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Quiescin sulfhydryl oxidase from Trypanosoma brucei: catalytic activity and mechanism of a QSOX family member with a single thioredoxin domain.

ABSTRACT: Quiescin sulfhydryl oxidase (QSOX) flavoenzymes catalyze the direct, facile, insertion of disulfide bonds into reduced unfolded proteins with the reduction of oxygen to hydrogen peroxide. To date, only QSOXs from vertebrates have been characterized enzymatically. These metazoan sulfhydryl oxidases have four recognizable domains: a redox-active thioredoxin (Trx) domain containing the first of three CxxC motifs (C(I)-C(II)), a second Trx domain with no obvious redox-active disulfide, a helix-rich domain, and then an Erv/ALR domain. This last domain contains the FAD moiety, a proximal C(III)-C(IV) disulfide, and a third CxxC of unknown function (C(V)-C(VI)). Plant and protist QSOXs lack the second Trx domain but otherwise appear to contain the same complement of redox centers. This work presents the first characterization of a single-Trx QSOX. Trypanosoma brucei QSOX was expressed in Escherichia coli using a synthetic gene and found to be a stable, monomeric, FAD-containing protein. Although evidently lacking an entire domain, TbQSOX shows catalytic activity and substrate specificity similar to the vertebrate QSOXs examined previously. Unfolded reduced proteins are more than 200-fold more effective substrates on a per thiol basis than glutathione and some 10-fold better than the parasite bisglutathione analogue, trypanothione. These data are consistent with a role for the protist QSOX in oxidative protein folding. Site-directed mutagenesis of each of the six cysteine residues (to serines) shows that the CxxC motif in the single-Trx domain is crucial for efficient catalysis of the oxidation of both reduced RNase and the model substrate dithiothreitol. As expected, the proximal disulfide C(III)-C(IV), which interacts with the flavin, is catalytically crucial. However, as observed with human QSOX1, the third CxxC motif shows no obvious catalytic role during the in vitro oxidation of reduced RNase or dithiothreitol. Pre-steady-state kinetics demonstrates that turnover in TbQSOX is limited by an internal redox step leading to 2-electron reduction of the FAD cofactor. In sum, the single-Trx domain QSOX studied here shows a striking similarity in enzymatic behavior to its double-Trx metazoan counterparts.

SUBMITTER: Kodali VK

PROVIDER: S-EPMC2831140 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Quiescin sulfhydryl oxidase from Trypanosoma brucei: catalytic activity and mechanism of a QSOX family member with a single thioredoxin domain.

Kodali Vamsi K VK Thorpe Colin C

Biochemistry 20100301 9

Quiescin sulfhydryl oxidase (QSOX) flavoenzymes catalyze the direct, facile, insertion of disulfide bonds into reduced unfolded proteins with the reduction of oxygen to hydrogen peroxide. To date, only QSOXs from vertebrates have been characterized enzymatically. These metazoan sulfhydryl oxidases have four recognizable domains: a redox-active thioredoxin (Trx) domain containing the first of three CxxC motifs (C(I)-C(II)), a second Trx domain with no obvious redox-active disulfide, a helix-rich ...[more]

PMID: 20121244

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Project description:BACKGROUND: The enzyme family Quiescin Sulfhydryl Oxidase (QSOX) is defined by the presence of an amino-terminal thioredoxin-fold (Trx) domain and a carboxy-terminal Erv family sulfhydryl oxidase domain. QSOX enzymes, which generate disulfide bonds and transfer them to substrate proteins, are present in a wide variety of eukaryotic species including metazoans and plants, but are absent from fungi. Plant and animal QSOXs differ in their active-site amino acid sequences and content of non-catalytic domains. The question arises, therefore, whether the Trx-Erv fusion has the same mechanistic significance in all QSOX enzymes, and whether shared features distinguish the functional domains of QSOX from other instances in which these domains occur independently. Through a study of QSOX phylogeny and an analysis of QSOX sequence diversity in light of recently determined three-dimensional structures, we sought insight into the origin and evolution of this multi-domain redox alliance. RESULTS: An updated collection of QSOX enzymes was used to confirm and refine the differences in domain composition and active-site sequence motif patterns of QSOXs belonging to various eukaryotic phyla. Beyond the expected phylogenetic distinction of animal and plant QSOX enzymes, trees based on individual redox-active QSOX domains show a particular distinction of the Trx domain early in plant evolution. A comparison of QSOX domains with Trx and Erv domains from outside the QSOX family revealed several sequence and structural features that clearly differentiate QSOXs from other enzymes containing either of these domains. Notably, these features, present in QSOXs of various phyla, localize to the interface between the Trx and Erv domains observed in structures of QSOX that model interdomain redox communication. CONCLUSIONS: The infrastructure for interdomain electron relay, previously identified for animal and parasite QSOXs, is found broadly across the QSOX family, including the plant enzymes. We conclude that the conserved three-dimensional framework of the QSOX catalytic domains accommodates lineage-specific differences and paralog diversification in the amino acid residues surrounding the redox-active cysteines. Our findings indicate that QSOX enzymes are characterized not just by the presence of the two defining domain folds but also by features that promote coordinated activity.

Dataset Information

Quiescin sulfhydryl oxidase from Trypanosoma brucei: catalytic activity and mechanism of a QSOX family member with a single thioredoxin domain.

Publications

Quiescin sulfhydryl oxidase from Trypanosoma brucei: catalytic activity and mechanism of a QSOX family member with a single thioredoxin domain.

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