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J. Biol. Chem., Vol. 282, Issue 42, 30827-30835, October 19, 2007

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Phenylalanine Biosynthesis in Arabidopsis thaliana

IDENTIFICATION AND CHARACTERIZATION OF AROGENATE DEHYDRATASES^*

Man-Ho Cho, Oliver R. A. Corea, Hong Yang, Diana L. Bedgar, Dhrubojyoti D. Laskar, Aldwin M. Anterola, Frances Anne Moog-Anterola, Rebecca L. Hood, Susanne E. Kohalmi, Mark A. Bernards, ChulHee Kang^¶, Laurence B. Davin, and Norman G. Lewis¹

From the Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, the Department of Biology, University of Western Ontario, London, Ontario N6A 5B7, Canada, and the ^¶School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4660

There is much uncertainty as to whether plants use arogenate, phenylpyruvate, or both as obligatory intermediates in Phe biosynthesis, an essential dietary amino acid for humans. This is because both prephenate and arogenate have been reported to undergo decarboxylative dehydration in plants via the action of either arogenate (ADT) or prephenate (PDT) dehydratases; however, neither enzyme(s) nor encoding gene(s) have been isolated and/or functionally characterized. An in silico data mining approach was thus undertaken to attempt to identify the dehydratase(s) involved in Phe formation in Arabidopsis, based on sequence similarity of PDT-like and ACT-like domains in bacteria. This data mining approach suggested that there are six PDT-like homologues in Arabidopsis, whose phylogenetic analyses separated them into three distinct subgroups. All six genes were cloned and subsequently established to be expressed in all tissues examined. Each was then expressed as a Nus fusion recombinant protein in Escherichia coli, with their substrate specificities measured in vitro. Three of the resulting recombinant proteins, encoded by ADT1 (At1g11790), ADT2 (At3g07630), and ADT6 (At1g08250), more efficiently utilized arogenate than prephenate, whereas the remaining three, ADT3 (At2g27820), ADT4 (At3g44720), and ADT5 (At5g22630) essentially only employed arogenate. ADT1, ADT2, and ADT6 had k_cat/K_m values of 1050, 7650, and 1560 M^-1 s^-1 for arogenate versus 38, 240, and 16 M^-1 s^-1 for prephenate, respectively. By contrast, the remaining three, ADT3, ADT4, and ADT5, had k_cat/K_m values of 1140, 490, and 620 M^-1 s^-1, with prephenate not serving as a substrate unless excess recombinant protein (>150 µg/assay) was used. All six genes, and their corresponding proteins, are thus provisionally classified as arogenate dehydratases and designated ADT1–ADT6.

Received for publication, March 28, 2007 , and in revised form, August 28, 2007.

^* This work was supported in part by National Science Foundation Grant MCB-0417291, United States Dept. of Energy Grant DE-FG-0397ER20259, National Institutes of Health Grant GM66173, and funds from the G. Thomas and Anita Hargrove Center for Plant Genomic Research and the Natural Sciences and Engineering Research Council of Canada (to M. A. B. and S. E. K.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed. Tel.: 509-335-8382; Fax: 509-335-8206; E-mail: lewisn{at}wsu.edu.

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