Abstract
After heterologous expression in Escherichia coli, the Azotobacter vinelandii rhodanese RhdA is purified in a persulfurated form (RhdA-SSH). We identified l-cysteine as the most effective sulfur source in producing RhdA-SSH. An E. coli soluble extract was required for in vitro persulfuration of RhdA, and the addition of pyridoxal-5'-phosphate increased RhdA-SSH production, indicating a likely involvement of a cysteine desulfurase. We were able to show the formation of a covalent complex between IscS and RhdA. By combining a time-course fluorescence assay and mass spectrometry analysis, we demonstrated the transfer of sulfur from E. coli IscS to RhdA.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Azotobacter vinelandii / enzymology
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Carbon-Sulfur Lyases / chemistry*
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Carbon-Sulfur Lyases / metabolism*
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Catalytic Domain
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Cysteine / chemistry
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Escherichia coli / genetics
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Histidine / chemistry
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Peptide Mapping
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Protein Structure, Tertiary
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Spectrometry, Fluorescence
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Spectrometry, Mass, Electrospray Ionization
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Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Sulfhydryl Compounds / chemistry
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Sulfhydryl Compounds / metabolism
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Sulfur / chemistry*
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Sulfur / metabolism
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Thiosulfate Sulfurtransferase / biosynthesis*
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Thiosulfate Sulfurtransferase / chemistry
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Thiosulfate Sulfurtransferase / genetics
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Thiosulfate Sulfurtransferase / isolation & purification
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Thiosulfate Sulfurtransferase / metabolism*
Substances
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Sulfhydryl Compounds
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Histidine
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Sulfur
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Thiosulfate Sulfurtransferase
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Carbon-Sulfur Lyases
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cysteine desulfurase
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Cysteine