Fumarase from Arabidospis thaliana: two paralogous genes codifying three biochemically divergent isoforms.

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

Rosario

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

The enzyme fumarate hydratase (FH) participates in the tricarboxylic acid (TCA) cycle in mitochondria which is essential for cellular respiration. However, a common theme conserved in eukaryotes isthe existence of cytosolic FH activity. In yeast and animals is well documented that the FH dual localization is based on a double distribution of a single translation product. However, Arabidopsis thaliana genome possesses a paralogous gene (FH2) for a cytosolic FH, different that the one encoding the mitochondrial isoform (FH1), which is required for fumarate accumulation. In addition, an in silico analysis indicates that FH2 would codify two different genetic products through alternative splicing. Here, we describe the first biochemical characterization of recombinant FH1, FH2 and FH2spl from A. thaliana. Unexpected for an enzyme catalyzing the reversible single substrate hydration reaction, all isoforms exhibited complex kinetics as they showed product inhibition, which was released by malic enzyme coupling. The cytosolic FH2 isoforms exhibited higher substrate affinity and lower kcat and pH optimum than the mitochondrial FH1. In addition, direct and reverse reactions were oppositely modulated by several metabolic effectors. In this regard, the oxaloacetate inhibited the fumarate hydration but strongly stimulated the L-malate dehydration catalyzed by FH1. Theses biochemical assays are essential for a comprehensive understanding of the mechanisms allowing fumarateor Lmalate accumulation in plants.