Title

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

Authors

Authors

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

Comments

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Abbreviated Journal Title

J. Biol. Chem.

Keywords

COENZYME-A CARBOXYLASE; PURIFICATION; EXPRESSION; GROWTH; DOMAIN; GENES; ACIDS; Biochemistry & Molecular Biology

Abstract

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 alpha 3-subunit (AccA3, Rv3285), the 59-kDa beta 5-subunit (AccD5, Rv3280), and the 56-kDa beta 4-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 beta 5-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 alpha 3-beta 5 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 alpha 3-beta 4 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.

Journal Title

Journal of Biological Chemistry

Volume

281

Issue/Number

7

Publication Date

1-1-2006

Document Type

Article

Language

English

First Page

3899

Last Page

3908

WOS Identifier

WOS:000235275300017

ISSN

0021-9258

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