Rules for cofactor specificity are conserved in mitochondrial plant malic enzymes beside their different evolutionary origin.

TRONCONI, M.; MARTINATTO, A; ZUBIMENDI, JP; DRINCOVICH, M.F.; ANDREO CS

Rosario

Congreso; 8TH INT. CONFERENCE FOR PLANT MITOCHONDRIAL BIOLOGY; 2013

Malic enzyme (ME) catalyzes the oxidative decarboxylation of L-malate to yield pyruvate and CO2, with NAD or NADP being essential cofactors for the reaction. In eukaryotic cells, NADP-dependent MEs are found in cytosol and plastids, while NAD-dependent MEs arelocated in mitochondria. In animals, NADP- and NAD-MEs show high sequence homology and are evolutionarily related. However, in plants these isoforms share only near 38 % of identity and appear to have evolved from different ancestors. So far, structural determinants for NAD or NADP usage were identified for animal MEs, where a limited numberof amino acidic replacements were necessary to completely change the cofactor specificity, supporting the related origins. Here, we describe the effect of residue replacement in the cofactor binding site of Arabidopsis mitochondrial NAD-ME2 for those found in NADP-ME isoforms. Kinetic characterization of simple, double and triple mutants reveals that the conserved Lys356 residue inNADP-ME isoforms is the major cofactor determinant, as NAD-ME mutants harboring this residue exhibit the highest fold increase in NADP-dependent catalytic efficiency. In addition, Ser341 and Lys342 also function in cofactor specificity and in the triple mutants work together and function synergistically with Lys356 to increase the binding affinity for NADP. These results suggest that the rules for cofactor specificity are universal and even may be applied to others Rossmann dehydrogenases.