Characterization of thiol-disulfide interchange of maize recombinant C4 and non-C4 NADP-malic enzyme

CLARISA ALVAREZ; DETARSIO, E.; ANDREO, CARLOS S; MARIA FABIANA DRINCOVICH

Cambridge, UK

Congreso; Satellite Meeting of XIV Internacional Congreso of Photosynthesis: C4 and CAM: from molecular diversity to ecological convergence; 2007

C4 photosynthetic NADP-malic enzyme has evolved from non-C4 isoforms during evolution. In maize, the plastidic non-photosynthetic NADP-ME represents the more recent and direct ancestor of the C4–isoform. Although the high sequence similarity (85%) between these two NADP-MEs, they display well distinct kinetic and structural properties. In order to identify possible differences in response to -SH oxidation, recombinant C4- and non-C4 NADP-ME were subjected to oxidation and further reduction. Incubation of maize C4 NADP-ME with the oxidant o-iodosobenzoate (IBZ) leads to the complete inactivation of the enzyme. The reversal of the inactivation by reductive agents suggests that the modification of the enzyme by IBZ occurs concomitant with the oxidation of one or more pairs of -SH groups to the disulfide state. On the other hand, maize non-C4 NADP-ME isoform was not inactivated at all by -SH oxidation. In order to identify Cys residues involved in C4 NADP-ME oxidation, site directed mutagenesis of four different Cys residues was performed. The four mutants obtained, C192A, C231A, C246A and C270A, showed significant lower catalytic efficiency than the wild-type C4 NADP-ME. Moreover, the four mutants exhibit different responses to oxidation and further reduction, which may allow to infer the possible disulfide bond/s that may take place during maize C4 NADP-ME oxidation. The results obtained suggest that the redox state of maize C4 NADP-ME is important for the expression of the maximal catalytic activity of this enzyme, although the non-photosynthetic counterpart of the enzyme seems to be not so sensitive to -SH oxidation