Dead-end complex, lipid interactions and catalytic mechanism of microsomal glutathione transferase 1, an electron crystallography and mutagenesis investigation

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.

Crystal structures of human MGST2 reveal synchronized conformational changes regulating catalysis

“…The GSH at full occupancy observed in the holo complex adopts a “crescent”-shaped binding mode (Fig. 2a ), similar to what has been reported for LTC 4 synthase 10 , 11 and mPGES-1 12 , while contrasting to the extended conformation described for MGST1 17 , 18 . In comparison, the GSH at partial occupancy displays several distinct features.…”

The mercapturic acid pathway

“…Electrospray mass spectrometric data obtained by Ålander et al (Ålander et al 2009b) showed, as also indicated by electron crystallographic data (Holm et al 2006;Kuang et al 2017), that each rMGST1 trimer binds three GSH molecules, rather than one as reported earlier (Sun and Morgenstern 1997;Lengqvist et al 2004). Equilibrium dialysis studies revealed that there is one high-affinity and two low-affinity sites for GSH and that only the high-affinity site is catalytically competent (Ålander et al 2009b); hence, MGST1 shows one-third-of-sites-reactivity.…”

“…Cryoelectron microscopic studies provided additional details about the structure of rat MGST1 (Kuang et al 2017). rMGST1 is present as a dimer of trimers with phospholipids at the interface between the two trimers.…”

MGST1, a GSH transferase/peroxidase essential for development and hematopoietic stem cell differentiation

“…The isoforms ( NM_001005957 , termed MGST1a; and NM_001002215 , termed MGST1b) are located on chromosome 4 and share ~ 56% identity at the amino acid level with the human homologue. Residues critical for activity [30] are conserved between fish and human (see Fig. S4 ) indicating similar functions.…”