Mutagenic analysis of Thr-232 in rhodanese from Azotobacter vinelandii highlighted the differences of this prokaryotic enzyme from the known sulfurtransferases

Silvia Pagani, Fabio Forlani, Aristodemo Carpen, Domenico Bordo, Rita Colnaghi

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)307-311
Number of pages5
JournalFEBS Letters
Volume472
Issue number2-3
DOIs
Publication statusPublished - Apr 28 2000

Keywords

  • Azotobacter vinelandii rhodanese
  • Site-directed mutagenesis
  • Sulfur donor substrate
  • Sulfurtransferase

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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