Cloning, expression, and characterization of a root-form phosphoenolpyruvate carboxylase from Zea mays: comparison with the C4-form enzyme.
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.
Dong
L Y
LY
Laboratory of Plant Physiology, Graduate School of Agriculture, Kyoto University, Japan.
Masuda
T
T
Kawamura
T
T
Hata
S
S
Izui
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D018076:DNA, Complementary; D018744:DNA, Plant; D015870:Gene Expression; D007527:Isoenzymes / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics*; D010766:Phosphorylation; D018515:Plant Leaves; D018517:Plant Roots / Q000201:enzymology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics
D018076:DNA, Complementary; D018744:DNA, Plant; D007527:Isoenzymes; D017868:Cyclic AMP-Dependent Protein Kinases; D010730:Phosphoenolpyruvate Carboxylase