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Title: Production and X-ray crystallographic analysis of fully deuterated human carbonic anhydrase II

Abstract

This article reports the production, crystallization and X-ray structure determination of perdeuterated human carbonic anhydrase (HCA II). The refined structure is shown to be highly isomorphous with hydrogenated HCA II, especially with regard to the active site architecture and solvent network. Human carbonic anhydrase II (HCA II) is a zinc metalloenzyme that catalyzes the reversible hydration and dehydration of carbon dioxide and bicarbonate, respectively. The rate-limiting step in catalysis is the intramolecular transfer of a proton between the zinc-bound solvent (H{sub 2}O/OH{sup −}) and the proton-shuttling residue His64. This distance (∼7.5 Å) is spanned by a well defined active-site solvent network stabilized by amino-acid side chains (Tyr7, Asn62, Asn67, Thr199 and Thr200). Despite the availability of high-resolution (∼1.0 Å) X-ray crystal structures of HCA II, there is currently no definitive information available on the positions and orientations of the H atoms of the solvent network or active-site amino acids and their ionization states. In preparation for neutron diffraction studies to elucidate this hydrogen-bonding network, perdeuterated HCA II has been expressed, purified, crystallized and its X-ray structure determined to 1.5 Å resolution. The refined structure is highly isomorphous with hydrogenated HCA II, especially with regard to the active-site architecture and solventmore » network. This work demonstrates the suitability of these crystals for neutron macromolecular crystallography.« less

Authors:
 [1];  [2];  [3];  [1];  [3];  [4];  [1];  [5];  [3];  [2]
  1. European Molecular Biology Laboratory Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble (France)
  2. (France)
  3. Department of Biochemistry and Molecular Biology, PO Box 100245, University of Florida, Gainesville, FL 32610 (United States)
  4. Department of Pharmacology and Therapeutics, PO Box 100267, University of Florida, Gainesville, FL 32610 (United States)
  5. (United States)
Publication Date:
OSTI Identifier:
22356243
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 62; Journal Issue: Pt 1; Other Information: PMCID: PMC2150928; PMID: 16511248; PUBLISHER-ID: fw5056; OAI: oai:pubmedcentral.nih.gov:2150928; Copyright (c) International Union of Crystallography 2006; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; ATOMS; BONDING; CARBON DIOXIDE; CHAINS; CRYSTAL STRUCTURE; CRYSTALLIZATION; CRYSTALLOGRAPHY; CRYSTALS; DEHYDRATION; HYDRATION; HYDROGEN; NEUTRON DIFFRACTION; RESOLUTION; ZINC

Citation Formats

Budayova-Spano, Monika, Institut Laue-Langevin, 6 Rue Jules Horowitz, BP 156, 38042 Grenoble, Fisher, S. Zoë, Dauvergne, Marie-Thérèse, Agbandje-McKenna, Mavis, Silverman, David N., Myles, Dean A. A., Oak Ridge National Laboratory, PO Box 2008, Oak Ridge, TN 37831, McKenna, Robert, E-mail: rmckenna@ufl.edu, and European Molecular Biology Laboratory Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble. Production and X-ray crystallographic analysis of fully deuterated human carbonic anhydrase II. United Kingdom: N. p., 2006. Web. doi:10.1107/S1744309105038248.
Budayova-Spano, Monika, Institut Laue-Langevin, 6 Rue Jules Horowitz, BP 156, 38042 Grenoble, Fisher, S. Zoë, Dauvergne, Marie-Thérèse, Agbandje-McKenna, Mavis, Silverman, David N., Myles, Dean A. A., Oak Ridge National Laboratory, PO Box 2008, Oak Ridge, TN 37831, McKenna, Robert, E-mail: rmckenna@ufl.edu, & European Molecular Biology Laboratory Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble. Production and X-ray crystallographic analysis of fully deuterated human carbonic anhydrase II. United Kingdom. doi:10.1107/S1744309105038248.
Budayova-Spano, Monika, Institut Laue-Langevin, 6 Rue Jules Horowitz, BP 156, 38042 Grenoble, Fisher, S. Zoë, Dauvergne, Marie-Thérèse, Agbandje-McKenna, Mavis, Silverman, David N., Myles, Dean A. A., Oak Ridge National Laboratory, PO Box 2008, Oak Ridge, TN 37831, McKenna, Robert, E-mail: rmckenna@ufl.edu, and European Molecular Biology Laboratory Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble. Sun . "Production and X-ray crystallographic analysis of fully deuterated human carbonic anhydrase II". United Kingdom. doi:10.1107/S1744309105038248.
@article{osti_22356243,
title = {Production and X-ray crystallographic analysis of fully deuterated human carbonic anhydrase II},
author = {Budayova-Spano, Monika and Institut Laue-Langevin, 6 Rue Jules Horowitz, BP 156, 38042 Grenoble and Fisher, S. Zoë and Dauvergne, Marie-Thérèse and Agbandje-McKenna, Mavis and Silverman, David N. and Myles, Dean A. A. and Oak Ridge National Laboratory, PO Box 2008, Oak Ridge, TN 37831 and McKenna, Robert, E-mail: rmckenna@ufl.edu and European Molecular Biology Laboratory Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble},
abstractNote = {This article reports the production, crystallization and X-ray structure determination of perdeuterated human carbonic anhydrase (HCA II). The refined structure is shown to be highly isomorphous with hydrogenated HCA II, especially with regard to the active site architecture and solvent network. Human carbonic anhydrase II (HCA II) is a zinc metalloenzyme that catalyzes the reversible hydration and dehydration of carbon dioxide and bicarbonate, respectively. The rate-limiting step in catalysis is the intramolecular transfer of a proton between the zinc-bound solvent (H{sub 2}O/OH{sup −}) and the proton-shuttling residue His64. This distance (∼7.5 Å) is spanned by a well defined active-site solvent network stabilized by amino-acid side chains (Tyr7, Asn62, Asn67, Thr199 and Thr200). Despite the availability of high-resolution (∼1.0 Å) X-ray crystal structures of HCA II, there is currently no definitive information available on the positions and orientations of the H atoms of the solvent network or active-site amino acids and their ionization states. In preparation for neutron diffraction studies to elucidate this hydrogen-bonding network, perdeuterated HCA II has been expressed, purified, crystallized and its X-ray structure determined to 1.5 Å resolution. The refined structure is highly isomorphous with hydrogenated HCA II, especially with regard to the active-site architecture and solvent network. This work demonstrates the suitability of these crystals for neutron macromolecular crystallography.},
doi = {10.1107/S1744309105038248},
journal = {Acta Crystallographica. Section F},
number = Pt 1,
volume = 62,
place = {United Kingdom},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}