the CXXC motif in the active site sequence of Erv2p is catalytically essential, reaction mechanism involving reactive cysteine residues C121 and C124 of the A subunit, and C176 and C178 of the B subunit
recombinantly expressed substrate amino acids 284-403, which is the C-terminal domain of Mia40, electron transfer between the shuttle and active site disulfides of Erv1p. Both intersubunit and intermolecular electron transfer can occur, overview
Erv2p functions in the generation of microsomal disulfide bonds acting in parallel with Ero1p, the essential FAD-dependent oxidase of protein disulfide isomerase
Erv2p is a modest catalyst of disulfide bond formation. None of the monothiols (at 10 mM), including beta-mercaptoethanol, N-acetylcysteamine, reduced glutathione and CoASH, prove detectable substrates of the yeast oxidase at pH 7.5. In contrast, dithiols are significant substrates
Erv1p contains three conserved disulfide bonds arranged in two CXXC motifs and one CX16C motif, the CX16C disulfide plays an important role in stabilizing the folding of Erv1p, both CXXC disulfides are required for Erv1 oxidase activity, but none of the disulfide is essential for FAD binding, overview
Erv2p functions in the generation of microsomal disulfide bonds acting in parallel with Ero1p, the essential FAD-dependent oxidase of protein disulfide isomerase
dithionite and photochemical reductions of Erv2p show full reduction of the flavin cofactor after the addition of 4 electrons with a midpoint potential of -200 mV at pH 7.5. No charge-transfer complex between a proximal thiolate and the oxidized flavin
upon coordination with Zn2+, full reduction of Erv2p requires 6 electrons. Strongly inhibits Erv2p when assayed using tris(2-carboxyethyl)phosphine as the reducing substrate of the oxidase. Restoration of 1 mM EDTA effects rapid recovery of 80% of the original activity
with 0.3 mM reduced thioredoxin as the substrate of the reductive half-reaction and an initial oxygen concentration of around 0.3 mM such that the reduced thioredoxin would be depleted by 10% during the course of the reaction
Erv1 requires the redox-regulated receptor Mia40 for its import into mitochondria. Interacts via disulfide bonds with Mia40. It does not need two CX2C motifs for import into mitochondria
Erv1p is a FAD-dependent sulfhydryl oxidase and is an essential component of the redox regulated Mia40/Erv1 import and assembly pathway used by many of the cysteine-containing intermembrane space proteins
Erv1p contains three conserved disulfide bonds arranged in two CXXC motifs and one CX16C motif in the highly conserved central catalytic core. The CX16C disulfide plays an important role in stabilizing the folding of Erv1p, both CXXC disulfides are required for Erv1 oxidase activity, but none of the disulfide is essential for FAD binding, overview
crystal structures at 2.0 A resolution of the C-terminal domain and at 3.0 A resolution of a C30S/C133S double mutant. The C-terminal domain exists as a homodimer, with each subunit consisting of a conserved four-helix bundle that accommodates the isoalloxazine ring of FAD and an additional single-turn helix. The N-terminal domain is an amphipathic helix flanked by two flexible loops. This structure also represents an intermediate state of electron transfer from the N-terminal domain to the C-terminal domain of another subunit. The four-helix bundle of the C-terminal domain forms a wide platform for the electron donor N-terminal domain. Moreover,the amphipathic helix close to the shuttle redox enter may be critical for the recognition of Mia40, the upstream electron donor
site-directed mutagenesis, the active site mutant shows no or very little activity, and the mutant shows a shifted protein-bound FAD spectrum compared to the wild-type enzyme Erv1p, the active site disulfide is located proximal to the isoalloxazine ring of FADa nd the mutation changes bound-FAD absorption slightly, the mutant is active in presence of DTT, but not with tris(2-carboxyethyl)phosphine
enhancement of catalytic activity for GSH, whereas the catalytic activity for gamma-glutamylcysteine remains unchanged. The mutant enzyme shows slightly decreased maximum temperatures at 55°C (compared to 60°C for the wild-type enzyme)
the mutant enzyme oxidizes GSH more efficiently (169%) than the wild-type enzyme. About 1.5fold increase in GSSG production compared to that of the parental ERV1 gene
the mutant enzyme oxidizes GSH more efficiently (240%) than the wild-type enzyme and shows comparable activity for gamma-glutamylcysteine (96%). The mutant enzyme shows slightly decreased maximum temperatures at 55°C (compared to 60°C for the wild-type enzyme)
the mutant enzyme shows almost the same activity for GSH (192%) compared to mutant enzyme S32A, S32T, and N34A, and high activity for gamma-glutamylcysteine (161%). The mutant enzyme shows slightly decreased maximum temperatures at 55°C (compared to 60°C for the wild-type enzyme)
construction of a His-tagged truncated enzyme form comprising the 15 kDa C-terminus, the mutant shows in vitro activity similar to the wild-type enzyme, dimerization behaviour of the mutant enzymes, overview
construction of a His-tagged truncated enzyme form comprising the 15 kDa C-terminus, the mutant shows in vitro activity similar to the wild-type enzyme, dimerization behaviour of the mutant enzymes, overview
the conserved C-terminal domain of the human Alrp can functionally replace the yeast domain in vivo, genetic system to study function of sulfhydryl oxidases, overview, enzyme-defective Erv1p mutant shows highly altered mitochondrial membrane morphology with loss of cristae, overview
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli strains DH5alpha and BL21(DE3) of wild-type enzyme and a His-tagged truncated enzyme form comprising the 15 kDa C-terminus, expression of full-length point mutants
truncated version of Erv2p, without the N-terminal 34 residues, cloned into a pET24(a+) plasmid carrying kanamycin resistance. Amplified in DH5alpha Escherichia coli strain. Expressed from the Escherichia coli strain BL21(DE3) or BL21(DE3 star)