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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Molecular Cloning and Characterization of a Vacuolar Class III Peroxidase Involved in the Metabolism of Anticancer Alkaloids in Catharanthus roseus^1,[C]

John Innes Centre, Norwich NR4 7UH, United Kingdom (M.M.R.C., F.H., M.L.); IBMC – Instituto de Biologia Molecular e Celular (P.D., L.G.P., I.A., M.S.) and Department of Botany of Faculty of Sciences (P.D., L.G.P., M.S.), Universidade do Porto, 4150–180 Porto, Portugal; Institute of Biology, Clusius Laboratory, Leiden University, 2333 AL Leiden, The Netherlands (I.A., J.M.); and Department of Plant Biology (Plant Physiology), University of Murcia, E–30100 Murcia, Spain (A.R.B.)

Catharanthus roseus produces low levels of two dimeric terpenoid indole alkaloids, vinblastine and vincristine, which are widely used in cancer chemotherapy. The dimerization reaction leading to -3',4'-anhydrovinblastine is a key regulatory step for the production of the anticancer alkaloids in planta and has potential application in the industrial production of two semisynthetic derivatives also used as anticancer drugs. In this work, we report the cloning, characterization, and subcellular localization of an enzyme with anhydrovinblastine synthase activity identified as the major class III peroxidase present in C. roseus leaves and named CrPrx1. The deduced amino acid sequence corresponds to a polypeptide of 363 amino acids including an N-terminal signal peptide showing the secretory nature of CrPrx1. CrPrx1 has a two-intron structure and is present as a single gene copy. Phylogenetic analysis indicates that CrPrx1 belongs to an evolutionary branch of vacuolar class III peroxidases whose members seem to have been recruited for different functions during evolution. Expression of a green fluorescent protein-CrPrx1 fusion confirmed the vacuolar localization of this peroxidase, the exact subcellular localization of the alkaloid monomeric precursors and dimeric products. Expression data further supports the role of CrPrx1 in -3',4'-anhydrovinblastine biosynthesis, indicating the potential of CrPrx1 as a target to increase alkaloid levels in the plant.

² These authors contributed equally to the article.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Mariana Sottomayor (msottoma{at}ibmc.up.pt).

^[C] Some figures in this article are displayed in color online but in black and white in the print edition.

www.plantphysiol.org/cgi/doi/10.1104/pp.107.107060

^* Corresponding author; e-mail msottoma{at}ibmc.up.pt.

Received August 9, 2007; accepted November 23, 2007; published December 7, 2007.

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