Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate.

Venu Gopal Vandavasi,Kevin L Weiss,Leighton Coates,Patricia S Langan,Jerry M Parks,Stephan L Ginell,Jonathan B Cooper

doi:10.1128/aac.01636-16

Abstract

ABSTRACTThe monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M β-lactamase but not for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A β-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, as previously proposed.

Highlights

The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum ␤-lactamases
The combination of neutron diffraction and 15 K X-ray diffraction enabled us to visualize most of the deuterium atoms within the active site of the monobactam antibiotic binding site
Using both X-ray and neutron crystallography, we have located the positions of most of the hydrogen atoms within the active site of Toho-1 ␤-lactamase in its acyl-enzyme intermediate state in complex with aztreonam. These structures suggest that Lys 73 can act as a general base in the acylation step of the ␤-lactam hydrolysis reaction, as has been previously proposed [11, 14, 17, 30]

Summary

Introduction

The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum ␤-lactamases. We used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum ␤-lactamases hydrolyze monobactam antibiotics In these first reported structures of a class A ␤-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Glu 166 has been proposed to act as the catalytic base in the acylation step of the reaction (Fig. 1, stages 1 to 3) in which this residue deprotonates the hydroxyl of Ser 70 via a water molecule before Ser 70 attacks the carbonyl carbon of the ␤-lactam ring [8, 12, 13]. When both Lys 73 and Glu 166 are mutated, the rate constants for the acylation reaction decrease by 10,000-fold [19]

Methods

Results

Conclusion