Abstract
The yeast and human mitochondrial sulfhydryl oxidases of the Erv1/Alr family have been shown to be essential for the biogenesis of mitochondria and the cytosolic iron sulfur cluster assembly. In this study we identified a likely candidate for the first mitochondrial flavin-linked sulfhydryl oxidase of the Erv1-type from a photosynthetic organism. The central core of the plant enzyme (AtErv1) exhibits all of the characteristic features of the Erv1/Alr protein family, including a redox-active YPCXXC motif, noncovalently bound FAD, and sulfhydryl oxidase activity. Transient expression of fusion proteins of AtErv1 and the green fluorescence protein in plant protoplasts showed that the plant enzyme preferentially localizes to the mitochondria. Yet AtErv1 has several unique features, such as the presence of a CXXXXC motif in its carboxyl-terminal domain and the absence of an amino-terminally localized cysteine pair common to yeast and human Erv1/Alr proteins. In addition, the dimerization of AtErv1 is not mediated by its amino terminus but by its unique CXXXXC motif. In vitro assays with purified protein and artificial substrates demonstrate a preference of AtErv1 for dithiols with a defined space between the thiol groups, suggesting a thioredoxin-like substrate.
Highlights
During evolution, the fusion of the catalytic ERV1 domain with a thioredoxin domain generated a new facile catalyst for disulfide formation with dramatically enhanced enzymatic activity [6, 10, 11]
Recent publications demonstrate that interdomain redox communication between the primary redox-active CXXC motif and additional cysteine pairs is another characteristic feature of sulfhydryl oxidases [11, 12, 14]
quiescin/sulfhydryl oxidases (QSOX) enzymes are not found in mitochondria but rather at many other subcellular locations and are excreted from cells [9]
Summary
The fusion of the catalytic ERV1 domain with a thioredoxin domain generated a new facile catalyst for disulfide formation with dramatically enhanced enzymatic activity [6, 10, 11] This new protein family of FAD-dependent sulfhydryl oxidases performs diverse and important functions in higher eukaryotes (for a review see Ref. 9). The first complete sequence of such an ERV1/thioredoxin fusion protein was derived from a human protein that was differentially expressed as fibroblasts enter quiescence [6, 10] Based on this phenotype, these proteins were termed quiescin/sulfhydryl oxidases (QSOX).. These proteins were termed quiescin/sulfhydryl oxidases (QSOX).1 In these enzymes, the ERV1 domain contains the FAD-binding site and the redox-active primary CXXC motif. This highly conserved ERV1 domain is the hallmark ʈ Supported by SFB (sonderforschungsbereich) 575 and by funds from the European Commission via MitEURO Program Grant QLG1-CT-2001-00966
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