Abstract
We have engineered a pathway for the formation of disulfide bonds. By imposing evolutionary pressure, we isolated mutations that changed thioredoxin, which is a monomeric disulfide reductase, into a [2Fe-2S] bridged dimer capable of catalyzing O2-dependent sulfhydryl oxidation in vitro. Expression of the mutant protein in Escherichia coli with oxidizing cytoplasm and secretion via the Tat pathway restored disulfide bond formation in strains that lacked the complete periplasmic oxidative machinery (DsbA and DsbB). The evolution of [2Fe-2S] thioredoxin illustrates how mutations within an existing scaffold can add a cofactor and markedly change protein function.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Motifs
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Amino Acid Sequence
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Amino Acid Substitution
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism
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Cell Membrane / metabolism
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Cysteine / analysis
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Cytoplasm / metabolism
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Dimerization
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Directed Molecular Evolution
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Disulfides / chemistry
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Disulfides / metabolism*
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Escherichia coli / genetics
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Escherichia coli / metabolism*
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Escherichia coli / physiology
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Hirudins / chemistry
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Hirudins / metabolism
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Iron / analysis
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Membrane Proteins / genetics
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Membrane Proteins / metabolism
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Movement
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Mutation
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Oxidation-Reduction
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Oxygen / metabolism
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Protein Disulfide-Isomerases / genetics
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Protein Disulfide-Isomerases / metabolism
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Protein Engineering*
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Protein Folding
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Proteins / chemistry
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Proteins / metabolism*
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Sulfides / analysis
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Thioredoxins / chemistry*
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Thioredoxins / genetics
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Thioredoxins / metabolism*
Substances
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Bacterial Proteins
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Disulfides
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DsbB protein, Bacteria
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Hirudins
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Membrane Proteins
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Proteins
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Sulfides
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Thioredoxins
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Iron
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Protein Disulfide-Isomerases
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Cysteine
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Oxygen