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JXB Advance Access published online on October 30, 2006
Journal of Experimental Botany, doi:10.1093/jxb/erl175
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© The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org
Received July 24, 2006
Accepted August 30, 2006

RESEARCH PAPER

Autophosphorylation of Solanum chacoense cytosolic nucleoside diphosphate kinase on Ser117

Sonia Dorion 1, France Dumas 2, and Jean Rivoal 1 *

1 IRBV, Université de Montréal, 4101 Rue Sherbrooke est, Montréal, QC, H1X 2B2 Canada
2 Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montréal, QC, H4P 2R2 Canada

* To whom correspondence should be addressed.
Jean Rivoal, E-mail: jean.rivoal{at}umontreal.ca


  Abstract

NDPK catalyses the interconversion of NTPs and NDPs using a phosphohistidine intermediate as part of its catalytic site. Recombinant Solanum chacoense cytosolic NDPK incubated with [{gamma}-32P]ATP was allowed to autophosphorylate and 32P-labelled P-Ser was identified in an acid hydrolysate of the protein by two-dimensional TLC. Further analysis of 32P-labelled recombinant NDPK by tryptic digestion followed by automated Edman sequencing of the radioactive peptide allowed the identification of a single and conserved P-Ser residue at position 117. Analysis of site-directed mutants where Ser117 was substituted to Asp indicated that the presence of a negative charge at position 117 dramatically lowered the enzyme's catalytic efficiency. Ser autophosphorylation was markedly reduced with increasing ADP concentrations in the autophosphorylation assay. These findings provide evidence that autophosphorylation of cytosolic NDPK on Ser117 could constitute a regulatory mechanism for this important enzyme and that autophosphorylation of Ser117 is modulated by NDP availability.

Keywords: Enzymology; nucleoside diphosphate kinase; plant; phosphoamino acid; phosphorylation; protein sequencing; site-directed mutagenesis.
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