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

Xiu Qing Zhang, Adrian A. Lund, Gautam Sarath, Ronald L. Cerny, Daniel M. Roberts, Raymond Chollet

Research output: Contribution to journalArticle

47 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)70-82
Number of pages13
JournalArchives of Biochemistry and Biophysics
Volume371
Issue number1
DOIs
StatePublished - Nov 1 1999

Keywords

  • Ca- dependent protein kinase (CDPK)
  • Nodulin-100
  • Protein phosphorylation
  • Root nodule
  • Soybean (Glycine max)
  • Sucrose synthase (SS)

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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