AccD6, a member of the Fas II locus, is a functional carboxyltransferase subunit of the acyl-coenzyme A carboxylase in Mycobacterium tuberculosis

The Mycobacterium tuberculosis acyl-coenzyme A (CoA) carboxylases provide the building blocks for de novo fatty acid biosynthesis by fatty acid synthase I (FAS 1) and for the elongation of FAS I end products by the FAS 11 complex to produce meromycolic acids. The M. tuberculosis genome contains three biotin carboxylase subunits (AccA1 to -3) and six carboxyltransferase subunits (AccD1 to -6), with accD6 located in a genetic locus that contains members of the FAS 11 complex. We found by quantitative real-time PCR analysis that the transcripts of accA3, accD4, accD5, and accD6 are expressed at high levels during the exponential growth phases of M. tuberculosis in vitro. Microarray analysis of M. tuberculosis transcripts indicated that the transcripts for accA3, accD4, accD5, accD6, and accE were repressed during later growth stages. AccD4 and AccD5 have been previously studied, but there are no reports on the function of AccD6. We expressed AccA3 (alpha(3)) and AccD6 (beta(6)) in E. coli and purified them by affinity chromatography. We report here that reconstitution of the alpha(3)-beta(6) complex yielded an active acyl-CoA carboxylase. Kinetic characterization of this carboxylase showed that it preferentially carboxylated acetyl-CoA (1.1 nmol/mg/min) over propionyl-CoA (0.36 nmol/mg/min). The activity of the alpha(3)-beta(6) complex was inhibited by the epsilon subunit. The alpha(3)-beta(6) carboxylase was inhibited significantly by dimethyl itaconate, C75, haloxyfop, cerulenin, and 1,2-cyclohexanedione. Our results suggest that the beta(6) subunit could play an important role in mycolic acid biosynthesis by providing malonyl-CoA to the FAS 11 complex.