carboxylation, thus enabling its decarboxylative condensation with a second fatty acid to afford mycolic acid synthesis. We now show that the acetyl-CoA carboxylase of C. glutamicum consists of the biotinylated alpha-subunit AccBC, the beta-subunit AccD1, and the small peptide AccE of 8.9 kDa, forming an active complex of approximately 812,000 Da. The carboxylase involved in mycolic acid synthesis is made up of the two highly similar beta-subunits AccD2 and AccD3 and of AccBC and AccE, the latter two identical to the subunits of the acetyl-CoA carboxylase complex. Since AccD2 and AccD3 orthologues are present in all Corynebacterianeae, these polypeptides are vital for mycolic acid synthesis forming the unique hydrophobic outer layer of these bacteria, and we speculate that the two beta-subunits present serve to lend specificity to this unique large multienzyme complex.

FIG. 1.

On top is shown the syntenic organization of accD1 as component of the acetyl-CoA carboxylase. Orthologous genes are colored similarly. Green is the biotin protein ligase birA; black is the paralogous accD2, which is named accD5 in M. tuberculosis (M. tub.); and red is the open reading frame encoding the ɛ-peptide. At the bottom is shown the syntenic organization of accD3, whose gene product together with accD2 of C. glutamicum is a component of the acyl carboxylase required for mycolic acid synthesis. The polyketide synthase, or condensase, ligating the two long-chain fatty acids to the mycolic acid backbone is pks in C. glutamicum (28) and pks13 in M. tuberculosis. C. glu, C. glutamicum; C. eff, C. efficiens; C. dip, C. diphtheriae; M. lep, M. leprae; M. tub, M. tuberculosis; M. sme, M. smegmatis.

FIG. 2.

Panel A shows the scheme to fuse in the chromosome of the wild-type of C. glutamicum (Cg) six His codons at the N-terminal end with accD1 to result in chromosomally encoded H6-AccD1 in the H6D1 strain. Panel B shows growth of the resulting strain on LB broth (⋄) compared to the wild type (▴). Also shown is growth of a similarly constructed strain (H6D2 strain) with six His codons fused in the chromosome at the 5′ end of accD2 (▴). Panel C shows the isolation of AccBC (upper arrow) together with H6-AccD1 via affinity chromatography (lower arrow).

FIG. 3.

Copurification of the ɛ-subunit AccE and α-subunit AccBC together with the His-tagged β-subunit AccD1 from C. glutamicum. Lane 3 shows the copurification from the C. glutamicum Δpyc paccBC p-H6D1-E strain. The arrowhead marks the small ɛ-peptide whose full sequence is given alongside. The peptides derived from the small ɛ-peptide and identified by mass fingerprinting are in bold, italics, and underlined. Lane 2 shows the copurification from an otherwise isogenic strain (the C. glutamicum Δpyc paccBC p-H6D1 strain) without accE overexpression. Silver-stained gels also confirmed in this strain AccE copurification (data not shown). Lane 1 shows standards with molecular masses in kDa.

FIG. 4.

Gel filtration of acetyl-CoA carboxylase complex isolated from the C. glutamicum Δpyc paccBC p-H6D1-E strain. On top is shown the elution profile with retention times of peaks marked, as well as the individual fractions collected and marked 9 to 15. At the bottom the silver-stained gel is shown where the individual fractions were separated. The majority of the protein elutes with a retention time of 47.4 min, and only this fraction contains the ɛ-peptide. Au, arbitrary units.

FIG. 5.

Substrate (S) saturation kinetics of acetyl-CoA carboxylase of C. glutamicum with either acetyl-CoA (▪) or propionyl-CoA (▴) as substrate.

FIG. 6.

Use of either H6-AccD2 or H6-AccD3 results in copurification of four peptides. In lane marked His-D2, peptides AccBC and AccD3 are copurified with AccD2 from an extract of the C. glutamicum paccBC p-H6D2 strain, and in the lane marked His-D3, AccBC and AccD2 are copurified with AccD3 from an extract of the C. glutamicum paccBC p-H6D3 strain. The copurification is evident from the peptide mass fingerprint analysis, with percentages given for the peptides recovered. The gel in the insert shows copurification of AccE with His-D3 (marked by E) from an extract of the C. glutamicum Δpyc paccBC p-D2-E-H6D3 strain. St, molecular weight standard.

FIG. 7.

Isolation of acyl carboxylase of mycolic acid synthesis. Shown is the elution profile with retention times of peaks marked, as well as the individual fractions collected and marked 5 to 10. At the bottom the Coomassie-stained gel is shown where the individual fractions were separated. Au, arbitrary units.

FIG. 8.

Substrate specificity of acyl carboxylase for mycolic acid synthesis. Carboxylation activity was followed by fixation of H¹⁴CO₃⁻ using various acyl substrates. Fixed radioactive carbon was measured by liquid scintillation counting after acidification of the reaction mixtures and evaporation to dryness.