Abstract
Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used electron crystallography of two-dimensional crystals in order to determine an atomic model of rat MGST1 in a lipid environment. The model comprises 123 of the 155 amino acid residues, two structured phospholipid molecules, two aliphatic chains and one glutathione (GSH) molecule. The functional unit is a homotrimer centered on the crystallographic three-fold axes of the unit cell. The GSH substrate binds in an extended conformation at the interface between two subunits of the trimer supported by new in vitro mutagenesis data. Mutation of Arginine 130 to alanine resulted in complete loss of activity consistent with a role for Arginine 130 in stabilizing the strongly nucleophilic GSH thiolate required for catalysis. Based on the new model and an electron diffraction data set from crystals soaked with trinitrobenzene, that forms a dead-end Meisenheimer complex with GSH, a difference map was calculated. The map reveals side chain movements opening a cavity that defines the second substrate site.
Highlights
Glutathione (GSH) is a γ−L-Glu-L-Cys-Gly tripeptide with a gamma peptide linkage between the amine group of cysteine and the carboxyl group of the glutamate side-chain
It is well established that MGST1, MGST2 and LTC4S belong to the membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG) superfamily[7]
As the residue identity between microsomal prostaglandin E synthase 1 (MPGES1) and MGST1 is partly high for the TM1-TM2 loop, it may be the case for MGST1 that this loop is not directly involved in GSH binding but serves other purposes
Summary
Glutathione (GSH) is a γ−L-Glu-L-Cys-Gly tripeptide with a gamma peptide linkage between the amine group of cysteine and the carboxyl group of the glutamate side-chain. It was observed that the most well studied membrane bound GST, microsomal glutathione transferase 1 (MGST1, EC number: 2.5.1.18) shared similarities with leukotriene C4 synthase (LTC4S, EC number: 4.4.1.20), catalyzing the conjugation reaction between leukotriene (LT) A4 and GSH. The enzyme stabilizes the thiolate anion at neutral pH and combines this capability with providing a binding pocket for hydrophobic electrophiles at a site adjacent to the bound GSH15. Apart from this important role in phase II detoxification, MGST1 has been shown to have GSH peroxidase activity against lipid hydroperoxides[16]. X-ray structures of LTC4S10, 11 and MPGES124, with better completeness of experimental data and more revealing contribution of high resolution information, confirmed these observations and could map further details with regard to binding of GSH
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