Differential fumarate binding to Arabidopsis NAD+-malic enzymes 1 and -2 produces an opposite activity modulation

Abstract

Arabidopsis mitochondria contain two NAD+-malic enzymes, NAD-ME1 and NAD-ME2. These proteinshave similar affinity for their substrates but display opposite regulation by fumarate, which stronglystimulates NAD-ME1 but inhibits NAD-ME2 activity. Here, the interaction of NAD-ME1 and -2 withfumarate was investigated by kinetic approaches, urea denaturation assays and intrinsic fluorescencequenching, in the absence and presence of NAD+. Fumarate inhibited NAD-ME2 at saturating, but not atlow, levels of NAD+, and it behaved as competitive inhibitor with respect to L-malate. In contrast, NADME1fumarate activation was higher at suboptimal NAD+ concentrations. In the absence of cofactor, thefluorescence of both NAD-ME1 and -2 is quenched by fumarate. However, for NAD-ME2 the quenchingarises from a collisional phenomenon, while in NAD-ME1 the fluorescence decay can be explained bya static process that involves fumarate binding to the protein. Furthermore, the residue Arg84 of NADME1is essential for fumarate binding, as the mutant protein R84A exhibits a collisional quenching bythis metabolite. Together, the results indicate that the differential fumarate regulation of ArabidopsisNAD-MEs, which is further modulated by NAD+ availability, is related to the gaining of an allosteric sitefor fumarate in NAD-ME1 and an active site-associated inhibition by this C4-organic acid in NAD-ME2.