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Supplementary teaching slides for:

Supplementary teaching slides for: Generating disulfides in multicellular organisms: emerging roles for a new flavoprotein family Colin Thorpe and Donald L. Coppock November 2006. 1. ct. Disulfides bonds are frequently found in secreted eukaryotic proteins – examples …. RNAse. Insulin.

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Supplementary teaching slides for:

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  1. Supplementary teaching slides for: Generating disulfides in multicellular organisms: emerging roles for a new flavoprotein family Colin Thorpe and Donald L. Coppock November 2006 1 ct

  2. Disulfides bonds are frequently found in secreted eukaryotic proteins – examples … RNAse Insulin Phospholipase A2 Laminin, four domains 2 ct

  3. Different cofactors support oxidative protein folding in prokaryotes and eukaryotes Escherichia coli periplasm: coenzyme Q (bound to DsbB) connects thiol oxidation with the respiratory chain. Note series of redox-active disulfides. Arrows represent flow of electron pairs. periplasm S H D s b A S H S S S S S S Electron transport chain Q cytosol O f u m a r a t e 2 D s b B In yeast, two flavoproteins, Ero1p and Erv2p, can cooperate with PDI (flavin ) Yeast endoplasmic reticulum: flavin involved in at least two alternate pathways. E r o 1 p S H O x y g e n P D I S H E r v 2 p 3 ct

  4. Multicellular organisms have additional options for inserting disulfide bonds In humans: Ero1 and QSOX (Erv2p is absent). Ero enzymes receive pairs of electrons from client proteins undergoing oxidative protein folding. PDIserves as a mediator. QSOXenzymes insert disulfides directly into the client proteins. PDI functions later. PDIacts both as a disulfide oxidoreductase and a disulfide isomerase(shuffling incorrect –S-S- bonds in a redox-neutral way). E r o 1 P D I S H O x y g e n S H Q S O X P D I Other pathways for oxidative protein folding? Q S O X Next: generating disulfides using flavin and oxygen 4 ct

  5. In eukaryotes, flavoenzymes are frequently used to generate disulfides. The net oxidant is molecular oxygen: Flavin-dependent sulfhydryl oxidases catalyze this reaction in a series of steps: sulfhydryl oxidase 5 ct

  6. Key stages of catalysis in a typical flavin-dependent sulfhydryl oxidase: sulfhydryl oxidase The first crystal structure of a sulfhydryl oxidase was of yeast Erv2p (Gross et al., 2002) • The numbered steps are shown above and to the left • 1. Input of 2-electrons from substrate by disulfide exchange. • A series of internal disulfide exchanges (here just one) leads to reduction of the disulfide proximal to the flavin (FAD). • Two-electron reduction of the flavin. • Reoxidation of reduced flavin by molecular oxygen generating H2O2 and reforming oxidized enzyme. 6 ct

  7. Key stages of sulfhydryl oxidase catalysis: steps 1 and 2 (multiple disulfide exchange steps are common) 2e- sulfhydryl oxidase 2e- Step 1: the initial reduction of the enzyme by the substrate dithiol. Flow of reducing equivalents = Donor dithiol (substrate) Acceptor disulfide (on oxidase) 7 ct

  8. Key stages of sulfhydryl oxidase catalysis: the flavin connects oxidation of thiols with the reduction of O2 2e- N N N N N N O O O N H N H N H N N N H H O O O S S S - S S H S H ( S ) sulfhydryl oxidase Step 3, reduction of the flavin prosthetic group (a transient cysteinyl-adduct forms with the flavin) 8 ct

  9. Key stages of sulfhydryl oxidase catalysis: step 4, reoxidation of the flavin prosthetic group Key stages of sulfhydryl oxidase catalysis: reoxidation of the enzyme and the generation of H2O2 2e- sulfhydryl oxidase Step 4, reoxidation of the flavin prosthetic group by oxygen (reaction proceeds via a diradical pair : not shown) 9 ct

  10. Structures of yeast Erv2p and Ero1p. Both use a series of disulfide exchanges and a flavin cofactor oxygen 4 E r o 1 p S S H H P P D D I I 1 S S H H E r v 2 p 10 ct

  11. QSOX - an ancient fusion of recognizable domains … metazoan QSOX depicted here -WCGHC- ERV/ALR domain An Erv homolog is called ALR (augmenter of liver regeneration) Thioredoxin domains Trx1 and 2 Resembling the first two thioredoxin domains in PDI 11 ct

  12. QSOX enzymes have 1 or 2 thioredoxin domains. Differential splicing at C-terminus yields a short form Mechanism: thioredoxin domain reacts with substrate dithiols. Steps 2-5 are by analogy with Erv2p H S O 2 H S P D I 1 5 - like 4 - C x x C - - C x x C - - C x x C - 3 2 12 ct

  13. QSOX oxidizes reduced protein substrate, PDI isomerizes incorrectly placed disulfides 13 1 ct

  14. Unresolved general questions for disulfide bond formation in multicellular organisms The relative importance of the pathways depicted here. The precise roles of Ero1a and Ero1b, and QSOX1 and QSOX2 … distinct or overlapping substrate specificities. How hydrogen peroxide, generated by these oxidases, is handled in the ER. How the redox state of PDI is maintained in the ER to optimize formation of correctly-paired disulfide bonds in client proteins. E r o 1 P D I S H O x y g e n S H Q S O X P D I Other pathways for oxidative protein folding? 14 ct

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