Structural Basis for Detoxification and Oxidative Stress Protection in Membranes

doi:10.1016/j.jmb.2006.05.056

Journal of Molecular Biology

Volume 360, Issue 5, 28 July 2006, Pages 934-945

https://doi.org/10.1016/j.jmb.2006.05.056 Get rights and content

Abstract

Synthesis of mediators of fever, pain and inflammation as well as protection against reactive molecules and oxidative stress is a hallmark of the MAPEG superfamily (membrane associated proteins in eicosanoid and glutathione metabolism). The structure of a MAPEG member, rat microsomal glutathione transferase 1, at 3.2 Å resolution, solved here in complex with glutathione by electron crystallography, defines the active site location and a cytosolic domain involved in enzyme activation. The glutathione binding site is found to be different from that of the canonical soluble glutathione transferases. The architecture of the homotrimer supports a catalytic mechanism involving subunit interactions and reveals both cytosolic and membraneous substrate entry sites, providing a rationale for the membrane location of the enzyme.

Introduction

Oxidative stress and exposure to toxic compounds are constant threats to living organisms. Efficient protection systems involving specific enzymes have emerged throughout evolution. Glutathione transferases (GSTs) are playing a crucial role in cellular biotransformation of electrophilic compounds through binding and positioning the tri-peptide glutathione (GSH), γ-l-glutamyl-l-cysteinyl-glycine, for nucleophilic attack. Based on sequence and structural similarity distinct, ancient glutathione transferase protein families of cytoplasmic, microsomal, mitochondrial and bacterial origin have been identified.¹ In addition to the detoxification role played by GSTs, homologous members within the families may carry out distinctly different functions. Soluble GSTs from mammals, plants, bacteria and insects have been well characterised structurally and subdivided into several classes. The canonical cytosolic enzymes are dimers and related by evolution to glutaredoxin having the unique thioredoxin βαβαββα fold.² Here we describe the first detailed structure of a GST from the microsomal family, MGST1 (microsomal glutathione transferase 1). Like most soluble GSTs, MGST1 catalyses conjugation of GSH to a number of electrophilic compounds and is therefore playing an important role in biotransformation of xenobiotic compounds.³ In addition, MGST1 protects biological membranes from degradation through GSH-dependent reduction of unspecifically peroxidised phospholipids.⁴ The microsomal GSTs, more recently termed MAPEG (membrane associated proteins in eicosanoid and glutathione metabolism), also include members that are crucial for synthesis of mediators of fever, pain and inflammation.⁵^,⁶ These pathophysiological responses are regulated by GSH-dependent transformations of specific oxidised lipid intermediates, prostaglandin H₂ endoperoxide and leukotriene A₄ epoxide, to prostaglandin E₂ and leukotriene C₄, respectively. Thus, variations of a hitherto unknown common protein structure have emerged to selectively catalyse distinct activities with strong physiological and pathophysiological significance. In the present work the structural basis for these reactions has been elucidated. By determining the atomic structure of a key enzyme we now begin to understand the specificity and function of these proteins. As they are potential targets for treatment of common diseases such as rheumatoid arthritis and asthma, the results should impact on drug development.

Section snippets

Structure of MGST1

Upon reconstitution into lipid bilayers at low lipid/protein ratio MGST1 forms two-dimensional crystals of two different two-sided plane groups, p22₁2₁ and p6, both suitable for analysis by electron crystallography. We have now solved the structure of the rat enzyme to 3.2 Å in plane resolution (4.0 Å perpendicular to the membrane plane) using data from both crystal forms (Figure 1(a) and Table 1). As previously described⁷^,⁸ both the p6 and the p22₁2₁ maps contain densities with three repeats

Discussion

A fundamental aspect of glutathione transferase catalysis involves the stabilisation of the reactive nucleophilic thiolate anion form of GSH. The presence of the GSH thiolate has been demonstrated by spectroscopy in both soluble glutathione transferases²⁶ and MGST1.²⁷ In soluble glutathione transferases a tyrosine or serine hydroxyl has been shown to hydrogen bond to the GSH thiolate and thereby lower its pK_a value by several units to ≈6.²⁸ In MGST1 GSH is surrounded by several residues that

Specimen preparation

MGST1 was purified from rat liver microsomes³ and two-dimensional crystals with p6 and p22₁2₁ two-dimensional plane group symmetry were prepared by reconstitution into phospholipid bilayers by dialysis at a molar lipid to protein ratio varying between 3 and 5 as previously.³⁶ The quality of the reconstituted crystals was evaluated by electron microscopy of negatively stained specimens. Excellent crystal preparations were selected for cryo-electron microscopy. These specimens were prepared on

Acknowledgements

We thank Gerd Lundqvist and Gudrun Tibbelin for excellent technical assistance. This work was supported by grants from Swedish Research Council (to H.H.) and from the Swedish Cancer Society, the Swedish National Board for Laboratory Animals and Funds from Karolinska Institutet (to R.M.). P. J. H. has been a recipient of a fellowship from the Swedish Foundation for Strategic Research.

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Microsomal glutathione transferase 1 (MGST1) is a member of the MAPEG family (membrane associated proteins in eicosanoid and glutathione metabolism), defined according to enzymatic activities, sequence motifs, and structural properties. MGST1 is a homotrimer which can bind three molecules of glutathione (GSH), with one modified to a thiolate anion displaying one-third-of-sites-reactivity. MGST1 has both glutathione transferase and peroxidase activities. Each is based on stabilizing the GSH thiolate in the same active site. MGST1 is abundant in the liver and displays a broad subcellular distribution with high levels in endoplasmic reticulum and mitochondrial membranes, consistent with a physiological role in protection from reactive electrophilic intermediates and oxidative stress. In this review paper, we particularly focus on recent advances made in understanding MGST1 activation, induction, broad subcellular distribution, and the role of MGST1 in apoptosis, ferroptosis, cancer progression, and therapeutic responses.
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Citation Excerpt :
The MAPEG superfamily was defined based on enzymatic activities, structural properties, and sequence similarities [19]. The first MAPEG structure solved, in 2006, was that of rat MGST1 [20], and it does not share structural similarity with the other family members. The microsomal enzymes are shorter than cytosolic ones, by around 150–160 amino acids, and some members of the family have been shown to be trimers [19,21,22].
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Eicosanoids are potent lipid mediators involved in central physiological processes such as hemostasis, renal function and parturition. When formed in excess, eicosanoids become critical players in a range of pathological conditions, in particular pain, fever, arthritis, asthma, cardiovascular disease and cancer. Eicosanoids are generated via oxidative metabolism of arachidonic acid along the cyclooxygenase (COX) and lipoxygenase (LOX) pathways. Specific lipid species are formed downstream of COX and LOX by specialized synthases, some of which reside on the nuclear and endoplasmic reticulum, including mPGES-1, FLAP, LTC4 synthase, and MGST2. These integral membrane proteins are members of the family “membrane-associated proteins in eicosanoid and glutathione metabolism” (MAPEG). Here we focus on this enzyme family, which encompasses six human members typically catalyzing glutathione dependent transformations of lipophilic substrates. Enzymes of this family have evolved to combat the topographical challenge and unfavorable energetics of bringing together two chemically different substrates, from cytosol and lipid bilayer, for catalysis within a membrane environment. Thus, structural understanding of these enzymes are of utmost importance to unravel their molecular mechanisms, mode of substrate entry and product release, in order to facilitate novel drug design against severe human diseases.
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In this study, the entire glutathione S-transferases (GSTs), the major phase II detoxification enzyme, were identified in two marine copepod species Tigriopus japonicus and Paracyclopina nana. The genome-wide identification of GSTs in T. japonicus and P. nana resulted in 32 and 20 GSTs in total, respectively. Among the identified GSTs, two specific classes of GSTs, specifically sigma and delta/epsilon GSTs were the dominant form of cytosolic GSTs in T. japonicus, while delta/epsilon and mu classes were dominant cytosolic GSTs in P. nana. In addition, Membrane-Associated Proteins in Eicosanoid and Glutathione metabolism (MAPEG) family were found in relatively higher proportion compared to other classes. Moreover, sigma, delta/epsilon, and microsomal GSTs have shown to expand through tandem duplication. To validate the detoxification function of the identified GSTs, both copepods were exposed to copper (Cu²⁺) and the reactive oxygen species (ROS) level and GST activity were measured. With integration of phylogenetic analysis and xenobiotic-mediated GST mRNA expression patterns along with previous enzymatic activities, the functional divergence among species-specific GST genes was clearly observed. This study covers full identification of GST classes in two marine copepod species and their important role in marine environmental ecotoxicology.

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^†: Present address: P.J. Holm, Aarhus University, Department of Molecular Biology, Gustav Wieds Vej 10C, DK - 8000 Aarhus C, Denmark.

^‡: Present address: K. Mitsuoka and N. Gyobu, Structural Analysis Team, Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, Aomi 2-41-6, Koto-Ku, Tokyo, 135-0064, Japan.

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Journal of Molecular Biology

Structural Basis for Detoxification and Oxidative Stress Protection in Membranes

Abstract

Introduction

Section snippets

Structure of MGST1

Discussion

Specimen preparation

Acknowledgements

Curr. Opin. Struct. Biol.

Biochem. Pharamcol.

Biochim. Biophys. Acta

J. Biol. Chem.

Structure

J. Biol. Chem.

J. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

Arch. Biochem. Biophys.

J. Biol. Chem.

Structure

J. Struct. Biol.

J. Struct. Biol.

Ultramicroscopy

J. Struct. Biol.

J. Mol. Graph.

J. Mol. Biol.

Microsomal glutathione S-transferase. Purification, initial characterization and demonstration that it is not identical to the cytosolic glutathione S-transferases A, B and C

Eur. J. Biochem.

Common structural features of MAPEG - a widespread superfamily of membrane associated proteins with highly divergent functions in eicosanoid and glutathione metabolism

Protein Sci.

Bioinformatic and enzymatic characterization of the MAPEG superfamily

FEBS J.