Cloning and analysis of the C4 photosynthetic NAD-dependent malic enzyme of amaranth mitochondria

In some C4 plant species, a mitochondrial NAD-dependent malic enzyme (EC 1.1.1.39) (NAD-ME) catalyzes the decarboxylation of 4 carbon malate in the bundle sheath cells, releasing CO₂ for the Calvin cycle of photosynthesis. In amaranth, a dicotyledonous NAD-ME-type C4 plant, the photosynthetic NAD- ME purified as two subunits of 65 and 60 kDa, designated α and β, respectively. Antiserum raised against the α subunit reacted only with the 65-kDa protein in immunoblot analysis. Immunogold electron microscopy using the α subunit antiserum demonstrated that this protein was localized specifically to the mitochondrial matrix of bundle sheath cells. The complete nucleotide sequence of a 2300-base pair α subunit cDNA clone showed that this gene encodes a protein that contains all of the motifs required for a complete and functional malic enzyme. The α subunit has significant similarity along its entire length to other known NAD- and NADP-dependent malic enzymes from plants, animals, and bacteria. The findings presented here provide new insights about the C4 photosynthetic NAD-ME and its evolutionary relationship to other forms of malic enzyme present in eukaryotic and prokaryotic organisms.