Biochemical characterization of the seryl-phosphorylation of soybean nodule phosphoenolpyruvate carboxylase and sucrose synthase, and molecular cloning and overexpression of a soybean nodule sucrose synthase cDNA in Escherichia coli

Phosphoenolpyruvate carboxylase (PEPC) and sucrose synthase (SS) are critical enzymes in legume nodule C/N-metabolism. In this dissertation it is documented that both of these enzymes in soybean root nodules are phosphorylated in situ on a serine residue(s). Stem-girdling or prolonged darkening of the parent plants significantly decreased the apparent phosphorylation state of nodule PEPC. The effect of darkness on PEPC phosphorylation was reversed by illuminating the darkened plants for more than 1 h. This reversal was prevented by concomitant stem girdling, suggesting that the phosphorylation of PEPC in soybean nodules is modulated by photosynthate translocated recently from the shoots. The partially purified PEPC-kinase is Ca$\sp{2+}$-independent and has an apparent native molecular mass of about 30 kDa. In-gel kinase assays revealed two PEPC-dependent, Ca$\sp{2+}$-independent protein kinase polypeptides with molecular masses of about 32 and 37 kDa. Moreover, this protein-Ser/Thr kinase is highly specific to plant PEPC. The Ca$\sp{2+}$-independent PEPC-kinase activity in nodules is up- and down-regulated by illumination and stem girdling or prolonged darkness, respectively, suggesting that control of the phosphorylation state of PEPC by current photosynthate in the nodule is mediated largely by this Ca$\sp{2+}$-independent protein kinase. In contrast, SS in soybean nodules (nodulin-100) is phosphorylated by a Ca$\sp{2+}$-dependent protein kinase (CDPK). The molecular mass of this SS-kinase is about 55 kDa under both native and denaturing conditions. Unlike nodule PEPC and PEPC-kinase, the phosphorylation state of SS and the total activity of its CDPK are not likely modulated by photosynthate supply from the shoots. A full-length cDNA encoding soybean nodule SS was cloned, sequenced (Accession No. AF030231), and overexpressed in E. coli as His-tagged and untagged constructs. This cDNA is 2842 bp long and has an open reading frame of 805 amino acid residues. The soluble recombinant enzymes are catalytically active and phosphorylatable in vitro by the partially purified SS-kinase from soybean nodules