Azotobacter vinelandii RhdA uses thiosulfate as the only sulfur donor in vitro, and this apparent selectivity seems to be a unique property among the characterized sulfurtransferases. To investigate the basis of substrate recognition in RhdA, we replaced Thr-232 with either Ala or Lys. Thr-232 was the target of this study since the corresponding Lys-249 in bovine rhodanese has been identified as necessary for catalytic sulfur transfer, and replacement of Lys-249 with Ala fully inactivates bovine rhodanese. Both T232K and T232A mutants of RhdA showed significant increase in thiosulfate-cyanide sulfurtransferase activity, and no detectable activity in the presence of 3-mercaptopyruvate as the sulfur donor substrate. Fluorescence measurements showed that wild-type and mutant RhdAs were overexpressed in the persulfurated form, thus conferring to this enzyme the potential of a persulfide sulfur donor compound. RhdA contains a unique sequence stretch around the catalytic cysteine, and the data here presented suggest a possible divergent physiological function of A. vinelandii sulfurtransferase. Copyright (C) 2000 Federation of European Biochemical Societies.
Mutagenic analysis of Thr-232 in rhodanese from Azotobacter vinelandii highlighted the differences of this prokaryotic enzyme from the known sulfurtransferases / S. Pagani, F. Forlani, A. Carpen, D. Bordo, R. Colnaghi. - In: FEBS LETTERS. - ISSN 0014-5793. - 472:2-3(2000 Apr 28), pp. 307-311.
Mutagenic analysis of Thr-232 in rhodanese from Azotobacter vinelandii highlighted the differences of this prokaryotic enzyme from the known sulfurtransferases
S. Pagani
;F. ForlaniSecondo
;A. Carpen;R. ColnaghiUltimo
2000
Abstract
Azotobacter vinelandii RhdA uses thiosulfate as the only sulfur donor in vitro, and this apparent selectivity seems to be a unique property among the characterized sulfurtransferases. To investigate the basis of substrate recognition in RhdA, we replaced Thr-232 with either Ala or Lys. Thr-232 was the target of this study since the corresponding Lys-249 in bovine rhodanese has been identified as necessary for catalytic sulfur transfer, and replacement of Lys-249 with Ala fully inactivates bovine rhodanese. Both T232K and T232A mutants of RhdA showed significant increase in thiosulfate-cyanide sulfurtransferase activity, and no detectable activity in the presence of 3-mercaptopyruvate as the sulfur donor substrate. Fluorescence measurements showed that wild-type and mutant RhdAs were overexpressed in the persulfurated form, thus conferring to this enzyme the potential of a persulfide sulfur donor compound. RhdA contains a unique sequence stretch around the catalytic cysteine, and the data here presented suggest a possible divergent physiological function of A. vinelandii sulfurtransferase. Copyright (C) 2000 Federation of European Biochemical Societies.File | Dimensione | Formato | |
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