A Persulfurated Cysteine Promotes Active Site Reactivity in Azotobacter vinelandii Rhodanese

Abstract

Active site reactivity and specificity of RhdA, a thiosulfate:cyanide sulfurtransferase (rhodanese) from Azotobacter vinelandii, have been investigated through ligand binding, site-directed mutagenesis, and X-ray crystallographic techniques, in a combined approach. In native RhdA the active site Cys230 is found persulfurated; fluorescence and sulfurtransferase activity measurements show that phosphate anions interact with Cys230 persulfide sulfur atom and modulate activity. Crystallographic analyses confirm that phosphate and hypophosphite anions react with native RhdA, removing the persulfide sulfur atom from the active site pocket. Considering that RhdA and the catalytic subunit of Cdc25 phosphatases share a common three-dimensional fold as well as active site Cys (catalytic) and Arg residues, two RhdA mutants carrying a single amino acid insertion at the active site loop were designed and their phosphatase activity tested. The crystallographic and functional results reported here show that specific sulfurtransferase or phosphatase activities are strictly related to precise tailoring of the catalytic loop structure in RhdA and Cdc25 phosphatase, respectively.

Enzymatic Detoxification of Cyanide: Clues from <i>Pseudomonas aeruginosa</i> Rhodanese

“…thiosulfate and cyanide) are suggestive of a marginal role of rhodanese in cyanide detoxification. A similar conclusion had also been drawn from biochemical investigation on A. vinelandii RhdA which resembles the P. aeruginosa homologue from both structural and functional viewpoints [Bordo et al, 2001;Cipollone et al, 2004Cipollone et al, , 2007aPagani et al, 1993]. However, since in vitro characterization cannot take into due account the complex cellular environment, the investigation of r-RhdA properties has been extended to the in vivo situation, in both homologous and heterologous systems.…”

“…Rhodanese from A zotobacter vinelandii (RhdA) has been crystallized and its biochemical properties have been investigated [Bordo et al, 2000[Bordo et al, , 2001Pagani et al, 1993]. Its structure matches the double-domain architecture of Rhobov, although sequence identity is very low (22%) [Bordo and Bork, 2002].…”

Selenophosphate synthetase genes from lung adenocarcinoma cells: Sps1 for recycling l -selenocysteine and Sps2 for selenite assimilation

“…NifS-like proteins have a conserved cysteine residue near the active site, and the residue is proposed to bind selenium and deliver it as a substrate for selenium metabolizing enzymes such as SPS (26). It seems likely that the human lung Sps1 also used the E. coli system that provides protein-bound selenium as the substrate.…”

Redox and Chemical Activities of the Hemes in the Sulfur Oxidation Pathway Enzyme SoxAX

“…This modification may arise from decomposition of a catalytic intermediate during enzyme purification. Alterna-tively, conjugation of a sulfur atom to Cys-222A with the formation of CysS Ϫ could form a step in the pathway for thiosulfate oxidation in a mechanism similar to that of sulfur transferases such as rhodanese (11,12). In such a mechanism, the oxidation of thiosulfate ( Ϫ S-SO 3 Ϫ ) would not occur as an inner sphere reaction of the heme iron.…”