Soybean nodule sucrose synthase (nodulin-100): Further analysis of its phosphorylation using recombinant and authentic root-nodule enzymes

Sucrose synthase (SS) is a known phosphoserine-containing enzyme in legume root nodules and various other plant 'sink' tissues. In order to begin to investigate the possible physiological significance of this posttranslational modification, we have cloned a full-length soybean nodule SS (nodulin-100) cDNA and overexpressed it in Escherichia coli. Authentic nodule SS and recombinant wild-type and mutant forms of the enzyme were purified and characterized. We document that a conserved serine near the N- terminus (Ser¹¹) is the primary phosphorylation site for a nodule Ca²⁺- dependent protein kinase (CDPK) in vitro. Related tryptic digestion and mass spectral analyses indicated that this target residue was also phosphorylated in planta in authentic nodulin-100. In addition, a secondary phosphorylation site(s) in recombinant nodule SS was implicated given that all active mutant enzyme forms (S11A, S11D, S11C, and N-terminal truncation between Ala² and Arg¹³) were phosphorylated, albeit weakly, by the CDPK. This secondary site(s) likely resides between Glu¹⁴ and Met¹⁹³ as evidenced by CNBr cleavage and phosphopeptide mapping. Phosphorylation of the recombinant and authentic nodule Ser¹¹ enzymes in vitro by the nodule CDPK had no major effect on the sucrose-cleavage activity and/or kinetic properties. However, phosphorylation de- creased the apparent surface hydrophobicity of the recombinant wild-type enzyme, suggesting that this covalent modification could potentially play some role in the documented partitioning of nodulin- 100 between the nodule symbiosome/plasma membranes and cytosol in planta.