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

Abstract

Lines of transgenic tobacco have been generated that are transformed with either the wild-type peanut peroxidase prxPNC2 cDNA, driven by the CaMV35S 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 35S: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 H 2O 2, while values of 11.9 mM and 1.12 mM were determined for the PNC2-M isozyme. A higher Arrenhius activation energy ( E a) 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.