title: Mutagenic analysis of Thr-232 in rhodanese from Azotobacter vinelandii highlighted the differences of this prokaryotic enzyme from the known sulfurtransferases.
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.
authors.affiliation: Dipartimento di Scienze Molecolari Agroalimentari, Università di Milano, Via Celoria n. 2, 20133, Milan, Italy. silvia.pagani@unimi.it
authors.forename: S
authors.initials: S
authors.lastname: Pagani
authors.forename: F
authors.initials: F
authors.lastname: Forlani
authors.forename: A
authors.initials: A
authors.lastname: Carpen
authors.forename: D
authors.initials: D
authors.lastname: Bordo
authors.forename: R
authors.initials: R
authors.lastname: Colnaghi
mesh_terms: D000818:Animals; D016948:Azotobacter vinelandii / Q000201:enzymology*; D002417:Cattle; D006801:Humans; D016296:Mutagenesis; D011387:Prokaryotic Cells; D013466:Sulfurtransferases / Q000145:classification / Q000235:genetics / Q000378:metabolism; D013884:Thiosulfate Sulfurtransferase / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D013912:Threonine / Q000235:genetics* / Q000378:metabolism
chemical_list: D013912:Threonine; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase