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

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

Angra dos Reis, RJ Brazil.

Congreso; 1st Latin American Protein Society Meeting; 2004

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 multi-drug resistance. Therefore, the identification of the pathways that are required for mycobacterial growth would provide new targets for the rational design of more effective tuberculosis therapeutics. 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 chemotherapies and contributes to the survival of this pathogen within the host. Large-scale transposon mutagenesis have 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 a specific b subunit (AccD4, AccD5, and AccD6) and share the same biotinylated a subunit (AccA3) and e 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 e subunit in M. tuberculosis. This unique e subunit is shared by different b subunits. We also solved the crystal structure of AccD5 to 2.0 Å. The surface property of AccD5 showed a dramatic difference from that of Streptomyces b 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 and will lead to novel tuberculosis therapeutics. 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 multi-drug resistance. Therefore, the identification of the pathways that are required for mycobacterial growth would provide new targets for the rational design of more effective tuberculosis therapeutics. 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 chemotherapies and contributes to the survival of this pathogen within the host. Large-scale transposon mutagenesis have 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 a specific b subunit (AccD4, AccD5, and AccD6) and share the same biotinylated a subunit (AccA3) and e 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 e subunit in M. tuberculosis. This unique e subunit is shared by different b subunits. We also solved the crystal structure of AccD5 to 2.0 Å. The surface property of AccD5 showed a dramatic difference from that of Streptomyces b 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 and will lead to novel tuberculosis therapeutics. 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 multi-drug resistance. Therefore, the identification of the pathways that are required for mycobacterial growth would provide new targets for the rational design of more effective tuberculosis therapeutics. 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 chemotherapies and contributes to the survival of this pathogen within the host. Large-scale transposon mutagenesis have 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 a specific b subunit (AccD4, AccD5, and AccD6) and share the same biotinylated a subunit (AccA3) and e 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 e subunit in M. tuberculosis. This unique e subunit is shared by different b subunits. We also solved the crystal structure of AccD5 to 2.0 Å. The surface property of AccD5 showed a dramatic difference from that of Streptomyces b 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 and will lead to novel tuberculosis therapeutics.