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-50-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.
The cysteine-desulfurase IscS promotes the production of the rhodanese RhdA in the persulfurated form / F. Forlani, A. Cereda, A. Freuer, M. Nimtz, S. Leimkuehler, S.G. Pagani. - In: FEBS LETTERS. - ISSN 0014-5793. - 579:30(2005), pp. 6786-6790. [10.1016/j.febslet.2005.11.013]
The cysteine-desulfurase IscS promotes the production of the rhodanese RhdA in the persulfurated form
F. ForlaniPrimo
;A. CeredaSecondo
;S.G. PaganiUltimo
2005
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-50-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.Pubblicazioni consigliate
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