Binding and Glutathione Conjugation of Porphyrinogens by Plant Glutathione Transferases

Abstract

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.

Selective Binding of Glutathione Conjugates of Fatty Acid Derivatives by Plant Glutathione Transferases

“…Plant Metabolite Profiling-Strep-tagged GSTs were transiently expressed in N. benthamiana using agroinfiltration (12). Before harvest, leaves were wounded by pinching with forceps and proteins extracted 1 h later.…”

“…For Arabidopsis studies, plants were stably transformed using floral dipping (16). In both cases the Strep-tagged GSTs were purified by affinity chromatography (12), with bound ligands recovered from the concentrated protein in 2 volumes of methanol. Extracts were then analyzed by liquid chromatography-MS as described for bacterial metabolite profiling but using a 1 ϫ 100-mm column running at 0.1 ml⅐min Ϫ1 to enhance sensitivity.…”

“…Here, we reasoned that the lack of compartmentalization in bacteria would allow the GST to interact with any endogenous metabolic intermediate with disruptions in metabolism then directly monitored. A similar approach has recently demonstrated that specific maize GSTUs derail heme metabolism in recombinant E. coli by selectively binding porphyrinogen intermediates (12). In the secondary screening, we have then taken Strep-tagged GSTs with interesting profiles of ligand binding activity in E. coli and transgenically overexpressed them in Nicotiana benthamiana and/or Arabidopsis.…”

Arsenic induced oxidative stress in plants

“…GSTs are dimeric proteins and up to six functional classes of this enzyme have been characterized in plants so far: phi, tau, theta, lambda, zeta and DHARs (Dixon et al 2008;Ghelfi et al 2011).…”

Characterization of a Phanerochaete chrysosporium Glutathione Transferase Reveals a Novel Structural and Functional Class with Ligandin Properties

“…Most studies have focused on the GSH-dependent catalytic activities involved both in detoxification processes and endogenous metabolism. However, some GSTs have also been identified as proteins that selectively bind organic anions such as tetrapyrroles in mammals and plants (3)(4)(5). This "ligandin" property has been defined as the capacity of the protein to bind nonsubstrate ligands (3).…”