IDENTIFICATION OF THE LONG CHAIN ACYL- COENZYME A CARBOXYLASE INVOLVED IN MYCOLIC ACID BIOSYNTHESIS OF Mycobacterium tuberculosis

BERNARDO BAZET LYONNET; FABIENNE BARDOU; ANNAIK QUEMARD; MAMADOU DAFFE; LAUTARO DIACOVICH; GABRIELA M. GAGO; HUGO GRAMAJO

Rosario

Congreso; IX Congreso Nacional de Microbiología General (SAMIGE 2013); 2013

Sociedad Argentina de Microbiología General (SAMIGE)

Mycolic acids, the major cell wall lipids of the human pathogen Mycobacterium tuberculosis, are synthesized by the condensation of a long-chain carboxyacyl-CoA and a meromycolil-AMP by the enzyme Pks13. So far, there are no conclusive results of the subunit composition of the ACCase responsible to generate the long-chain carboxyacyl-CoA. To solve this issue, we performed an in vitro assay in which we mixed different ACCase subunits together with the enzymes FadD32 and Pks13, and looked for the condensation products. We found that the subunits AccA3, AccD4, AccD5 and AccE5 form the active ACCase that generates the long-chain carboxyacyl-CoA. Furthermore, we identified by MALDI-TOF the condensation products using C16-CoA, C20-CoA and C24-CoA as substrates of the ACCase. Also, by generating an accD5 conditional mutant in M. smegmatis we demonstrated that accD5 is essential in this bacterium. 14C acetate labeling and TLC analysis of FAMEs and MAMEs of this mutant showed that it is defective in mycolic acid biosynthesis. Also, the mutant accumulates an unknown lipid, which is now under study.