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J. Biol. Chem., Vol. 283, Issue 29, 20268-20276, July 18, 2008

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Binding and Glutathione Conjugation of Porphyrinogens by Plant Glutathione Transferases^*

David P. Dixon, Adrian Lapthorn, Panagiotis Madesis^¶, Elisabeth A. Mudd^¶, Anil Day^¶, and Robert Edwards¹

From the Centre for Bioactive Chemistry, School of Biological and Biomedical Sciences, Durham University, Durham DH1 3LE, Department of Chemistry, Glasgow University, Glasgow G12 8QQ, Scotland, and ^¶Faculty of Life Sciences, The University of Manchester, Manchester, M13 9PT, United Kingdom

Overexpression in Escherichia coli of a tau (U) class glutathione transferase (GST) from maize (Zea mays L.), termed ZmGSTU1, caused a reduction in heme levels and an accumulation of porphyrin precursors. This disruption was highly specific, with the expression of the closely related ZmGSTU2 or other maize GSTs having little effect. Expression in E. coli of a series of chimeric ZmGSTU1/ZmGSTU2 proteins identified domains responsible for disrupting porphyrin metabolism. In addition to known heme precursors, expression of ZmGSTU1 led to the accumulation of a novel glutathione conjugate of harderoporphyrin(ogen) (2,7,12,18-tetramethyl-3-vinylporphyrin-8,13,17-tripropionic acid). Using the related protoporphyrinogen as a substrate, conjugation could be shown to occur on one vinyl group and was actively catalyzed by the ZmGSTU. In plant transgenesis studies, the ZmGSTUs did not perturb porphyrin metabolism when expressed in the cytosol of Arabidopsis or tobacco. However, expression of a ZmGSTU1-ZmGSTU2 chimera in the chloroplasts of tobacco resulted in the accumulation of the harderoporphyrin(ogen)-glutathione conjugate observed in the expression studies in bacteria. Our results show that the well known ability of GSTs to act as ligand binding (ligandin) proteins of porphyrins in vitro results in highly specific interactions with porphyrinogen intermediates, which can be demonstrated in both plants and bacteria in vivo.

Received for publication, March 13, 2008 , and in revised form, May 6, 2008.

^* This work was supported by the Biotechnology and Biological Sciences Research Council Grant BBC51227X1 and a research development fellowship (to R. E.). 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 Figs. S1–S3.

¹ To whom correspondence should be addressed. Tel.: 44-191-3341318; Fax: 44-191-3341201; E-mail: Robert.Edwards{at}durham.ac.uk.

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