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

Azotobacter vinclandii RhdA uses thiosulfate? as the only sulfur donor in v itro, and this apparent selectivity seems to be a unique property among the characterized sulfurtransferases. To investigate the basis of substrate re cognition in RhdA, we replaced Thr-232 with tither hla or Lys. Thr-232 was the target of this study since the corresponding Lys-249 in bovine rhodanes e has been identified as necessary for catalytic sulfur transfer, and repla cement of Lys-249 with Ala fully inactivates bovine rhodanese, Both T232K a nd T232A mutants of RhdA shelved significant increase in thiosulfate-cyanid e sulfurtransferase activity, and no detectable activity in the presence of 3-mercaptopyruvate as the sulfur donor substrate. Fluorescence measurement s showed that wild-type and mutant RhdAs tt cre overexpressed in the persul furated form, thus conferring to this enzyme the potential of a persulfide sulfur donor compound. RhdA contains a unique sequence stretch around the c atalytic cysteine, and the data here presented suggest a possible divergent physiological function of A. vinelandii sulfurtransferase, (C) 2000 Federa tion of European Biochemical Societies.