Kinetic and structural analysis of a group of acyl-CoA carboxylases from Mycobacterium tuberculosis.

GAGO G., KURTH D., DIACOVICH L., TSAI, S., GRAMAJO, H.

Angra dos Reis, RJ Brazil.

Congreso; 1st Latin American Protein Society Meeting.; 2004

Latin American Protein Society.

It is estimated that one third of the world population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis, a widespread disease that is responsible for 3 million deaths annually. Although effective drugs exist, current therapy requires prolonged treatment with several drugs, leading to compliance problems and the emergence of multidrug resistance. Therefore, the identification of the pathways that are required for mycobacterial growth would provide new targets for the rational design of more effective antimycobacterial agents. It has been widely recognized that the unusually complex cell wall of the organism plays a major role in the exceptional ability of M. tuberculosis to be a successful pathogen. The cell wall, rich in unusual lipids, constitutes an effective barrier to antimycobacterial therapies and contributes to the survival of this pathogen within the host. Large-scale transposon mutagenesis has recently identified many essential biosynthetic pathways and among them are those for the synthesis of the complex lipids present in the mycobacterial cell wall. Here we present the biochemical and structural characterization of a group of essential acyl-CoA carboxylases from M. tuberculosis. These are key enzymes for the synthesis of lipids as they provide the elongating units malonyl- and methylmalonyl-CoA. Three acyl-CoA carboxylases have been successfully reconstituted from their purified components. The three complexes consist of a specificc beta subunit (AccD4, AccD5, and AccD6) and share the same biotinylated alpha subunit (AccA3) and epsilon subunit (AccE5). For all complexes the addition of AccE5 dramatically increased the specific activity of the enzymes. The kinetic properties of the acyl-CoA carboxylases showed that they are able to carboxylate both acetyl and propionyl-CoA, although a clear preference for propionyl-CoA was evident. This is the first report of the presence of a functional epsilon subunit in M. tuberculosis and it is the first case described were a unique epsilon subunit is shared by different beta subunits. We also solved the crystal structure of AccD5 to 2.0 A. The surface property of AccD5 showed a dramatic difference from that of Streptomyces beta subunits, indicating different protein-protein interaction and biological roles. The active site is L-shaped, with a predominant hydrophobic biotin binding pocket and a hydrophilic acyl-CoA binding pocket. Our functional and structural study provides a novel drug design target for tuberculosis that could lead to novel tuberculosis therapeutics.