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Scientific article
English

Identification of a Ca(2+)-pectate binding site on an apoplastic peroxidase

Contributors

Carpin, Sabine; Crèvecoeur, Michèle; De Meyer-Fague, Mireille Anne; Simon, Patrice; Greppin, Hubert; Penel, Claude

Published in

The Plant cell, vol. 13, no. 3, p. 511-520

Publication date

2001

Abstract

An apoplastic isoperoxidase from zucchini (APRX) was shown to bind strongly to polygalacturonic acid in their Ca(2)+-induced conformation. By homology modeling, we were able to identify a motif of four clustered arginines (positions 117, 262, 268, and 271) that could be responsible for this binding. To verify the role of these arginine residues in the binding process, we prepared three mutants of APRX (M1, R117S; M2, R262Q/R268S; and M3, R262Q/R268S/R271Q). APRX and the three mutants were expressed as recombinant glycoproteins by the baculovirus-insect cell system. This procedure yielded four active enzymes with similar molecular masses that were tested for their ability to bind Ca(2)+-pectate. Recombinant wild-type APRX exhibited an affinity for the pectic structure comparable to that of the native plant isoperoxidase. The mutations impaired binding depending on the number of arginine residues that were replaced. M1 and M2 showed intermediate affinities, whereas M3 did not bind at all. This was demonstrated using an in vitro binding test and on cell walls of hypocotyl cross-sections. It can be concluded that APRX bears a Ca(2)+-pectate binding site formed by four clustered arginines. This site could ensure that APRX is properly positioned in cell walls, using unesterified domains of pectins as a scaffold.

Keywords

  • Arginine/metabolism
  • Binding Sites/genetics
  • Calcium-Binding Proteins/genetics/metabolism
  • Cell Wall/metabolism
  • Cucurbitaceae/cytology/genetics/metabolism
  • Electrophoresis
  • Hypocotyl/cytology
  • Models, Molecular
  • Mutation
  • Pectins/metabolism
  • Peroxidase/chemistry/genetics/metabolism
  • Peroxidases/metabolism
  • Protein Conformation
  • Static Electricity

UNIGE affiliation entities

Citation (ISO format)

CARPIN, Sabine et al. Identification of a Ca(2+)-pectate binding site on an apoplastic peroxidase. In: The Plant cell, 2001, vol. 13, n° 3, p. 511–520. doi: 10.2307/3871403

Main files (1)

Article (Published version)

Identifiers

Journal ISSN

1040-4651
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Technical informations

Creation

07/06/2010 16:11:00

First validation

07/06/2010 16:11:00

Update

14/03/2023 15:29:45

Status update

14/03/2023 15:29:45

Last indexation

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