A biochemical and molecular characterization of LEP1, an extensin peroxidase from lupin.
An analysis of apoplastic extensin cross-linking activity in vegetative organs of Lupinus albus indicated that leaves contained the highest specific activity. Assays of peroxidases fractionated from this material demonstrated that this activity could be largely attributed to a soluble and apoplastic 51-kDa peroxidase, denoted LEP1. Relative to other purified peroxidases, LEP1 demonstrates high extensin cross-linking activity and can be classified as an extensin peroxidase (EP). Optimal conditions for the in vitro oxidation of other phenolic substrates included 1.5-3.0 mm peroxide at pH 5.0. EP activity of LEP1 was low under these conditions but optimal and substantially higher with 100 microm peroxide and neutral pH, suggesting that physiological changes in pH and peroxide in muro could heavily influence the extensin cross-linking activity of LEP1 in vivo. Analysis of LEP1 glycans indicated 11-12 N-linked glycans, predominantly the heptasaccharide Man3XylFucGlcNAc2, but also larger structures showing substantial heterogeneity. Comparative assays with horseradish peroxidase isoform C and peanut peroxidases suggested the high level of glycosylation in LEP1 may be responsible for the high solubility of this EP in the apoplastic space. A full-length cDNA corresponding to LEP1 was cloned. Quantitative reverse transcriptase-PCR demonstrated LEP1 induction in apical portions of etiolated hypocotyls 30-60 min after exposure to white light, prior to the onset of growth inhibition. Comparative modeling of the translated sequence indicated an unusually unobstructed equatorial cleft across the substrate access channel, which might facilitate interaction with extensin and confer higher EP activity.
Price
Nicholas J
NJ
Instituto de Tecnologia Química e Biológica, Apartado 127, 2781-901 Oeiras, Portugal.
Pinheiro
Carla
C
Soares
Claudio M
CM
Ashford
David A
DA
Ricardo
Cândido Pinto
CP
Jackson
Phil A
PA
D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D006735:Horseradish Peroxidase / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008027:Light; D031297:Lupinus / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009195:Peroxidase / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D018515:Plant Leaves / Q000201:enzymology; D010940:Plant Proteins / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011134:Polysaccharides / Q000737:chemistry; D011189:Potassium Chloride / Q000494:pharmacology; D020033:Protein Isoforms; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D013997:Time Factors
D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D006023:Glycoproteins; D007477:Ions; D010940:Plant Proteins; D011134:Polysaccharides; D020033:Protein Isoforms; C005488:extensin protein, plant; D011189:Potassium Chloride; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D010544:Peroxidases; D009195:Peroxidase