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Mutagenesis of Asp25 or Arg27 in PSPE Destabilizes the Rhodanese Fold
Author(s) -
Shih IChia,
Volini Marguerite
Publication year - 2007
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.21.5.a275-d
Subject(s) - rhodanese , sulfurtransferase , thiosulfate , biochemistry , mutagenesis , site directed mutagenesis , biology , recombinant dna , chemistry , enzyme , gene , sulfur , mutation , mutant , organic chemistry , cysteine
In studies of features of the Rhodanese Fold that give rise to thiosulfate sulfurtransferase activity, we have shown that positioning of the cysteinyl residue, which is the site of substrate‐sulfur binding, is essential for enzyme activity. In our model of the protein product of the E. coli pspE gene, this residue is Cys67. We have further shown that Arg71 of PSPE is the important determinant of thiosulfate substrate specificity. The K m for thiosulfate is increased more than 7‐fold upon mutagenesis of Arg71 to Gly71. In the present work, we have investigated the role of residues that are necessary for maintenance of the rhodanese fold in PSPE and likely in all other proteins that exhibit rhodanese sulfurtransferase activity. Asp25 and Arg27 are close to the active‐site sulfhydryl group in our 3‐D model of PSPE, but they are also conserved in rhodanese proteins that we have tested for sulfurtransferase activity with negative results. Hence, we have selected these residues as potentially critical for proper folding rather than as participants in the catalytic mechanism. Arg27 was replaced with glycine by site‐directed mutagenesis using PCR. The mutated gene was cloned into pET22b+ and the recombinant plasmid was transformed into the E. coli expression strain, BL21 (DE3). SDS‐PAGE gels showed that the recombinant protein was expressed but the recombinant clone extracts exhibited no sulfurtransferase activity. Asp25 was similarly mutagenized to Gly25 and again the expressed protein exhibited no activity. In our model of PSPE and other rhodanese proteins the side‐chains of Asp25 and Arg27 form an ion pair. This pair appears to be important for stabilization of the Rhodanese Fold. (Supported by UHF#434138).