Maize C4 NADP-Malic Enzyme

Abstract

Malic enzymes catalyze the oxidative decarboxylation of L-malate to yield pyruvate, CO2, and NAD(P)H in the presence of a bivalent metal ion. In plants, different isoforms of the NADP-malic enzyme (NADP-ME) are involved in a wide range of metabolic pathways. The C4-specific NADP-ME has evolved from C3-type malic enzymes to represent a unique and specialized form of NADP-ME as indicated by its particular kinetic and regulatory properties. In the present study, the mature C4-specific NADP-ME of maize was expressed in Escherichia coli. The recombinant enzyme has essentially the same physicochemical properties and Km for the substrates as those of the naturally occurring NADP-ME previously characterized. However, the kcat was almost 7-fold higher, which may suggest that the previously purified enzyme from maize leaves was partially inactive. The recombinant NADP-ME also has a very low intrinsic NAD-dependent activity. Five mutants of NADP-ME at the postulated putative NADP-binding site(s) (Gsite5V, Gsite2V, A392G, A387G, and R237L) were constructed by site-directed mutagenesis and purified to homogeneity. The participation of these residues in substrate binding and/or the catalytic reaction was inferred by kinetic measurements and circular dichroism and intrinsic fluorescence spectra. The results obtained were compared with a predicted three-dimensional model of maize C4 NADP-ME based on crystallographic studies of related animal NAD(P)-MEs. The data presented here represent the first prokaryotic expression of a plant NADP-ME and reveals valuable insight regarding the participation of the mutated amino acids in the binding of substrates and/or catalysis.