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Title: Preliminary neutron diffraction studies of Escherichia coli dihydrofolate reductase bound to the anticancer drug methotrexate

Abstract

The contribution of H atoms in noncovalent interactions and enzymatic reactions underlies virtually all aspects of biology at the molecular level, yet their 'visualization' is quite difficult. To better understand the catalytic mechanism of Escherichia coli dihydrofolate reductase (ecDHFR), a neutron diffraction study is under way to directly determine the accurate positions of H atoms within its active site. Despite exhaustive investigation of the catalytic mechanism of DHFR, controversy persists over the exact pathway associated with proton donation in reduction of the substrate, dihydrofolate. As the initial step in a proof-of-principle experiment which will identify ligand and residue protonation states as well as precise solvent structures, a neutron diffraction data set has been collected on a 0.3 mm{sup 3} D{sub 2}O-soaked crystal of ecDHFR bound to the anticancer drug methotrexate (MTX) using the LADI instrument at ILL. The completeness in individual resolution shells dropped to below 50% between 3.11 and 3.48 {angstrom} and the I/{sigma}(I) in individual shells dropped to below 2 at around 2.46 {angstrom}. However, reflections with I/{sigma}(I) greater than 2 were observed beyond these limits (as far out as 2.2 {angstrom}). To our knowledge, these crystals possess one of the largest primitive unit cells (P6{sub 1}, amore » = b = 92, c = 73 {angstrom}) and one of the smallest crystal volumes so far tested successfully with neutrons.« less

Authors:
 [1];  [2]; ORCiD logo [3];  [1];  [1]
  1. University of Tennessee, Knoxville (UTK)
  2. Institut Laue-Langevin (ILL)
  3. ORNL
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1003253
DOE Contract Number:  
AC05-00OR22725
Resource Type:
Journal Article
Journal Name:
Acta Crystallographica Section D: Biological Crystallography
Additional Journal Information:
Journal Volume: D; Journal Issue: 61
Country of Publication:
United States
Language:
English
Subject:
72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS; 59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; ATOMS; BIOLOGY; ESCHERICHIA COLI; HYDROGEN; METHOTREXATE; NEUTRON DIFFRACTION; OXIDOREDUCTASES; PROTONS; RESIDUES; RESOLUTION; SOLVENTS

Citation Formats

Bennett, Brad C., Meilleur, Flora, Myles, Dean A A, Howell, Elizabeth E., and Dealwis, Chris G. Preliminary neutron diffraction studies of Escherichia coli dihydrofolate reductase bound to the anticancer drug methotrexate. United States: N. p., 2005. Web. doi:10.1107/S0907444905004804.
Bennett, Brad C., Meilleur, Flora, Myles, Dean A A, Howell, Elizabeth E., & Dealwis, Chris G. Preliminary neutron diffraction studies of Escherichia coli dihydrofolate reductase bound to the anticancer drug methotrexate. United States. https://doi.org/10.1107/S0907444905004804
Bennett, Brad C., Meilleur, Flora, Myles, Dean A A, Howell, Elizabeth E., and Dealwis, Chris G. Tue . "Preliminary neutron diffraction studies of Escherichia coli dihydrofolate reductase bound to the anticancer drug methotrexate". United States. https://doi.org/10.1107/S0907444905004804.
@article{osti_1003253,
title = {Preliminary neutron diffraction studies of Escherichia coli dihydrofolate reductase bound to the anticancer drug methotrexate},
author = {Bennett, Brad C. and Meilleur, Flora and Myles, Dean A A and Howell, Elizabeth E. and Dealwis, Chris G.},
abstractNote = {The contribution of H atoms in noncovalent interactions and enzymatic reactions underlies virtually all aspects of biology at the molecular level, yet their 'visualization' is quite difficult. To better understand the catalytic mechanism of Escherichia coli dihydrofolate reductase (ecDHFR), a neutron diffraction study is under way to directly determine the accurate positions of H atoms within its active site. Despite exhaustive investigation of the catalytic mechanism of DHFR, controversy persists over the exact pathway associated with proton donation in reduction of the substrate, dihydrofolate. As the initial step in a proof-of-principle experiment which will identify ligand and residue protonation states as well as precise solvent structures, a neutron diffraction data set has been collected on a 0.3 mm{sup 3} D{sub 2}O-soaked crystal of ecDHFR bound to the anticancer drug methotrexate (MTX) using the LADI instrument at ILL. The completeness in individual resolution shells dropped to below 50% between 3.11 and 3.48 {angstrom} and the I/{sigma}(I) in individual shells dropped to below 2 at around 2.46 {angstrom}. However, reflections with I/{sigma}(I) greater than 2 were observed beyond these limits (as far out as 2.2 {angstrom}). To our knowledge, these crystals possess one of the largest primitive unit cells (P6{sub 1}, a = b = 92, c = 73 {angstrom}) and one of the smallest crystal volumes so far tested successfully with neutrons.},
doi = {10.1107/S0907444905004804},
url = {https://www.osti.gov/biblio/1003253}, journal = {Acta Crystallographica Section D: Biological Crystallography},
number = 61,
volume = D,
place = {United States},
year = {2005},
month = {3}
}