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.z 2000 Federation of European Biochemical Societies.

Enzymatic and genetic profiles in environmental strains grown on polycyclic aromatic hydrocarbons

“…The active-site loop of E. coli SseA, shared by a number of rhodanese-like proteins listed in databases, represents a signature for MST activity, while the active-site loop of A. vinelandii RhdA, found in a limited number of rhodanese-like proteins is discriminant for TST activity Pagani et al 2000;Colnaghi et al 2001;Bordo et al 2001). No presence of RhdA-like proteins, nor significant level of TST activity were found in any of the analysed strains.…”

“…RhdA from A. vinelandii and SseA from E. coli, are prototypes of rhodanese-like proteins with TST and MST activity, respectively (Bordo et al 2001(Bordo et al , 2002Pagani et al 2000;Colnaghi et al 2001). Taking advantage of the finding that the antibody raised against RhdA did not recognize SseA, and vice versa, a preliminary screening for the presence of RhdA-like and or SseA-like proteins in the strains here studied was carried out by Western blot analysis.…”

Enzymatic Activity of the Arabidopsis Sulfurtransferase Resides in the C-Terminal Domain But Is Boosted by the N-Terminal Domain and the Linker Peptide in the Full- Length Enzyme

“…Only the Azotobacter ST showed unique properties among the characterized STs because it accepted almost exclusively thiosulfate with a 1×10 3 fold higher specific activity than with 3-MP (Colnaghi et al, 1996). Probably the unique stretch of amino acids around the active site is responsible for this substrate specificity Pagani et al, 2000).…”

Structural rearrangements of the two domains of Azotobacter vinelandii rhodanese upon sulfane sulfur release: essential molecular dynamics, NMR relaxation and deuterium exchange on the uniformly labeled protein

“…Recently it was found that the replacement of Thr232 with Ala in the active site of RhdA increased its sulfur-transfer activity [36]. Conversely, the corresponding mutation in the bovine enzyme (Lys249 mutated in Ala) inactivated the enzyme [16].…”

“…Moreover, the use of thiosulfate during the purification steps was not required for RhdA, as it was for the bovine enzyme [37], since its E form was very sensitive to the oxidative reactions [38]. In addition, the functional stability of A. vinelandii rhodanese was not affected by the presence of thiosulfate and the protein appeared to be more stable compared to the bovine enzyme [36]. These results may be interpreted as evidence of differences in the catalytic properties of RhdA compared with those of vertebrate rhodanese.…”