A full-length cDNA for maize root-form phosphoenolpyruvate carboxylase (PEPC) was isolated. In the coding region, the root-form PEPC showed 76 and 77% identity with the C4- and C3-form PEPCs of maize, respectively, at the nucleotide level. At the amino acid level, the root-form was 81 and 85% identical to the C4- and C3-form PEPCs, respectively. The entire coding region was inserted into a pET32a expression vector so that it was expressed under the control of T7 promoter. The purified recombinant root-form PEPC had a Vmax value of about 28 mumol min-1 (mg protein)-1 at pH 8.0. The K(m) values of root-form PEPC for PEP and Mg2+ were one-tenth or less of those of C4-form PEPC when assayed at either pH 7.3 or 8.0, while the value for HCO3- was about one-half of that of C4-form PEPC at pH 8.0. Glucose 6-phosphate and glycine had little effect on the root-form PEPC at pH 7.3; they caused two-fold activation of the C4-form PEPC. The Ki (L-malate) values at pH 7.3 were 0.12 and 0.43 mM for the root- and C4-form PEPCs, respectively. Comparison of hydropathy profiles among the maize PEPC isoforms suggested that several stretches of amino acid sequences may contribute in some way to their characteristic kinetic properties. The root-form PEPC was phosphorylated by both mammalian cAMP-dependent protein kinase and maize leaf protein kinase, and the phosphorylated enzyme was less sensitive to L-malate.
Phosphoenolpyruvate carboxylase is an enzyme involved in a wide variety of important metabolic pathways of plants such as anaplerotic reactions and C4 and CAM photosynthetic pathways. The accumulation of molecular sequence data of phosphoenolpyruvate carboxylases has enabled us to investigate the function and molecular evolution of the enzymes by computer‐assisted sequence comparison. Here we report the results of sequence comparison of phosphoenolpyruvate carboxylases: (1) Phosphoenofpyruvate carboxylases were classified into four groups; a subgroup of bacterial enzymes and three subgroups of plants enzymes. (2) The divergence time of the monocot enzymes involved in the C4 pathways was roughly estimated to be 150—300 million years. On the other hand, the phylogenetic tree of the enzymes suggested that those for the dicot enzymes involved in the C4 and CAM pathways might be close to the divergence time between the monocots and the dicots. (3) The evolutionary positions of the enzymes prevalent in roots or root nodules were identified. (4) Although sorghum and maize contained at least three genes for the enzymes in their genomes, the rates of amino acid substitution of the enzymes were different from gene to gene. The difference could not be explained by either lineage effects nor bias in base contents.
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