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Structure And Function of YRKF, a Rhodanese‐Domain Protein From B. subtilis
Author(s) -
Shih IChia,
Volini Marguerite
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a906-c
Subject(s) - rhodanese , sulfurtransferase , thiosulfate , biochemistry , chemistry , enzyme , biology , sulfur , cysteine , organic chemistry
We are investigating the structural determinants that give rise to thiosulfate sulfurtransferase (TST) activity in the Rhodanese Superfamily of Proteins. Our previous studies contrasted the structural and kinetic properties of PSPE, GLPE, YIBN and SSEA, all rhodanese‐domain proteins from E. coli. The results implicated the positioning of a cysteinyl residue as critical for enzyme activity and the positioning of an arginyl residue as essential for thiosulfate sulfur‐donor specificity. PSPE, GLPE and SSEA all have enzyme activity but only PSPE exhibits a K m value for thiosulfate that is comparable to the bovine liver rhodanese, the prototype rhodanese protein. In the present work we have studied the properties of a rhodanese‐domain protein from B. subtilis . This protein, YRKF (185 amino acids), consists of two domains but only the C‐terminal domain is a rhodanese module. Superposition of a 3‐D molecular model of the rhodanese module of YRKF on the C‐terminal domain of the bovine rhodanese as well as on the rhodanese module of PSPE predicts that YRKF should exhibit TST activity with a K m value for thiosulfate similar to that of the other two proteins (~5 mM). To test the validity of this prediction we have cloned the yrkF gene into pET22b. The hybrid plasmid was used to transform the E. coli expression strain BL21(DE3). Kinetic analyses of culture extracts show that YRKF has ample TST activity and that it exhibits a low K m value for thiosulfate, ~2mM. Furthermore, changing Arg153 of YRKF to Gly153 by overlap extension mutagenesis increases the K m value for thiosulfate some 10–fold, supporting the proposal that the positioning of an arginyl residue in the 3‐D structure of the protein is a major determinant of thiosulfate substrate specificity. (Supported by UHF#43418).