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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}
}
  • Neutron protein crystallography allows H-atom positions to be located in biological structures at the relatively modest resolution of 1.5-2.0 {angstrom}. A difficulty of this technique arises from the incoherent scattering from hydrogen, which considerably reduces the signal-to-noise ratio of the data. This can be overcome by preparing fully deuterated samples. Efficient protocols for routine and low-cost production of in vivo deuterium-enriched proteins have been developed. Here, the overexpression and crystallization of highly (>99%) deuterium-enriched cytochrome P450cam for neutron analysis is reported. Cytochrome P450cam from Pseudomonas putida catalyses the hydroxylation of camphor from haem-bound molecular O{sub 2} via a mechanism thatmore » is thought to involve a proton-shuttle pathway to the active site. Since H atoms cannot be visualized in available X-ray structures, neutron diffraction is being used to determine the protonation states and water structure at the active site of the enzyme. Analysis of both hydrogenated and perdeuterated P450cam showed no significant changes between the X-ray structures determined at 1.4 and 1.7 {angstrom}, respectively. This work demonstrates that the fully deuterated protein is highly isomorphous with the native (hydrogenated) protein and is appropriate for neutron protein crystallographic analysis.« less
  • Human carbonic anhydrases (CAs) are well studied targets for the development of inhibitors for pharmaceutical applications. The crystal structure of human CA II has been determined in complex with two CA inhibitors (CAIs) containing conventional sulfonamide and thiadiazole moieties separated by a -CF{sub 2}- or -CHNH{sub 2}- spacer group. The structures presented here reveal that these spacer groups allow novel binding modes for the thiadiazole moiety compared with conventional CAIs.
  • Carbonic anhydrase II (CA II) deficiency has been shown to be the primary defect in the recessively inherited syndrome of osteopetrosis with renal tubular acidosis. Until now, the absence of CA II in kidney of CA II-deficient patients has not been shown directly, and the status of the membrane-associated CA in kidney of CA II-deficient patients has been unclear. To address these questions, the authors analyzed urinary membranes and soluble fractions from normal and CA II-deficient subjects. The CA activity in membrane fractions of normal urine was found to comprise two components-(i) a vesicle-enclosed, sodium dodecyl sulfate (SDS)-sensitive fraction, whichmore » was shown immunochemically to be the 29-kDa CA II, and (ii) an SDS-resistant fraction, which was due to native and cleaved forms of the 35-kDa, membrane-anchored isozyme CA IV. Urinary membranes from CA II-deficient patients showed little or no SDS-sensitive activity and no immunoreactivity for CA II, providing direct evidence that their mutation, which produces CA II deficiency in erythrocytes, also affects CA II in kidney. CA IV activity and immunoreactivity were present in normal amounts in urinary membranes from CA II-deficient patients. They conclude from the enzymatic and immunological evidence presented that both CA II and CA IV are present in urinary membranes from normal subjects, that renal CA IV is present but renal CA II is absent in urinary membranes from patients with the CA II-deficiency syndrome, and that the methods presented should be useful in studying renal CA II and renal CA IV in other disorders of impaired bicarbonate reabsorption.« less
  • A detailed analysis of the stability and activity of Mn(II) human carbonic anhydrase I and the kinetics and mechanism of its catalysis of the HCO/sub 3//sup -//CO/sub 2/ exchange have been performed at pH 8.5. The analysis was based on the paramagnetic relaxation rates R/sub 1p/ and R/sub 2p/ of the /sup 13/C atom of HCO/sub 3//sup -/ in the Mn/sup 2 +//apoenzyme/HCO/sub 3//sup -//CO/sub 2/ system and the HCO/sub 3//sup -/ in equilibrium CO/sub 2/ interconversion rate obtained by the magnetization-transfer technique. The R/sub 1p/ and R/sub 2p/ rates were measured as functions of the temperature, magnetic field strength,more » and substrate and apoenzyme concentrations and were interpreted on the basis of Solomon-Bloembergen-Morgan theories and general equations for the liquid exchange. From the analysis of the data, a formation constant for the Mn(II) enzyme of log K/sub MA//sup M/ = 5.8 +/- 0.4 was obtained while the activity of the Mn(II) enzyme, measured as the HCO/sub 3//sup -//CO/sub 2/ interconversion rate at (HCO/sub 3//sup -/) = 0.100 M and pH 8.5, was found to be about 4% of that of the native Zn(II) enzyme. Most conspicuously, the resulting distance of only 2.71 +/- 0.03 A between the Mn/sup 2 +/ ion of the enzyme and the /sup 13/C atom of HCO/sub 3//sup -/ in the enzyme-bicarbonate complex indicates that the bicarbonate is bound to the metal ion by two of its oxygen atoms in the central catalytic step, thereby supporting the modified Zn(II)-OH mechanism. In contrast, this binding mode differs from the structure of the complexes suggested in the rapid-equilibrium kinetic model.« less