Novel insights into Arabidopsis thaliana NADP-ME2 regulation by fumarate

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

Mar del Plata

Congreso; LI Reunión Anual SAIB; 2015

SAIB

Arabidopsis thaliana is a plant species thataccumulates high levels of organic acids and uses them as carbon, energy andreducing power sources. NADP-ME2 is the only cytosolic malic enzyme (ME) presentin all Arabidopsis organs providing mostof the total NADP-ME activity. NADP-ME2 has a complex regulation by fumaratethat depends on pH and substrates/fumarate concentrations. Kinetics experimentsshow activation of NADP-ME2 possibly caused by the binding to a putativeallosteric binding site. However, at high fumarate concentrations seems tocompete with the substrate malate at the active site and causes inhibition. Untilnow there is not any plant ME structure solved yet. In this work, we performed homologymodeling, molecular docking and dynamics studies trying to discover theputative residues and/or motives involved in this complex fumarate behavior. Theresults confirmed the presence of two fumarate binding sites: an allostericsite (activation) and the active site (inhibition). Besides, they show a novel fumarateinteraction with the backbone of the residue Leu62, present in the activating sitenot shown before in any structure of MEs. In order to study the participationof Leu62 in the activation of ME2 we expressed and characterized the mutantL62W-ME2. By kinetics assays we found out that, as predicted by moleculardynamics, this mutant is no longer activated by fumarate.