Basic residues play key roles in catalysis and NADP(+)-specificity in maize (Zea mays L.) photosynthetic NADP(+)-dependent malic enzyme

Abstract

C(4)-specific (photosynthetic) NADP(+)-dependent malic enzyme (NADP(+)-ME) has evolved from C(3)-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.

Figure 1. Three-dimensional model view of the maize chloroplastic C₄-NADP⁺-ME corresponding to the active site of the enzyme

(A) The predicted model was obtained using human mitochondrial NAD(P)⁺-ME complexed with their substrates as templates. The position of Lys-255 and Arg-237 relative to tartronate (analogue of malate) is shown. (B) The predicted model was obtained using pigeon liver NADP⁺-ME in order to analyse the position of the 2′-phosphate in NADP⁺ relative to Lys-435 and Lys-436. The Figures were created by using Swiss Protein Data Bank Viewer version 3.7.

Figure 2. Sequence alignment around Lys-435/6 of maize photosynthetic NADP⁺-ME

Maize NADP⁺-ME sequences (C₄ and non-C₄) were compared with the sequences of the three NAD(P)⁺-MEs that have been crystallized. This comparison was performed using Clustal W (1.81). :, conserved substitution are observed; ., semi-conserved substitutions are observed.