ACCase 6 is the essential acetyl-CoA carboxylase involved in fatty acid and mycolic acid biosynthesis in mycobacteria

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

Keystone, Colorado, USA

Congreso; Keystone Symposia: “Tuberculosis: Biology, Pathology and Therapy”; 2009

Fatty acid biosynthesis is an emerging target for the development of novel antibacterial chemotherapeutics. In addition to the usual fatty acids found in membrane lipids, mycobacteria have a wide variety of very long-chain saturated (C18-C32) and mono-unsaturated (up to C26) n-fatty acids as well as multimethyl-branched fatty acids present in several cell wall lipids that have been implicated in the ability of the organism to resist the host defences (1, 2, 3). Despite the essential role that fatty acids play in the formation of the lipids of the mycobacterial cell wall, little is known about the biochemistry and physiological role of the AcCCs from Mycobacterium tuberculosis. We have recently characterized at the biochemical and structural levels the first AcCC complex of  M. tuberculosis (4). The enzyme complex, called AcCC5, is composed of the a biotinylated subunit AccA3, the carboxyltransferase b subunit AccD5 and the e subunit AccE5 (Rv3281). The addition of the e subunit was essential for gaining maximal enzyme activity. This complex 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 and extensive in silico screening resulted in the identification of two inhibitors whose Ki are 1-10 mM (5).Heterologous protein expression and purification of the individual enzyme subunits allowed the successful reconstitution of the essential AcCC6 complex, whose main role appears to be the synthesis of malonyl-CoA. The enzyme complex was reconstituted from the biotinylated subunit AccA3 and the carboxyltransferase subunit AccD6. A complex with similar kinectics parameters was also reconstituted using AccD6 and AccA1 as the alpha subunit. The kinetic properties of this enzyme suggests that AcCC6 could provide the substrate, malonyl-CoA, for the biosynthesis of straight fatty acids in this microorganism. Interestingly, one of the inhibitors identified for the AcCC5 complex, NCI 170233, also inhibited AcCC6 at low mM concentrations. Moreover, this compound also inhibited growth of M. smegmatis at mM concentrations. 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.