Study of the structure-function relationship in maize NADP-malic enzyme.

DETARSIO E.; ALVAREZ C.E.; SAIGO M.; DRINCOVICH M.F; ANDREO C.S.

Energy from the Sun

Springer Publishing.

Año: 2007; p. 841 - 844

NADP-malic enzyme catalyses the reversible oxidative decarboxilation of L-malate to yield carbon dioxide and pyruvate with the concomitant reduction of NADP. Maize presents at least three isoforms of this enzyme, one of them, the photosynthetic isoform is responsible for providing CO2 to the Calvin Cycle for carbon fixation. We studied different kinetic and structural aspects of this enzyme by recombination protein engineering. Site directed mutagenesis showed that the residues A392, A387 and K435/6 contribute to the specificity of NADP over NAD as substrate of the enzyme and that the conserved basic residues K255 y R237 are implicated in substrate binding and catalysis, probably acting as a base, accepting a proton in the malate oxidation step. The atructural basis of the differences between the photosynthetic and non-photosynthetic isoforms were studied by constructing and analyzing chimeras between these isoforms. Their analysis showed that the region between residues 102 and 247 of the photosynthetic isoform is responsible for its tetramerization capacity, and the region between residues 248 and C-terminus, for the malate inhibition phenomena. This photosynthetic isoform is activated by the reduction of intramolecular disulfide bonds. The role of conserved cysteine residues, analyzed by mutagenesis, indicated that cys 192, but not cys 232, is necessary for the catalytic reaction, however, both affect the affinity for the substrate malat