A persulfurated cysteine promotes active site reactivity in Azotobacter vinelandii Rhodanese.
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.
Bordo
D
D
National Cancer Research Institute, Advanced Biotechnology Center, Genova, Italy.
Forlani
F
F
Spallarossa
A
A
Colnaghi
R
R
Carpen
A
A
Bolognesi
M
M
Pagani
S
S
D016948:Azotobacter vinelandii / Q000201:enzymology*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry*; D005453:Fluorescence; D008958:Models, Molecular; D018716:Molecular Mimicry; D009154:Mutation; D010710:Phosphates; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D013431:Sulfates / Q000737:chemistry; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020687:cdc25 Phosphatases / Q000737:chemistry / Q000378:metabolism
D010710:Phosphates; D013431:Sulfates; C012036:sodium sulfate; D013884:Thiosulfate Sulfurtransferase; D020687:cdc25 Phosphatases; D003545:Cysteine