Binding and glutathione conjugation of porphyrinogens by plant glutathione transferases.
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.
Dixon
David P
DP
Centre for Bioactive Chemistry, School of Biological and Biomedical Sciences, Durham University, Durham, UK.
Lapthorn
Adrian
A
Madesis
Panagiotis
P
Mudd
Elisabeth A
EA
Day
Anil
A
Edwards
Robert
R
D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D013058:Mass Spectrometry; D008958:Models, Molecular; D015394:Molecular Structure; D011165:Porphyrinogens / Q000737:chemistry / Q000378:metabolism*; D011166:Porphyrins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics
D008024:Ligands; D011165:Porphyrinogens; D011166:Porphyrins; D005982:Glutathione Transferase; D005978:Glutathione