"Novel insights into Arabidopsis thaliana NADP-ME2 regulation by fumarate."

FONSECA REZENDE E MELLO J.; WHEELER M. G; ARIAS C.L., ; DRINCOVICH M. F., ; ANDREO C. S., ; MENDONÇA TELES DE SOUZA A. ; ALVAREZ C.E.

Mar del Plata

Congreso; VL CONGRESO DE LA SOCIEDAD ARGENTINA DE BIOQUÍMICA Y BIOLOGÍA MOLECULAR (SAIB); 2015

SAIB

Arabidopsis thaliana is a plant species that accumulates high levels of organic acids and uses them as carbon, energy and reducing power sources. NADP-ME2 is the only cytosolic malic enzyme (ME) present in all Arabidopsis organs providing most of the total NADP-ME activity. NADP-ME2 has a complex regulation by fumarate that depends on pH and substrates/fumarate concentrations. Kinetics experiments show activation of NADP-ME2 possibly caused by the binding to a putative allosteric binding site. However, at high fumarate concentrations seems to compete with the substrate malate at the active site and causes inhibition. Until now there is not any plant ME structure solved yet. In this work, we performed homology modeling, molecular docking and dynamics studies trying to discover the putative residues and/or motives involved in this complex fumarate behavior. The results confirmed the presence of two fumarate binding sites: an allosteric site (activation) and the active site (inhibition). Besides, they show a novel fumarate interaction with the backbone of the residue Leu62, present in the activating site not shown before in any structure of MEs. In order to study the participation of Leu62 in the activation of ME2 we expressed and characterized the mutant L62W-ME2. By kinetics assays we found out that, as predicted by molecular dynamics, this mutant is no longer activated by fumarate.