Biochemical and structural characterization of an essential acyl-CoA carboxilase of tuberculosis: finding new drug targets for treting mycobacterial diseases

GABRIELA MARISA GAGO; KURTH, D.; TSAI, S.; GRAMAJO, H.

Lisboa, Portugal

Congreso; 12th International Congress on Infectious Diseases; 2006

ISID

The unusual lipid-rich cell wall of M. tuberculosis contains several components essential for both mycobacterial viability and pathogenicity, and also plays an active role in modulating the host-immune response. This complex structure, which is a major contributor to its intrinsic resistance to most commonly-used antibiotics also represents the most successful target of antimycobacterial chemotherapy. Although the structures of these unique lipids have been well characterized and the enzymology and genetics leading to their biosynthesis has started to emerge, almost no information is available regarding the pathways involved in the biosynthesis of the precursors of the fatty-acid synthases or polyketide synthases that synthesize this complex molecules. Our working hypothesis is that the alpha-carboxy acyl-CoAs utilized in the biosynthesis of the membrane and cell-wall fatty acids are the product of the Acyl-CoA Carboxylase complexes (ACCase) present in M. tuberculosis. Thus, these enzymes, whose gene structure appears to be unique within actinomycetes, are an attractive target for the development of new and specific anti-mycobacterial agents. Heterologous protein expression and purification of the individual enzyme subunits allowed the successful reconstitution of an essential ACCase, ACC6, 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. The kinetic properties of this enzyme showed a clear substrate preference for acetyl-CoA, suggesting that ACC6 could provide the substrate, malonyl-CoA, for the biosynthesis of straight fatty acids in this microorganism. We have recently determined the crystal structure of the beta subunit AccD5 and extensive in silico screening of the UC Irvine ChemDB database resulted in the identification of two ligands (NCI 65828 and 170233) whose Ki are 1-10 mM. Interestingly, the ligand NCI 170233 also inhibited ACC6 (whose crystal structure is currently being solved) at low mM concentrations. Moreover, this compound also inhibited growth of M. bovis BCG 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.