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Plant and Cell Physiology Advance Access originally published online on April 13, 2009
Plant and Cell Physiology 2009 50(5):1012-1016; doi:10.1093/pcp/pcp053
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© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Short Communication

Detection of DOPA 4,5-Dioxygenase (DOD) Activity Using Recombinant Protein Prepared from Escherichia coli Cells Harboring cDNA Encoding DOD from Mirabilis jalapa

Nobuhiro Sasaki1,*, Yutaka Abe1, Yukihiro Goda2, Taiji Adachi3, Kichiji Kasahara4 and Yoshihiro Ozeki1

1Department of Biotechnology and Life Science, Faculty of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo, 184-8588 Japan
2Division of Pharmacognosy, Phytochemistry and Narcotics, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo, 158-8501 Japan
3Institute for Plant Biotechnology Research & Development, Ltd., 1-46-901, Hama 5-Chome, Tsurumi-Ku, Osaka, 538-0035 Japan
41128 Moro-oka, Kohoku-ku, Yokohama, Kanagawa, 222-0002 Japan

*Corresponding author: E-mail, nove{at}cc.tuat.ac.jp; Fax, +81-42-388-7239.


  Abstract

Betalains are synthesized in flowers, fruits and other tissues of the plant order Caryophyllales. Betalamic acid is the chromophore of betalain pigments synthesized by a ring-cleaving enzyme reaction on L-dihydroxyphenylalanine (DOPA). Although reverse genetic evidence has proven that DOPA 4,5-dioxygenase (DOD) is a key enzyme of betalain biosynthesis, all attempts to detect recombinant plant DOD activity in vitro have failed. Here, we report on the formation of betalamic acid from DOPA under suitable assay conditions using recombinant MjDOD produced by Escherichia coli. This is the first report showing biochemical evidence for DOD activity in vitro.

Keywords: Betalain - Betalamic acid - DOPA dioxygenase - Mirabilis jalapa

Abbreviations: CCD, carotenoid cleavage dioxygenase; c, ; DOPA5GT, cyclo-DOPA 5-O-glucosyltransferase; DAD, diode array detector; DOD, DOPA 4,5-dioxygenase; DOPA, L-dihydroxyphenylalanine; GUS, β-glucuronidase; LB, Luria–Bertani; MS, mass spectrometry; ORF, open reading frame.

(Received March 17, 2009; Accepted April 8, 2009)
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