1we4
From Proteopedia
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Crystal Structure of Class A beta-Lactamase Toho-1 G238C mutant
Overview
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.
About this Structure
1WE4 is a Single protein structure of sequence from Escherichia coli with SO4 as ligand. Active as Beta-lactamase, with EC number 3.5.2.6 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 Wed Nov 21 05:20:28 2007
