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

DIACOVICH L; VRANYCH C; GRAMAJO H; TOMASSETTI M; OTTADO J; GARAVAGLIA B; GOTTIG N

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias - LIII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB); 2017

Sociedades de Biociencias - LIII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB)

The phytopatogen Xanthomonas axonopodis pv. citri (Xcc) produce canker, pathology that affects citric. Xcc, as an obliged plant parasite, is unable to survive out of its host for long periods of time. Nevertheless, during the epiphytic survival state, over the leaf, many metabolic and nutritional aspects remain unknown. Branched-chain aminoacids catabolism prevents their over-accumulation, facilitates branched-chain fatty acids biosynthesis and provides energy for the cell. The enzymatic complex 3-methyl-crotonyl-CoA carboxylase (MCC) is essential for leucine degradation pathway. MCC carboxylates 3-methyl-crotonyl, as a substrate, to finally generate acetyl-CoA and acetoacetate, which are latter incorporated into different metabolic pathways. MCC complexes belong to the biotin-dependent acyl-CoA carboxylases (ACCases) group. The objective of this work was characterized a MCC complex from Xcc. We identified by bioinformatics two genes that encode two subunits of an ACCase complex in Xanthomonas, and others gram negative bacterial pathogens. We purified the two ACCases subunits, AccC and AccD, by affinity columns and reconstituted the enzymatic activity in vitro using different 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 these proteins conform a MCC complex. Furthermore, we had shown, by using a mutant analysis, that this MCC complex is relevant for the survival of Xcc in planta.