Neutron and X-ray Crystal Structures of a Perdeuterated Enzyme Inhibitor Complex Reveal the Catalytic Proton Network of the Toho-1 β-Lactamase for the Acylation Reaction

• Published in: The Journal of biological chemistry Vol. 288; no. 7; pp. 4715 - 4722
• Main Authors: Tomanicek, Stephen J., Standaert, Robert F., Weiss, Kevin L., Ostermann, Andreas, Schrader, Tobias E., Ng, Joseph D., Coates, Leighton
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.02.2013; American Society for Biochemistry and Molecular Biology
• Subjects: Acylation; beta-Lactamases - chemistry; Catalysis; Catalytic Domain; Class A Beta-Lactamase; Crystal Structure; Crystallography; Crystallography, X-Ray - methods; Drug Resistance, Bacterial; Enzyme Inhibitors - pharmacology; Enzyme Mechanisms; Escherichia coli - enzymology; Escherichia coli Proteins - chemistry; Hydrogen - chemistry; Hydrogen Bonding; Ligands; Models, Chemical; Molecular Conformation; Neutron Diffraction; Neutron Scattering; Neutrons; Nitrogen - chemistry; Protein Structure and Folding; Protons; Thiophenes - chemistry; Transition State Analog; X-ray Crystallography; Enzyme Mechanisms; Crystal Structure; X-ray Crystallography; Neutron Scattering; Neutron Diffraction; Crystallography; Class A Beta-Lactamase; Transition State Analog
• DOI: 10.1074/jbc.M112.436238
• ISSN: 0021-9258; 1083-351X

The mechanism by which class A β-lactamases hydrolyze β-lactam antibiotics has been the subject of intensive investigation using many different experimental techniques. Here, we report on the novel use of both neutron and high resolution x-ray diffraction to help elucidate the identity of the catalytic base in the acylation part of the catalytic cycle, wherein the β-lactam ring is opened and an acyl-enzyme intermediate forms. To generate protein crystals optimized for neutron diffraction, we produced a perdeuterated form of the Toho-1 β-lactamase R274N/R276N mutant. Protein perdeuteration, which involves replacing all of the hydrogen atoms in a protein with deuterium, gives a much stronger signal in neutron diffraction and enables the positions of individual deuterium atoms to be located. We also synthesized a perdeuterated acylation transition state analog, benzothiophene-2-boronic acid, which was also isotopically enriched with 11B, as 10B is a known neutron absorber. Using the neutron diffraction data from the perdeuterated enzyme-inhibitor complex, we were able to determine the positions of deuterium atoms in the active site directly rather than by inference. The neutron diffraction results, along with supporting bond-length analysis from high resolution x-ray diffraction, strongly suggest that Glu-166 acts as the general base during the acylation reaction. Background: Antibiotic resistance from extended-spectrum β-lactamases (ESBLs) makes infections more dangerous and difficult to treat. Results: Neutron and x-ray crystal structures were determined for an ESBL in complex with an acylation transition state analog. Conclusion: Glu-166 is implicated as the general base in the acylation reaction. Significance: Understanding the catalytic mechanism of β-lactamases will lead to improved antibiotics and β-lactamase inhibitors.