Folding of the four domains and dimerization are impaired by the Gly446.fwdarw.Glu exchange in human glutathione reductase. Implications for the design of antiparasitic drugs

Abstract

Glutathione reductase (NADPH+GSSG+H+-->NADP(+) + 2GSH) is a homodimeric flavoenzyme of known geometry. Each subunit contains four well-defined domains and contributes essential residues to the active sites; consequently, the monomer is expected to be inactive. As part of our program to develop dimerization inhibitors of human glutathione reductase (hGR) as antimalarial agents, we mutagenized the residues 446 and 447 which, together with their counterparts on the other subunit, represent the tightest contact between the subunits [Karplus, P. A., & Schulz, G. E. (1987) J. Mol. Biol. 195, 701-729]. Wild-type human glutathione reductase and mutants of this protein were produced in plasmid-transformed Escherichia coli SG5 cells. Active enzyme species, namely, wild-type hGR, N-terminally truncated delta(1-15)hGR, and the point mutant F447P-hGR, were purified by 2',5'-ADP-Sepharose chromatography and crystallization. Inactive mutants such as G446E-hGR or the double mutants G446E/F447P-hGR and G446P/F447P-hGR were isolated by immunoadsorption chromatography. G446E/F447P-hGR was studied in detail. This mutant behaved like a poorly folded monomeric protein, as indicated by the following properties: absence of the intersubunit disulfide bridge, Cys90-Cys90'; failure to bind FAD; failure to bind NADPH and analogues thereof; a short half-life (< 4 min) in E. coli cells; and high susceptibility to trypsin in vitro. The results suggest that the sequence around G446 can control dimerization as well as domain folding. This is unexpected since the FAD-binding domain and the NADPH-binding domain occur in many different enzymes and have been regarded as autonomous folding units.(ABSTRACT TRUNCATED AT 250 WORDS)

The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield

“…Recombinant human GR was prepared as described in ref. 21. To measure GR activity, 0.5 g of plant material was mixed with 2 ml of 0.1 M Tris, 1 mM EDTA, and 7.5% wt͞vol polyvinylpyrrolidone (pH 7.8).…”

“…After centrifugation, protein was determined in the supernatant according to Bradford (22) by using BSA as the standard. GR activity was measured spectrophotometrically (for NADPH, 340 ϭ 6.22 mM Ϫ1 ⅐cm Ϫ1 ) in 47 mM potassium phosphate, 200 mM KCl, and 1 mM EDTA (pH 6.9) at 25°C (21). A baseline was monitored in the presence of plant extract and 100 M NADPH to account for NADPH oxidase activity.…”

Glutathione Reductase and Glutamate Dehydrogenase of Plasmodium Falciparum, The Causative Agent of Tropical Malaria

“…Recombinant human glutathione reductase was prepared as described previously (Nordhoff et al, 1993). Becf heart glutamate dehydrogenase was purchased froin Boehringer Mannheim.…”

“…Becf heart glutamate dehydrogenase was purchased froin Boehringer Mannheim. The immunoadsorption gel was prepared by covalently coupling polyclonal rabbit antibodies against human glutathione reductase to protein-A -Sepharose CL-4B (Nordhoff et al, 1993). Ado(2',5')P,-Sephilro~e, Q-Sepharose, and a Superose 12 column were obtained from Pharmacia, Ccntricon 10 concentrators were from Amicon, bicinchoninic acid and the micro-bicinchoninic acid protein assay kit were from Pierce, the quick silver protein stain kit was from Amersham, and cellulose acetate sheets (Cellogel) were from Biotec Fischer.…”

Intersubunit Interactions in Plasmodium falciparumThioredoxin Reductase

“…Another reason might be the synthesis of monomers that do not dimerize co-translationally and are therefore degraded rapidly by the bacterial cell. It has been shown previously that monomeric mouse glutathione reductase is degraded rapidly by bacterial cells (22). The generation of E. coli glutathione reductase hybrid protein species resulted in high yields of proteins, and there was no mention about a differential expression pattern concerning the different protein species that were generated (23).…”