Engineering of an intersubunit disulphide bridge in glutathione reductase from Escherichia coli

Abstract

By site-directed mutagenesis, Thr-75 was converted to Cys-75 in the glutathione reductase (EC 1.6.4.2) of Escherichia coli. This led to the spontaneous formation of an intersubunit disulphide bridge across the 2-fold axis of the dimeric enzyme. The disulphide bridge had no deleterious effect on the catalytic activity, but nor did it increase the thermal stability of the enzyme, possibly because of local conformational flexibility on the dimer interface. The T75C mutant, like the wild-type enzyme, was inactivated by NADPH, proving that this inactivation cannot be due to simple dissociation of the dimer.

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

“…Figure 7 shows a superposition of this &sheet gg' onto the GRhum structure. Scrutton et al (1988) produced the mutant Thr 75 + Cys of G Q , designed to form the disulfide bridge of GRhum. From the geometry of the Thr 75 side chain, it seems likely that the formation of the disulfide bridge disrupts the 8-sheet.…”

“…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 (Henderson et al, 1991) and the reverse (Sullivan et al, 1991). Because the 2 enzyme species have only 52% amino acid residues in common (Greer & Perham, 1986), understanding the engineering results on the GR,,, necessitates detailed structural knowledge of this enzyme.…”

“…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 (Henderson et al, 1991) and the reverse (Sullivan et al, 1991). Because the 2 enzyme species have only 52% amino acid residues in common (Greer & Perham, 1986), understanding the engineering results on the GR,,, necessitates detailed structural knowledge of this enzyme.…”

“…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 (Henderson et al, 1991) and the reverse (Sullivan Noncrystallographic symmetry in crystal form-P. A: Symmetry elements of parent space group B2 in 1 unit cell (dashed line). Breaking the 2-fold axes yields the actual space group P21 with shifted origin (solid line), whereas breaking of the screw axes leads to space group P2 that was assumed by Ermler and Schulz (1991) at medium resolution.…”

Thermal stabilization of thymidylate synthase by engineering two disulfide bridges across the dimer interface

“…Covalent bridging by means of multiple, engineered disulfide bonds should impede subunit dissociation. There have been few attempts at analyzing the effect of disulfide crosslinks across protein-protein interfaces (Shirakawa et al, 1991;Scrutton et al, 1988;Sauer et al, 1986). We have engineered two disulfide bridges across the dimer interface of the enzyme, Lactobacillus casei thymidylate synthase (TSt) t Abbreviations used: TS, thymidylate synthase; WT, wild-type; TSM, thymidylate synthase mutant.…”