Characterization of an essential acyl-CoA carboxylase from Mycobacterium tuberculosis

KURTH, D., GAGO, G., DIACOVICH, L. AND GRAMAJO, H.

Pinamar, BA, Argentina

Congreso; XLI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular-X Congress of Panamerican Association for Biochemistry and Molecular Biology; 2005

Pathogenic mycobacteria contain a variety of unique fatty acids which have methyl branches at even-numbered position at the carboxyl end, and a long n-aliphatic chain. One such group of acids are the mycocerosic acids and their biosynthesis is essential for growth and pathogenesis. Therefore, the biosynthetic pathway of the unique precursor of such lipids, methylmalonyl-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 M. tuberculosis. The enzyme complex was reconstituted from the a biotinylated subunit AccA3, the carboxyltransferase b subunit AccD5 and the e subunit AccE5 (Rv3281). The kinetic properties of this enzyme showed a clear substrate preference for propionyl-CoA compared with acetyl-CoA (specificity constant five fold 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 crystal structure of AccD5 was determined at 2.9 Å. Extensive in silico screening resulted in the identification of two inhibitors whose Ki are 1-10 μM. Our results pave the first step towards understanding the biological roles of the key ACCases that commits acyl-CoAs to the biosynthesis of cell wall fatty acids, as well as providing a new structure-based drug design target for tuberculosis therapeutic development.