| An engineered disulfide bond between residues 69 and 238 in extended-spectrum beta-lactamase Toho-1 reduces its activity toward third-generation cephalosporins. | |
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MedLine Citation:
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PMID: 15595829 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Previous crystallographic structural analysis of extended-spectrum beta-lactamase Toho-1 predicted that the high flexibility of beta-strand B3, the region that contains a conserved KTG motif and forms one wall of the substrate-binding site, could be one of the key features contributing to Toho-1 activity toward third-generation cephalosporins. To investigate whether this possible flexibility really affects the substrate profile of this enzyme, two Toho-1 mutants have been produced, G238C and G238C/G239in, in which the glycine residue at position 238 was replaced with a cysteine and an additional glycine residue was inserted. Our intent was to introduce a disulfide bond between the cysteine residues at positions 69 and 238, and thus to lock the position of beta-strand B3. The results of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) titration indicated formation of a new disulfide bridge in the G238C mutant, although disulfide bond formation was not confirmed in the G238C/G239in mutant. Kinetic analysis showed that the activity of the G238C mutant decreased drastically against third-generation cephalosporins, while its catalytic efficiency against penicillins and first-generation cephalosporins was almost identical to that of the wild-type enzyme. This result was consistent with the prediction that flexibility in beta-strand B3 was critical for activity against third-generation cephalosporins in Toho-1. Furthermore, we have determined the crystal structure of the G238C mutant enzyme to analyze the structural changes in detail. The structural model clearly shows the introduction of a new disulfide bridge and that there is no appreciable difference between the overall structures of the wild-type enzyme and the G238C mutant, although the introduced disulfide bond slightly influenced the positions of Ser237 on beta-strand B3 and Asn170 on the Omega loop. The results of our kinetic and structural analyses suggest that the flexibility of beta-strand B3, as well as the positions of Ser237 and the Omega loop, is critical for the substrate specificity expansion of Toho-1. |
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Authors:
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Akiko Shimizu-Ibuka; Hiroshi Matsuzawa; Hiroshi Sakai |
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Publication Detail:
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Type: Journal Article |
Journal Detail:
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Title: Biochemistry Volume: 43 ISSN: 0006-2960 ISO Abbreviation: Biochemistry Publication Date: 2004 Dec |
Date Detail:
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Created Date: 2004-12-14 Completed Date: 2005-02-07 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 0370623 Medline TA: Biochemistry Country: United States |
Other Details:
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Languages: eng Pagination: 15737-45 Citation Subset: IM |
Affiliation:
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Department of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan. aibuka@mail.ecc.u-tokyo.ac.jp |
| Data Bank Information | |
Bank Name/Acc. No.:
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PDB/1WE4 |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Sequence Binding Sites / genetics Cephalosporins / chemistry, metabolism* Crystallography, X-Ray Disulfides / chemistry* Dithionitrobenzoic Acid / chemistry Escherichia coli Proteins / chemistry*, genetics, metabolism* Molecular Sequence Data Point Mutation / genetics Protein Engineering Protein Structure, Secondary Sequence Alignment Substrate Specificity beta-Lactamases / chemistry*, genetics, metabolism* |
| Chemical | |
Reg. No./Substance:
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0/Cephalosporins; 0/Disulfides; 0/Escherichia coli Proteins; 69-78-3/Dithionitrobenzoic Acid; EC 3.5.2.6/beta-Lactamases; EC 3.5.2.6/beta-lactamase Toho-1, E coli |
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