1WE4

Crystal Structure of Class A beta-Lactamase Toho-1 G238C mutant

Summary for 1WE4

• Entry DOI: 10.2210/pdb1we4/pdb
• Related: 1BZA 1IYS
• Descriptor: Beta-lactamase Toho-1, SULFATE ION (3 entities in total)
• Functional Keywords: hydrolase, antibiotic resistance
• Biological source: Escherichia coli
• Total number of polymer chains: 1
• Total formula weight: 29040.62

Authors

Shimizu-Ibuka, A.,Matsuzawa, H.,Sakai, H. (deposition date: 2004-05-24, release date: 2005-03-15, Last modification date: 2024-11-13)

Primary citation

Shimizu-Ibuka, A.,Matsuzawa, H.,Sakai, H.
An Engineered Disulfide Bond between Residues 69 and 238 in Extended-Spectrum beta-Lactamase Toho-1 Reduces Its Activity toward Third-Generation Cephalosporins
Biochemistry, 43:15737-15745, 2004

PubMed Abstract: 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.

PubMed: 15595829
DOI: 10.1021/bi048488u

Experimental method

X-RAY DIFFRACTION (1.7 Å)