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| | {{STRUCTURE_1we4| PDB=1we4 | SCENE= }} | | {{STRUCTURE_1we4| PDB=1we4 | SCENE= }} |
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| - | '''Crystal Structure of Class A beta-Lactamase Toho-1 G238C mutant'''
| + | ===Crystal Structure of Class A beta-Lactamase Toho-1 G238C mutant=== |
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| - | ==Overview==
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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.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_15595829}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 15595829 is the PubMed ID number. |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Antibiotic resistance]] | | [[Category: Antibiotic resistance]] |
| | [[Category: Hydrolase]] | | [[Category: Hydrolase]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 13:31:33 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Mon Jul 28 10:15:31 2008'' |
Revision as of 07:15, 28 July 2008
Template:STRUCTURE 1we4
Crystal Structure of Class A beta-Lactamase Toho-1 G238C mutant
Template:ABSTRACT PUBMED 15595829
About this Structure
1WE4 is a Single protein structure of sequence from Escherichia coli. Full crystallographic information is available from OCA.
Reference
An engineered disulfide bond between residues 69 and 238 in extended-spectrum beta-lactamase Toho-1 reduces its activity toward third-generation cephalosporins., Shimizu-Ibuka A, Matsuzawa H, Sakai H, Biochemistry. 2004 Dec 21;43(50):15737-45. PMID:15595829
Page seeded by OCA on Mon Jul 28 10:15:31 2008
