CHARACTERIZATION OF ESSENTIAL ACYL- COENZYME A CARBOXYLASES OF MYCOBACTERIUM TUBERCULOSIS

BAZET LYONNET, B; DIACOVICH, L.; CABRUJA, M.; GAGO, G.; GRAMAJO, H

Rosario

Congreso; L Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2014

SAIB

The two Fatty Acid Synthase (FAS) systems of the human pathogen M. tuberculosis work in concert to synthesize mycolic acids. FAS I builds a long-chain acyl-CoA (C24-CoA) and FAS II generate a very long-chain fatty acid (meromycolic acid). A long-chain acyl-CoA carboxylase activates the C24 acyl-CoA, and the acyl-AMP ligase FadD32 activates the meromycolic acid chain. These two chains are later condensed by Pks13 to yield the final mycolic acid. Besides its importance in mycolic acid biosynthesis, there are no conclusive results of the subunit composition of the acyl-CoA carboxylase responsible to generate the long-chain carboxyacyl-CoA. We found that an accD5-accE5 conditional mutant in Mycobacterium smegmatis have reduced levels of long-chain acyl-CoA carboxylase activity. Also, 14C-acetate labeling and TLC analysis of FAMEs and MAMEs of this mutant showed accumulation of previously uncharacterized compounds. LC-MS analysis of these compounds showed that they were meromycolic acids methyl esters. These results allow us to speculate that the subunits AccD5 and AccE5, that are part a of well-characterized propionyl-CoA carboxylase, are also involved in the long-chain acyl-CoA carboxylation. To solve this issue, we performed in vitro assays and we found evidences that the subunits AccD5 and AccE5 are part of the active long-chain acyl-CoA carboxylase.