C4-specific (photosynthetic) NADP+-dependent malic enzyme (NADP+-ME) has evolved from C3-malic enzymes and represents a unique and specialized form, as indicated by its particular kinetic and regulatory properties. In the present paper, we have characterized maize (Zea mays L.) photosynthetic NADP+-ME mutants in which conserved basic residues (lysine and arginine) were changed by site-directed mutagenesis. Kinetic characterization and oxaloacetate partition ratio of the NADP+-ME K255I (Lys-255→Ile) mutant suggest that the mutated lysine residue is implicated in catalysis and substrate binding. Moreover, this residue could be acting as a base, accepting a proton in the malate oxidation step. At the same time, further characterization of the NADP+-ME R237L mutant indicates that Arg-237 is also a candidate for such role. These results suggest that both residues may play ‘back-up’ roles as proton acceptors. On the other hand, Lys-435 and/or Lys-436 are implicated in the coenzyme specificity (NADP+ versus NAD+) of maize NADP+-ME by interacting with the 2′-phosphate group of the ribose ring. This is indicated by both the catalytic efficiency with NADP+ or NAD+, as well as by the reciprocal inhibition constants of the competitive inhibitors 2′-AMP and 5′-AMP, obtained when comparing the double mutant K435/6L (Lys-435/436→Ile) with wild-type NADP+-ME. The results obtained in the present work indicate that the role of basic residues in maize photosynthetic NADP+-ME differs significantly with respect to its role in non-plant MEs, for which crystal structures have been resolved. Such differences are discussed on the basis of a predicted three-dimensional model of the enzyme.

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