The effects of the site-directed removal of N-glycosylation from cationic peanut peroxidase on its function.
Peanut peroxidase has been diffracted. The location of its heme and calcium moieties have been shown and their role demonstrated. However, the structure and role of its glycans is only now being elucidated. The role of three N-linked complex glycans on cationic peroxidase (cPrx) of peanut (Arachis hypogaea L cv. Valencia), as expressed by prxPNC1 in transgenic tobacco, was analyzed by site-directed replacement of each of the three glycosylation sites, N-60, N-144, and N-185 with Q, individually. The mutant prxPNC1 cDNAs with a 3' histidine-tag were expressed in transgenic tobacco. The effect on the catalytic ability, thermal stability, and unfolding properties of the mutant peroxidases, isolated from the medium of transgenic tobacco cell suspension cultures were compared with those of the wild cPrx from peanut. It was found that the ablation of the glycans at N-60 and N-144 influences the full expression of the cPrx catalytic ability. The glycan at N-185 is important for the thermostability, as is the removal of the carbohydrate chain at N-185, resulting in rapid enzymatic decrease at temperatures of 50 degrees C. All three glycans appeared to influence the folding of the protein.
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Department of Plant Sciences, University of Western Ontario, London, Canada.
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van Huystee
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D010367:Arachis / Q000201:enzymology*; D001665:Binding Sites; D015153:Blotting, Western; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D019791:Guanidine / Q000494:pharmacology; D006639:Histidine / Q000378:metabolism; D007700:Kinetics; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009195:Peroxidase / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011134:Polysaccharides / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D013696:Temperature; D013997:Time Factors; D014026:Nicotiana / Q000235:genetics; D014170:Transformation, Genetic; D014357:Trypsin / Q000494:pharmacology
D011134:Polysaccharides; D006639:Histidine; D009195:Peroxidase; D014357:Trypsin; D019791:Guanidine