The novel disulfide reductase bis-gamma-glutamylcystine reductase and dihydrolipoamide dehydrogenase from Halobacterium halobium: purification by immobilized-metal-ion affinity chromatography and properties of the enzymes

Abstract

An NADPH-specific disulfide reductase that is active with bis-'y-glutamylcystine has been purified 1,900-fold from Halobacterium halobium to yield a homogeneous preparation of the enzyme. Purification of this novel reductase, designated bis-y-glutamylcystine reductase (GCR), and purification of halobacterial dihydrolipoamide dehydrogenase (DLD) were accomplished with the aid of immobilized-metal-ion affinity chromatography in high-salt buffers. Chromatography of GCR on immobilized Cu2" resin in buffer containing 1.23 M (NH4)2SO4 and on immobilized Ni2O resin in buffer containing 4.0 M NaCl together effected a 120-fold increase in purity. Native Disulfide-specific reductases play a central role in the maintenance of aerobic cell cytoplasms in a chemically reduced state. Glutathione reductase (EC 1.6.4.2) is an NADPH-dependent disulfide reductase that is present in aerobic eubacteria and eucaryotic cells and maintains the peptide glutathione (y-Glu-Cys-Gly) predominantly in its reduced or thiol form (13, 39). Reduced glutathione is found at millimolar levels in cells and is a coenzyme for several enzymes that reduce intracellular disulfides and detoxify potent oxidants, such as peroxides (8,15,21). These processes yield the disulfide form of glutathione, which is cycled back to the thiol by glutathione reductase.Glutathione may not be the only thiol antioxidant utilized in aerobic cells; several aerobic bacteria that lack glutathione have been shown to produce other low-molecularweight thiols and disulfide reductase activities (23,32,35). One such group of bacteria are the halophilic archaebacteria, which have been shown to lack reduced glutathione but contain millimolar levels of -y-glutamylcysteine (,y-Glu-Cys) (23). This finding suggested that the halobacteria may employ a redox metabolism similar to that established for glutathione but based on -y-Glu-Cys. Central to this metabolism would be a disulfide reductase that is active with -y-Glu-Cys disulfide (bis-y-glutamylcystine), and the detection of an NADPH-dependent reductase activity in crude extracts of Halobacterium halobium indicated that this enzyme was present (23). In this paper we report the complete purification of an NADPH-specific bis-y-glutamylcystine reductase (GCR) from extracts of H. halobium. The characteristics of GCR and the possible functions of -y-Glu-Cys in the halobacteria are also discussed.Purification of most halobacterial enzymes requires the use of high-ionic-strength buffers (,u > 2 M) to retain enzyme activity (17). Chromatographic methods have been devel-* Corresponding author.oped that function well in buffers containing molar levels of NaCl or (NH4) 2SO4 (14,18,22,24), and a small number of halobacterial enzymes have been purified to homogeneity. Our limited success in purifying the GCR from H. halobium by established methods led us to examine additional approaches to purification in high-salt buffers. Immobilizedmetal-ion affinity chromatography (also known as metal chelate affinity chromatography) (26, 27), a technique used...

Purification and characterization of the reduced-nicotinamide-dependent 2,2'-dithiodiethanesulfonate reductase from Methanobacterium thermoautotrophicum delta H

“…The CoMDSR is also a flavoprotein, binding most likely one molecule of FAD per 64,000 Da. In archaebacteria, a dimeric flavoprotein with an Mr of 122,000 which reduces bis-yglutamylcystine via NADPH oxidation has been purified from Halobacterium halobium (21). The nature of its flavin was not determined.…”

“…It seems that methanogens lack glutathione and could use HS-CoM or (S-CoM)2 as a major thiol-disulfide redox regulator (11). In this respect, the CoMDSR from M. thermoautotrophicum would be analogous to the bis--y-glutamylcystine reductase from H. halobium, an archaebacterium which lacks glutathione and contains -y-glutamylcysteine as the major low-molecular-weight thiol (21). Recently, Bobik and Wolfe showed that HS-CoM and HS-HTP are the substrates for fumarate reductase in M. thermoautotrophicum (3); HS-CoM may be involved in other redox reactions in the cell.…”

Isolation of an atypically small lipoamide dehydrogenase involved in the glycine decarboxylase complex from Eubacterium acidaminophilum

“…No (1,21) and from results of studies in our laboratory (Dietrichs, thesis of diploma) (Fig. 2) (11,16,28,37,46,47,54 …”

Purification and comparative studies of dihydrolipoamide dehydrogenases from the anaerobic, glycine-utilizing bacteria Peptostreptococcus glycinophilus, Clostridium cylindrosporum, and Clostridium sporogenes

“…The enzyme dihydrolipoamide dehydrogenase belongs to the group of flavin-containing pyridine nucleotide disulfide oxidoreductases (7,21,50), like thioredoxin reductase (19), glutathione reductase (30), trypanothione reductase (34), bisy-glutamylcystine reductase (38), pantethine 4',4"-diphosphate reductase (39), and mercuric reductase (15). The enzyme is known to be an integral component of the pyruvate, 2-oxoglutarate (7,20,21,50), and branched-chain 2-oxoacid dehydrogenase (28,36) and of the glycine decarboxylase complex (16,17,22,35,46).…”