Removal of the N-linked glycan structure from the peanut peroxidase prxPNC2: Influence on protein stability and activity

Pathirana, Ranjith, Watson, Lyn, Chen, Balance, Leung, Susanna, Voisey, Christine, Murray, Trish and McManus, Michael T. (2005) Removal of the N-linked glycan structure from the peanut peroxidase prxPNC2: Influence on protein stability and activity. Phytochemistry, 66 16: 1869-1879. doi:10.1016/j.phytochem.2005.06.027


Author Pathirana, Ranjith
Watson, Lyn
Chen, Balance
Leung, Susanna
Voisey, Christine
Murray, Trish
McManus, Michael T.
Title Removal of the N-linked glycan structure from the peanut peroxidase prxPNC2: Influence on protein stability and activity
Journal name Phytochemistry   Check publisher's open access policy
ISSN 0031-9422
Publication date 2005-08
Sub-type Article (original research)
DOI 10.1016/j.phytochem.2005.06.027
Volume 66
Issue 16
Start page 1869
End page 1879
Total pages 11
Editor D. Strack
G.P. Blwell
N. G. Lewis
Place of publication Oxford, England
Publisher Pergamon-Elsevier Science Ltd
Collection year 2005
Language eng
Subject C1
270400 Botany
620500 Sustainable Plant Production Systems
Abstract Lines of transgenic tobacco have been generated that are transformed with either the wild-type peanut peroxidase prxPNC2 cDNA, driven by the CaMV3 5S promoter (designated 35S::prxPNC2-WT) or a mutated PNC2 cDNA in which the asparagine residue (Asn(189)) associated with the point of glycan attachment (Asn(189)) has been replaced with alanine (designated 35S::prxPNC2-M). PCR, using genomic DNA as template, has confirmed the integration of the 35S::prxPNC2-WT and 35::prxPNC2-M constructs into the tobacco genome, and western analysis using anti-PNC2 antibodies has revealed that the prxPNC2-WT protein product (PNC2-WT) accumulates with a molecular mass of 34,670 Da, while the prxPNC2-M protein product (PNC2-M) accumulates with a molecular mass of 32,600 Da. Activity assays have shown that both PNC2-WT and PNC2-M proteins accumulate preferentially in the ionically-bound cell wall fraction, with a significantly higher relative accumulation of the PNC2-WT isoenzyme in the ionically-bound fraction when compared with the PNC2-M isoform. Kinetic analysis of the partially purified PNC2-WT isozyme revealed an affinity constant (apparent K-m) of 11.2 mM for the reductor substrate guaiacol and 1.29 mM for H2O2, while values of 11.9 mM and 1.12 mM were determined for the PNC2-M isozyme. A higher Arrenhius activation energy (E,,) was determined for the PNC2-M isozyme (22.9 kJ mol(-1)), when compared with the PNC2-WT isozyme (17.6 kJ mol(-1)), and enzyme assays have determined that the absence of the glycan influences the thermostability of the PNC2-M isozyme. These results are discussed with respect to the proposed roles of N-linked glycans attached to plant peroxidases. (c) 2005 Elsevier Ltd. All rights reserved.
Keyword Arachis hypogaea (L.)
Leguminosae
peanut
N-linked glycosylation
peroxidase
Horseradish-peroxidase
Partial Deglycosylation
Cationic Peroxidase
Covalent Structure
Escherichia-coli
Amino-acid
Cells
Secretion
Sequence
Enzyme
Q-Index Code C1

Document type: Journal Article
Sub-type: Article (original research)
Collections: 2006 Higher Education Research Data Collection
School of Chemistry and Molecular Biosciences
 
Versions
Version Filter Type
Citation counts: TR Web of Science Citation Count  Cited 6 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 6 times in Scopus Article | Citations
Google Scholar Search Google Scholar
Created: Wed, 15 Aug 2007, 05:52:50 EST