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J. Biol. Chem., Vol. 278, Issue 42, 41389-41399, October 17, 2003

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A Biochemical and Molecular Characterization of LEP1, an Extensin Peroxidase from Lupin^*

Nicholas J. Price  , Carla Pinheiro , Claudio M. Soares , David A. Ashford ¶, Cândido Pinto Ricardo  || and Phil A. Jackson  **

From the Instituto de Tecnologia Química e Biológica, Apartado 127, 2781-901 Oeiras, Portugal, ^¶Research and Analytical Glycobiology, Department of Biology, University of York, P. O. Box 373, York YO10 5YW, United Kingdom, and ^||Instituto de Agronomia, Tapada da Ajuda, 1349-017 Lisbon, Portugal

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 µM 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.

Received for publication, April 30, 2003 , and in revised form, July 4, 2003.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF403735.

^* This work was supported by Fundação para a Ciência e a Tecnologia Research Grants POCTI/1999/BCI/3311/99 and POCTI/33201/BME/2000 (to P. A. J.), Personal Grant PRAXIS XXI/BD/17106 (to N. J. P.), and PRAXIS XXI/BPD/16316/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Botany and Plant Sciences and the Center for Plant Cell Biology, 2130 Batchelor Hall, University of California, Riverside, CA 92521.

^** To whom correspondence should be addressed: Instituto de Tecnologia Química e Biológica, Apartado 127, 2781-901 Oeiras, Portugal. Tel.: 351-214469634; Fax: 351-214411277; E-mail: Phil{at}itqb.unl.pt.

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