Kinetic and structural characterization of NADP-malic enzyme family from Arabidopsis thaliana

GERRARD WHEELER, M. C.; MAURINO, V. G.; ANDREO, C. S.; DRINCOVICH, M. F.

Pinamar, Argentina

Congreso; XLI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2005

SAIB

The A. thaliana genome contains four genes encoding putative NADP-malic enzymes (ME1-4). This enzyme catalyzes the oxidative decarboxylation of L-malate to form pyruvate, CO2 and NADPH. ME4 is localized to plastids, whereas the other three do not possess any organellar targeting sequence. The cDNAs of each isoform were expressed in E. coli and the purified recombinant proteins used for kinetic and structural studies. All the A. thaliana isozymes showed NADP-ME activity, but with significant differences in kcat values, affinities for both substrates and pH optimum suggesting different biological roles for each protein. On the other hand, ME4 exists in equilibrium of active dimers and tetramers, while the cytosolic counterparts are present as hexamers or octamers. All of them showed different native isoelectric points with the same pI under denaturing conditions, probably due to a change in the net charge in the native enzymes due to oligomerization. Unexpectedly, ME3 y ME4 exhibited inhibition by malate a pH 7, a particular feature of the enzyme in C4 metabolism, as well as a fast reverse reaction (pyruvate carboxylation). The recombinant proteins were also active with NAD, although the specific activities were lower than the values obtained in the presence of NADP. In addition, the response of the purified enzymes to several key compounds involved in distinct metabolisms was also tested suggesting that some members of the AtNADP-ME family may be subject to metabolite control in vivo.