A Flavin-Dependent Sulfhydryl Oxidase in Bovine Milk

Jaje, Jennifer; Wolcott, Holly N.; Fadugba, Olajumoke; Cripps, Diane; Yang, Austin J.; Mather, Ian H.; Thorpe, Colin

doi:10.1021/bi7016975

Cited by 33 publications

(53 citation statements)

References 47 publications

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“…The UV-visible spectrum of recombinant HsQSOX1 shows a typical unresolved flavin envelope (Figure 4) that was seen for both the rat seminal vesicle (RnQSOX1), the avian egg white (GgQSOX1) and the bovine milk (BtQSOX1) enzymes (1,18,19). The experimentally-determined extinction coefficient is 12.5 ± 0.6 mM −1 cm −1 at 456 nm (see Methods), comparable to that obtained for GgQSOX1.…”

Section: Expression Of Human Qsox1 In Escherichia Colisupporting

confidence: 69%

Human Quiescin-Sulfhydryl Oxidase, QSOX1: Probing Internal Redox Steps by Mutagenesis

et al. 2008

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The flavoprotein Quiescin-sulfhydryl oxidase (QSOX) rapidly inserts disulfide bonds into unfolded, reduced proteins with the concomitant reduction of oxygen to hydrogen peroxide. This study reports the first heterologous expression and enzymological characterization of a human QSOX1 isoform. Like QSOX isolated from avian egg white, recombinant HsQSOX1 is highly active towards reduced ribonuclease A (RNase) and dithiothreitol but shows a >100-fold lower k cat /K m for reduced glutathione. Previous studies on avian QSOX led to a model in which reducing equivalents were proposed to relay through the enzyme from the first thioredoxin domain (C70-C73) to a distal disulfide (C509-C512), then across the dimer interface to the FADproximal disulfide (C449-C452), and finally to the FAD. The present work shows that, unlike the native avian enzyme, HsQSOX1 is monomeric. The recombinant expression system enabled construction of the first cysteine mutants for mechanistic dissection of this enzyme family. Activity assays with mutant HsQSOX1 indicated that the conserved distal C509-C512 disulfide is dispensable for the oxidation of reduced RNase or dithiothreitol. The four other cysteine residues chosen for mutagenesis, C70, C73, C449, and C452, are all crucial for efficient oxidation of reduced RNase. C452, of the proximal disulfide, is shown to be the charge-transfer donor to the flavin ring of QSOX, and its partner, C449, is expected to be the interchange thiol, forming a mixed disulfide with C70 in the thioredoxin domain. These data demonstrate that all the internal redox steps occur within the same polypeptide chain of mammalian QSOX and commence with a direct interaction between the reduced thioredoxin domain and the proximal disulfide of the Erv/ ALR domain.All eukaryotic sulfhydryl oxidases yet described utilize either metal ions or a flavin cofactor to couple the oxidation of thiols with the reduction of oxygen to hydrogen peroxide:The copper-and iron-dependent enzymes are relatively poorly understood; there are no sequences, structures, or detailed analyses of the catalytic role of the metal (1-4). In contrast, a growing literature describes the structures, mechanisms, and roles in oxidative † This work was supported in part by National Institutes of Health Grant GM26643 (C.T.) and the United States -Israel Binational Science Foundation (D.F.). * Author for correspondence. Phone: (302) 831-2689; FAX: (302) 831-6335; cthorpe@udel.edu. ‡ University of Delaware § Weizmann Institute of Science Figures S1-4 show sequence alignments of selected QSOXs; the full sequence of the human QSOX1b construct used for most of this work; the sequence of the construct used for the analytical ultracentrifugation; and analysis of ultracentrifugation data. This material is available free of charge via the Internet at http:// pubs.acs.org. SUPPORTING INFORMATION AVAILABLE Supplemental NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2013 January 11. (3,4,16,17), which are the subject of this cont...

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Section: Expression Of Human Qsox1 In Escherichia Colisupporting

confidence: 69%

Human Quiescin-Sulfhydryl Oxidase, QSOX1: Probing Internal Redox Steps by Mutagenesis

et al. 2008

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“…Although this monothiol is a poor substrate of both protist and mammalian QSOX enzymes (28,53,54), communication between GSH and the C I XXC II disulfide is rapid enough to ensure equilibration with the FAD after several sec- 24 and thus runs as a higher molecular weight band than the oxidized protein. Minor intermediate bands corresponding to protein containing a mixed disulfide species with glutathione (*) were excluded from analysis.…”

Section: Resultsmentioning

confidence: 99%

“…Thiol Substrates-The trypanosomal enzyme (28), like other QSOXs (3,18,32,54), is capable of oxidizing a very wide range of mono-, di-, and multithiol substrates. Conformationally flexible peptides and proteins containing two or more cysteine residues appear to be excellent substrates of the enzyme, although there is comparatively little data regarding their redox potentials.…”

Section: Tbqsox In Thermodynamic Context: Redox Potentials Ofmentioning

confidence: 99%

“…Third, in both systems, a charge transfer interaction between a thiolate and the oxidized cofactor (flavin in QSOX and a quinone in DsbB) is the first observable intermediate in rapid reaction studies (28,30,68). Fourth, decomposition of this charge transfer intermediate to yield dihydroflavin or hydroquinone is rate-limiting in overall catalysis in both QSOX and DsbB, respectively (25,27,54). Finally, accumulation of these reduced enzyme species is believed to occur in both instances via thiol-cofactor covalent adducts (1,69,70).…”

Section: Coupled Disulfide Exchange Reactions Promote Efficientmentioning

confidence: 99%

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Israel¹,

Kodali²,

Thorpe

2014

Journal of Biological Chemistry

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Background: QSOX enzymes are medically important catalysts of disulfide bond generation. Results: Mechanistic studies of the three redox centers in QSOX revealed an unexpected thermodynamic mismatch in the reaction coordinate. Conclusion: QSOX circumvents this thermodynamic barrier using a mixed disulfide intermediate that links distal redox centers. Significance: The strategy of coupling disulfide exchanges appears to be exploited by other enzymes of oxidative protein folding.

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“…34,35 These clusters finally aggregate into larger and not active nanoparticles. The reaction mechanism has been theoretically studied and it is shown that isolated gold atoms cannot activate O 2 , while small gold clusters are excellent catalysts for O 2 activation and formation of disulfide.…”

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confidence: 99%

Exceptional oxidation activity with size-controlled supported gold clusters of low atomicity

et al. 2013

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Nature Publishing GroupCorma Canós, A.; Concepción Heydorn, P.; Boronat Zaragoza, M.; Sabater Picot, MJ.; Navas Escrig, J.; Yacaman, MJ.; Larios, E.... (2013). Exceptional oxidation activity with sizecontrolled supported gold clusters of low atomicity. Nature Chemistry. 5 (9) SummaryThe catalyticic activity of gold depends on particle size, with reactivity increasing as particle diameter decreases. Investigation of the trends in the subnanometer regime, where gold exists as small clusters of a few atoms, is now starting thanks to recent advances in synthesis and characterization techniques. An easy method to prepare isolated gold atoms supported on functionalized carbon nanotubes and their performance in the oxidation of thiophenol with O 2 are described. Single gold atoms are not active and they aggregate under reaction conditions into gold clusters of low atomicity, which show a catalytic activity comparable to that sulfhydryl oxidase enzymes. When clusters grow into larger nanoparticles, catalyst activity drops to zero.Theoretical calculations show that gold clusters are able to simultaneously activate thiophenol and O 2 , while larger nanoparticles become passivated by strongly adsorbed thiolates. The combination of an optimum for reactants activation and product desorption makes gold clusters excellent catalysts. Main TextGold has attracted wide interest as catalyst in the last years due to its unexpected activity and, specially, to its high selectivity in organic reactions. [1][2][3] The catalytic properties of gold depend on several factors that in some cases are intimately related:gold particle size and morphology, metal-oxide support interaction, oxidation state of the active sites, etc. 4-8 The influence of particle size has been extensively investigated, and a volcano type curve with a maximum in activity at an optimum diameter has been reported for CO oxidation, 7 alkane oxidation, 9 or propene epoxidation with O 2 and H 2 , 10 while in other cases an exponential increase in activity with decreasing particle size has been observed. 5,11,12 However, the trends in catalytic activity when the particle diameter While it appears that in order to control reactivity, the atomicity control of the gold clusters is crucial, the synthesis of size-selected metal clusters and their deposition over a solid support is a challenging task. 26 The wet-chemistry methods for preparing supported metal clusters involve the anchoring of well defined precursors to an adequate support, 27,28 followed by removal of the ligands by post-synthesis treatments, trying to prevent cluster agglomeration during these steps. 9,[29][30][31]32 Soft landing of monodisperse metal clusters grown in the gas phase and with precise size selection by mass spectrometry is a more straightforward method, but it requires sophisticated equipment, and the scaling up of the process is a major drawback. 20,23,33 In The chemical nature of these isolated atoms has been investigated by X-ray absorption spectroscopy (XAS) and X-ray photoelectron spe...

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A Flavin-Dependent Sulfhydryl Oxidase in Bovine Milk

Cited by 33 publications

References 47 publications

Human Quiescin-Sulfhydryl Oxidase, QSOX1: Probing Internal Redox Steps by Mutagenesis

Human Quiescin-Sulfhydryl Oxidase, QSOX1: Probing Internal Redox Steps by Mutagenesis

Going through the Barrier

Exceptional oxidation activity with size-controlled supported gold clusters of low atomicity

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