Structure of glutathione reductase from escherichia coli at 1.86 Å resolution: Comparison with the enzyme from human erythrocytes

Abstract

The crystal structure of the dimeric flavoenzyme glutathione reductase from Escherichia coli was determined and refined to an R-factor of 16.8% at 1.86 A resolution. The molecular 2-fold axis of the dimer is local but very close to a possible crystallographic 2-fold axis; the slight asymmetry could be rationalized from the packing contacts.The 2 crystallographically independent subunits of the dimer are virtually identical, yielding no structural clue on possible cooperativity. The structure was compared with the well-known structure of the homologous enzyme from human erythrocytes with 52% sequence identity. Significant differences were found at the dimer interface, where the human enzyme has a disulfide bridge, whereas the E. coli enzyme has an antiparallel P-sheet connecting the subunits. The differences at the glutathione binding site and in particular a deformation caused by a LeuIle exchange indicate why the E. coli enzyme accepts trypanothione much better than the human enzyme. The reported structure provides a frame for explaining numerous published engineering results in detail and for guiding further ones.Keywords: asymmetries; crystal packing contacts; crystal structure; disulfide oxidoreductases; glutathione; trypanothione Glutathione reductase (EC 1.6.4.2) catalyzes the reduction of oxidized glutathione according to: GSSG + NADPH + H + 2GSH + NADP+. The enzyme is important in maintaining a reducing environment within the cell (Akerboom et al., 1982); glutathione is involved in various cellular functions (Meister, 1989).Glutathione reductase from Escherichia coli is a homodimer with 450 amino acid residues and 1 FAD per subunit (M, 49,560). It belongs to the family of FAD-dependent disulfide oxidoreductases, which also includes lipoamide dehydrogenase (Mattevi et al., 1991), trypanothione reductase (Kuriyan et al., 1991a), mercuric ion reductase (Schiering et al., 1991), and thioredoxin reductase (Kuriyan et al., 1991b).The structure of glutathione reductase from human erythrocytes is known in great detail (Karplus & Schulz, 1987, 1989) and served as a guide for the design of several site-directed mutagenesis experiments on the enzyme GR,,,. Among them were the Reprint requests to: Georg E. Schulz, Institut fur Organische Chemie und Biochemie, Albertstr. 21, 79104 Freiburg im Breisgau, Germany; e-mail: schulz@bio2.chemie.uni-freiburg.de.Abbreviations: B-factor, crystallographic temperature factor; GK,,, glutathione reductase from Escherichia coli; GR,,,, human glutathione reductase; GSSG, oxidized glutathione; NCS, noncrystallographic symmetry; R-factor, crystallographic reliability factor; u, standard deviation; MIR, multiple isomorphous replacement.insertion of an intersubunit disulfide bridge (Scrutton et al., 1988), the identification of catalytically important residues (Deonarain et al., 1989; Scrutton et al., 1990aScrutton et al., , 1992, the switch of the coenzyme specificity from NADP to NAD (Scrutton et al., 1990b), and the specificity change from glutathione to trypanothione (Hender...

Cloning, Sequencing, and Regulation of the Glutathione Reductase Gene from the Cyanobacterium Anabaena PCC 7120

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Comparative Investigation of Techniques Used for Mtr Homology Model Refinement

“…The top hit 1GER was obtained (E. coli glutathione reductase, Gtr) having crystal structure resolution of 1.86Å, showing good alignment i.e. 30% sequence identity (abundance of exact amino acid at particular position in query and target sequences) and an E-value (expectation value: signifying error that may occur by chance) of 5e-48 with Mtr sequence [37]. Generally, a lower E-value indicates that an alignment is real.…”