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The Use of a β-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions
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Acyl-intermediate structures of the extended-spectrum class A beta-lactamase, Toho-1, in complex with cefotaxime,

Tatsuro Shimamura1, Akiko Ibuka, Shinya Fushinobu

  • 1Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

The Journal of Biological Chemistry
|September 11, 2002
PubMed

Insights

Toho-1 beta-lactamase efficiently degrades advanced cephalosporin antibiotics. Structural analysis reveals unique adaptations in Toho-1, including an Omega-loop displacement and specific residue interactions, explaining its broad-spectrum resistance mechanism.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Microbiology

Background:

  • Bacterial resistance to beta-lactam antibiotics poses a significant clinical challenge.
  • Beta-lactamases are the primary mechanism by which bacteria inactivate these drugs.
  • Extended-spectrum beta-lactamases (ESBLs) like Toho-1 confer resistance to a wide range of beta-lactam antibiotics, including newer cephalosporins.

Purpose of the Study:

  • To elucidate the structural basis for the extended-spectrum activity of the Toho-1 beta-lactamase.
  • To understand the molecular mechanisms by which Toho-1 hydrolyzes expanded-spectrum cephalosporins.

Main Methods:

  • X-ray crystallography was used to determine the acyl-intermediate structures of Toho-1.
  • Structures were solved in complex with cefotaxime, cephalothin, and benzylpenicillin at high resolutions (1.8–2.1 Å).

Main Results:

  • The Omega-loop of Toho-1 undergoes displacement to accommodate the bulky side chain of cefotaxime.
  • Conserved residues Asn(104) and Asp(240) form specific interactions that tightly bind the cefotaxime side chain.
  • A unique interaction involving Ser(237) positions the beta-lactam carbonyl group for efficient hydrolysis, enhancing cephalosporinase activity.

Conclusions:

  • The structural features of Toho-1, including Omega-loop flexibility and specific residue interactions, explain its ability to hydrolyze expanded-spectrum cephalosporins.
  • Understanding these resistance mechanisms is crucial for developing new antibiotics and combating antimicrobial resistance.

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