Identification and characterization of Rv3281 as a novel subunit of a biotin-dependent acyl-CoA Carboxylase in Mycobacterium tuberculosis H37Rv.
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.
Oh
Tae-Jin
TJ
Burnett College of Biomedical Sciences, University of Central Florida, Orlando, 32816, USA.
Daniel
Jaiyanth
J
Kim
Hwa-Jung
HJ
Sirakova
Tatiana D
TD
Kolattukudy
Pappachan E
PE
D000595:Amino Acid Sequence; D001710:Biotin / Q000494:pharmacology*; D019735:Carbon-Carbon Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D021122:Protein Subunits; D020449:Repetitive Sequences, Amino Acid
D021122:Protein Subunits; D001710:Biotin; D019735:Carbon-Carbon Ligases; C018757:acyl-CoA carboxylase