Active-site protonation states in an acyl-enzyme intermediate of a class A β-lactamase with a monobactam substrate

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

doi:10.1128/AAC.01636-16

Active-site protonation states in an acyl-enzyme intermediate of a class A β-lactamase with a monobactam substrate

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

Research output: Contribution to journal › Article › peer-review

28 Scopus citations

Abstract

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 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.

Original language	English
Article number	e01636-16
Journal	Antimicrobial Agents and Chemotherapy
Volume	61
Issue number	1
DOIs	https://doi.org/10.1128/AAC.01636-16
State	Published - Jan 1 2017

Bibliographical note

Publisher Copyright:
Copyright © 2016 Vandavasi et al.

Funding

This research was sponsored by the Laboratory Directed Research and Development Program at Oak Ridge National Laboratory (ORNL), which is managed by UT-Battelle, LLC, for the U.S. Department of Energy (DOE). Research at ORNL's Spallation Neutron Source was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. The Office of Biological and Environmental Research supported research at Oak Ridge National Laboratory's Center for Structural Molecular Biology (CSMB), using facilities supported by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. Results shown in this report are derived from work performed at Argonne National Laboratory (ANL), Structural Biology Center at the Advanced Photon Source. ANL is operated by UChicago Argonne, LLC, for the U.S. Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357.

Funders	Funder number
Oak Ridge National Laboratory
Scientific User Facilities Division
U.S. Department of Energy
Basic Energy Sciences
Biological and Environmental Research	DE-AC02-06CH11357
Argonne National Laboratory
Oak Ridge National Laboratory
Canadian Society for Molecular Biosciences

Keywords

Acyl-enzyme complex
Aztreonam
Neutron structure
X-ray structure
β-lactamase

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1128/AAC.01636-16

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title = "Active-site protonation states in an acyl-enzyme intermediate of a class A β-lactamase with a monobactam substrate",

abstract = "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 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.",

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AU - Langan, Patricia S.

AU - Weiss, Kevin L.

AU - Parks, Jerry M.

AU - Cooper, Jonathan B.

AU - Ginell, Stephan L.

AU - Coates, Leighton

PY - 2017/1/1

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N2 - 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 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.

AB - 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 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.

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Active-site protonation states in an acyl-enzyme intermediate of a class A β-lactamase with a monobactam substrate

Abstract

Bibliographical note

Funding

Keywords

UN SDGs

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