Publications

• Identification and characterization of Rv3281 as a novel subunit of a biotin-dependent acyl-CoA carboxylase in Mycobacterium tuberculosis H37Rv.

  Oh T.J., Daniel J., Kim H.J., Sirakova T.D., Kolattukudy P.E.

  Mycobacterium tuberculosis produces a large number of structurally diverse lipids generated from the carboxylation products of acetyl-CoA and propionyl-CoA. A biotin-dependent acyl-CoA carboxylase was purified from M. tuberculosis H37Rv by avidin affinity chromatography, and the three major protei ... >> More

  Mycobacterium tuberculosis produces a large number of structurally diverse lipids generated from the carboxylation products of acetyl-CoA and propionyl-CoA. A biotin-dependent acyl-CoA carboxylase was purified from M. tuberculosis H37Rv by avidin affinity chromatography, and the three major protein components were determined by N-terminal sequencing to be the 63-kDa alpha3-subunit (AccA3, Rv3285), the 59-kDa beta5-subunit (AccD5, Rv3280), and the 56-kDa beta4-subunit (AccD4, Rv3799). A minor protein of about 24 kDa that co-purified with the above subunits was identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry to be the product of Rv3281 that is located immediately downstream of the open reading frame encoding the beta5-subunit. This protein displays identity over a short stretch of amino acids with the recently discovered epsilon-subunits of Streptomyces coelicolor, suggesting that it might be an epsilon-subunit of the mycobacterial acyl-CoA carboxylase. To test this hypothesis, the carboxylase subunits were expressed in Escherichia coli and purified. Acyl-CoA carboxylase activity was successfully reconstituted for the first time from purified subunits of the acyl-CoA carboxylase of M. tuberculosis. The reconstituted alpha3-beta5 showed higher activity with propionyl-CoA than with acetyl-CoA, and the addition of the epsilon-subunit stimulated the carboxylation by 3.2- and 6.3-fold, respectively. The alpha3-beta4 showed very low activity with the above substrates but carboxylated long chain acyl-CoA. This epsilon-subunit contains five sets of tandem repeats at the N terminus that are required for maximal enhancement of carboxylase activity. The Rv3281 open reading frame is co-transcribed with Rv3280 in the mycobacterial cell, and the level of epsilon-protein was highest during the log phase and decreased during the stationary phase. << Less

  J. Biol. Chem. 281:3899-3908(2006) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

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

  Daniel J., Oh T.J., Lee C.M., Kolattukudy P.E.

  The Mycobacterium tuberculosis acyl-coenzyme A (CoA) carboxylases provide the building blocks for de novo fatty acid biosynthesis by fatty acid synthase I (FAS I) and for the elongation of FAS I end products by the FAS II complex to produce meromycolic acids. The M. tuberculosis genome contains th ... >> More

  The Mycobacterium tuberculosis acyl-coenzyme A (CoA) carboxylases provide the building blocks for de novo fatty acid biosynthesis by fatty acid synthase I (FAS I) and for the elongation of FAS I end products by the FAS II 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 II 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 (alpha3) and AccD6 (beta6) in E. coli and purified them by affinity chromatography. We report here that reconstitution of the alpha3-beta6 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 alpha3-beta6 complex was inhibited by the epsilon subunit. The alpha3-beta6 carboxylase was inhibited significantly by dimethyl itaconate, C75, haloxyfop, cerulenin, and 1,2-cyclohexanedione. Our results suggest that the beta6 subunit could play an important role in mycolic acid biosynthesis by providing malonyl-CoA to the FAS II complex. << Less

  J. Bacteriol. 189:911-917(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.