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Journal of Bacteriology, January 2006, p. 477-486, Vol. 188, No. 2
0021-9193/06/$08.00+0     doi:10.1128/JB.188.2.477-486.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Biochemical and Structural Characterization of an Essential Acyl Coenzyme A Carboxylase from Mycobacterium tuberculosis

Gabriela Gago,1 Daniel Kurth,1 Lautaro Diacovich,1 Shiou-Chuan Tsai,2 and Hugo Gramajo1*

Microbiology Division, Instituto de Biología Molecular y Celular de Rosario (IBR), Universidad Nacional de Rosario, Rosario, Argentina,1 Department of Molecular Biology and Biochemistry and Department of Chemistry, University of California, Irvine, California 926122

Received 4 July 2005/ Accepted 24 October 2005

Pathogenic mycobacteria contain a variety of unique fatty acids that have methyl branches at an even-numbered position at the carboxyl end and a long n-aliphatic chain. One such group of acids, called mycocerosic acids, is found uniquely in the cell wall of pathogenic mycobacteria, and their biosynthesis is essential for growth and pathogenesis. Therefore, the biosynthetic pathway of the unique precursor of such lipids, methylmalonyl coenzyme A (CoA), represents an attractive target for developing new antituberculous drugs. Heterologous protein expression and purification of the individual subunits allowed the successful reconstitution of an essential acyl-CoA carboxylase from Mycobacterium tuberculosis, whose main role appears to be the synthesis of methylmalonyl-CoA. The enzyme complex was reconstituted from the {alpha} biotinylated subunit AccA3, the carboxyltransferase ß subunit AccD5, and the {varepsilon} subunit AccE5 (Rv3281). The kinetic properties of this enzyme showed a clear substrate preference for propionyl-CoA compared with acetyl-CoA (specificity constant fivefold higher), indicating that the main physiological role of this enzyme complex is to generate methylmalonyl-CoA for the biosynthesis of branched-chain fatty acids. The {alpha} and ß subunits are capable of forming a stable {alpha}6-ß6 subcomplex but with very low specific activity. The addition of the {varepsilon} subunit, which binds tightly to the {alpha}-ß subcomplex, is essential for gaining maximal enzyme activity.

* Corresponding author. Mailing address: Microbiology Division, Instituto de Biología Molecular y Celular de Rosario (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, (S2002LRK) Rosario, Argentina. Phone: 54-341-4350661. Fax: 54-341-4390465. E-mail: gramajo{at}ibr.gov.ar.

Journal of Bacteriology, January 2006, p. 477-486, Vol. 188, No. 2
0021-9193/06/$08.00+0     doi:10.1128/JB.188.2.477-486.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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