BIOCHEMICAL AND PHYSIOLOGICAL CHARACTERIZATION OF A 3-METHYLCROTONYL-COA CARBOXYLASE COMPLEX FROM Xanthomonas

LAUTARO DIACOVICH, BETIANA GARAVAGLIA (1), MAURO TOMASSETTI (1), CECILIA VRANYCH (1), NATALIA GOTTIG (1), JORGELINA OTTADO (1), HUGO GRAMAJO (1)

Congreso; SAIB 2017; 2017

The phytopatogen Xanthomonas axonopodis pv. citri (Xcc) producecanker, pathology that affects citric. Xcc, as an obliged plantparasite, is unable to survive out of its host for long periods of time.Nevertheless, during the epiphytic survival state, over the leaf, manymetabolic and nutritional aspects remain unknown. Branched-chainaminoacids catabolism prevents their over-accumulation, facilitatesbranched-chain fatty acids biosynthesis and provides energy for thecell. The enzymatic complex 3-methyl-crotonyl-CoA carboxylase(MCC) is essential for leucine degradation pathway. MCC carboxylates3-methyl-crotonyl, as a substrate, to finally generate acetyl-CoA and acetoacetate, which are latter incorporated into differentmetabolic pathways. MCC complexes belong to the biotin-dependentacyl-CoA carboxylases (ACCases) group. The objective of thiswork was characterized a MCC complex from Xcc. We identified bybioinformatics two genes that encode two subunits of an ACCasecomplex in Xanthomonas, and others gram negative bacterial pathogens.We purified the two ACCases subunits, AccC and AccD, byaffinity columns and reconstituted the enzymatic activity in vitro usingdifferent acyl-CoAs as substrate. For this we used two methods,including fixation of radiolabeled bicarbonate and a coupled assay.We determined the kinetic parameters and confirmed that theseproteins conform a MCC complex. Furthermore, we had shown, byusing a mutant analysis, that this MCC complex is relevant for thesurvival of Xcc in planta.Keywords: Xanthomomas, Methyl-crotonyl-CoA carboxylases,phytopatogen, leucine catabolism, canker