The cysteine-desulfurase IscS promotes the production of the rhodanese RhdA in the persulfurated form.
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.
Forlani
Fabio
F
Dipartimento di Scienze Molecolari Agroalimentari, Facoltà di Agraria, Università di Milano, Via Celoria 2, Milano 20133, Italy.
Cereda
Angelo
A
Freuer
Andrea
A
Nimtz
Manfred
M
Leimkühler
Silke
S
Pagani
Silvia
S
D016948:Azotobacter vinelandii / Q000201:enzymology; D013437:Carbon-Sulfur Lyases / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D010449:Peptide Mapping; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D013455:Sulfur / Q000737:chemistry* / Q000378:metabolism; D013884:Thiosulfate Sulfurtransferase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D013438:Sulfhydryl Compounds; D006639:Histidine; D013455:Sulfur; D013884:Thiosulfate Sulfurtransferase; D013437:Carbon-Sulfur Lyases; C080546:cysteine desulfurase; D003545:Cysteine