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Title: Direct visualization of critical hydrogen atoms in a pyridoxal 5'-phosphate enzyme

Abstract

Enzymes dependent on pyridoxal 5'-phosphate (PLP, the active form of vitamin B6) perform a myriad of diverse chemical transformations. They promote various reactions by modulating the electronic states of PLP through weak interactions in the active site. Neutron crystallography has the unique ability of visualizing the nuclear positions of hydrogen atoms in macromolecules. Here we present a room-temperature neutron structure of a homodimeric PLP-dependent enzyme, aspartate aminotransferase, which was reacted in situ with α-methylaspartate. In one monomer, the PLP remained as an internal aldimine with a deprotonated Schiff base. In the second monomer, the external aldimine formed with the substrate analog. We observe a deuterium equidistant between the Schiff base and the C-terminal carboxylate of the substrate, a position indicative of a low-barrier hydrogen bond. As a result, quantum chemical calculations and a low-pH room-temperature X-ray structure provide insight into the physical phenomena that control the electronic modulation in aspartate aminotransferase.

Authors:
 [1]; ORCiD logo [2]; ORCiD logo [2]; ORCiD logo [3]; ORCiD logo [4]; ORCiD logo [2]; ORCiD logo [5]; ORCiD logo [2];  [6]
  1. Univ. of Toledo, Toledo, OH (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  3. Institut Laue Langevin, Grenoble Cedex (France)
  4. Rutherford Appleton Lab., Didcot (United Kingdom)
  5. Univ. of Tennessee, Knoxville, TN (United States)
  6. Univ. of Toledo, Toledo, OH (United States)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1407771
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Accepted Manuscript
Journal Name:
Nature Communications
Additional Journal Information:
Journal Volume: 8; Journal Issue: 1; Journal ID: ISSN 2041-1723
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY

Citation Formats

Dajnowicz, Steven, Johnston, Ryne C., Parks, Jerry M., Blakeley, Matthew P., Keen, David A., Weiss, Kevin L., Gerlits, Oksana, Kovalevsky, Andrey, and Mueser, Timothy C. Direct visualization of critical hydrogen atoms in a pyridoxal 5'-phosphate enzyme. United States: N. p., 2017. Web. doi:10.1038/s41467-017-01060-y.
Dajnowicz, Steven, Johnston, Ryne C., Parks, Jerry M., Blakeley, Matthew P., Keen, David A., Weiss, Kevin L., Gerlits, Oksana, Kovalevsky, Andrey, & Mueser, Timothy C. Direct visualization of critical hydrogen atoms in a pyridoxal 5'-phosphate enzyme. United States. doi:10.1038/s41467-017-01060-y.
Dajnowicz, Steven, Johnston, Ryne C., Parks, Jerry M., Blakeley, Matthew P., Keen, David A., Weiss, Kevin L., Gerlits, Oksana, Kovalevsky, Andrey, and Mueser, Timothy C. Mon . "Direct visualization of critical hydrogen atoms in a pyridoxal 5'-phosphate enzyme". United States. doi:10.1038/s41467-017-01060-y. https://www.osti.gov/servlets/purl/1407771.
@article{osti_1407771,
title = {Direct visualization of critical hydrogen atoms in a pyridoxal 5'-phosphate enzyme},
author = {Dajnowicz, Steven and Johnston, Ryne C. and Parks, Jerry M. and Blakeley, Matthew P. and Keen, David A. and Weiss, Kevin L. and Gerlits, Oksana and Kovalevsky, Andrey and Mueser, Timothy C.},
abstractNote = {Enzymes dependent on pyridoxal 5'-phosphate (PLP, the active form of vitamin B6) perform a myriad of diverse chemical transformations. They promote various reactions by modulating the electronic states of PLP through weak interactions in the active site. Neutron crystallography has the unique ability of visualizing the nuclear positions of hydrogen atoms in macromolecules. Here we present a room-temperature neutron structure of a homodimeric PLP-dependent enzyme, aspartate aminotransferase, which was reacted in situ with α-methylaspartate. In one monomer, the PLP remained as an internal aldimine with a deprotonated Schiff base. In the second monomer, the external aldimine formed with the substrate analog. We observe a deuterium equidistant between the Schiff base and the C-terminal carboxylate of the substrate, a position indicative of a low-barrier hydrogen bond. As a result, quantum chemical calculations and a low-pH room-temperature X-ray structure provide insight into the physical phenomena that control the electronic modulation in aspartate aminotransferase.},
doi = {10.1038/s41467-017-01060-y},
journal = {Nature Communications},
number = 1,
volume = 8,
place = {United States},
year = {2017},
month = {10}
}

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Cited by: 9 works
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Figures / Tables:

Fig. 1 Fig. 1: Structure of AAT with $α$-methylaspartate. The PLP cofactor in the external aldimine (chain A in light green) and the internal aldimine (chain B in dark green) forms are shown in yellow and gray carbon scheme, respectively. In the external aldimine, R292 and R386 interact directly with the carboxylatemore » groups of $α$-methylaspartate. These interactions are not present in the internal aldimine. Here and in Figs. 2, 3, and 5, the deuterium atoms are colored green and the external aldimine is labeled as PLA« less

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Works referenced in this record:

Balanced basis sets of split valence, triple zeta valence and quadruple zeta valence quality for H to Rn: Design and assessment of accuracy
journal, January 2005

  • Weigend, Florian; Ahlrichs, Reinhart
  • Physical Chemistry Chemical Physics, Vol. 7, Issue 18, p. 3297-3305
  • DOI: 10.1039/b508541a

Use of 1 H− 15 N Heteronuclear Multiple-Quantum Coherence NMR Spectroscopy To Study the Active Site of Aspartate Aminotransferase
journal, January 1997

  • Mollova, Emilia T.; Metzler, David E.; Kintanar, Agustin
  • Biochemistry, Vol. 36, Issue 3
  • DOI: 10.1021/bi9615811

Refinement and Comparisons of the Crystal Structures of Pig Cytosolic Aspartate Aminotransferase and Its Complex with 2-Methylaspartate
journal, July 1997

  • Rhee, Sangkee; Silva, Mercedes M.; Hyde, C. Craig
  • Journal of Biological Chemistry, Vol. 272, Issue 28
  • DOI: 10.1074/jbc.272.28.17293

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography
journal, September 2015

  • Wan, Qun; Parks, Jerry M.; Hanson, B. Leif
  • Proceedings of the National Academy of Sciences, Vol. 112, Issue 40
  • DOI: 10.1073/pnas.1504986112

LAUEGEN version 6.0 and INTLDM
journal, June 1998

  • Campbell, J. W.; Hao, Q.; Harding, M. M.
  • Journal of Applied Crystallography, Vol. 31, Issue 3
  • DOI: 10.1107/S0021889897016683

Conformation and reaction specificity in pyridoxal phosphate enzymes.
journal, April 1966

  • Dunathan, H. C.
  • Proceedings of the National Academy of Sciences, Vol. 55, Issue 4
  • DOI: 10.1073/pnas.55.4.712

Neutron macromolecular crystallography with LADI-III
journal, October 2010

  • Blakeley, Matthew P.; Teixeira, Susana C. M.; Petit-Haertlein, Isabelle
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 11
  • DOI: 10.1107/S0907444910019797

Aspartate aminotransferase: An old dog teaches new tricks
journal, February 2014


The IMAGINE instrument: first neutron protein structure and new capabilities for neutron macromolecular crystallography
journal, September 2013

  • Meilleur, Flora; Munshi, Parthapratim; Robertson, Lee
  • Acta Crystallographica Section D Biological Crystallography, Vol. 69, Issue 10
  • DOI: 10.1107/S0907444913019604

Critical hydrogen bonds and protonation states of pyridoxal 5′-phosphate revealed by NMR
journal, November 2011

  • Limbach, Hans-Heinrich; Chan-Huot, Monique; Sharif, Shasad
  • Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol. 1814, Issue 11
  • DOI: 10.1016/j.bbapap.2011.06.004

X-ray and NMR Crystallography in an Enzyme Active Site: The Indoline Quinonoid Intermediate in Tryptophan Synthase
journal, January 2011

  • Lai, Jinfeng; Niks, Dimitri; Wang, Yachong
  • Journal of the American Chemical Society, Vol. 133, Issue 1
  • DOI: 10.1021/ja106555c

Conformational Change in Aspartate Aminotransferase on Substrate Binding Induces Strain in the Catalytic Group and Enhances Catalysis
journal, December 2002

  • Hayashi, Hideyuki; Mizuguchi, Hiroyuki; Miyahara, Ikuko
  • Journal of Biological Chemistry, Vol. 278, Issue 11
  • DOI: 10.1074/jbc.M209235200

Natural localized molecular orbitals
journal, August 1985

  • Reed, Alan E.; Weinhold, Frank
  • The Journal of Chemical Physics, Vol. 83, Issue 4
  • DOI: 10.1063/1.449360

HKL -3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes
journal, July 2006

  • Minor, Wladek; Cymborowski, Marcin; Otwinowski, Zbyszek
  • Acta Crystallographica Section D Biological Crystallography, Vol. 62, Issue 8
  • DOI: 10.1107/S0907444906019949

Features and development of Coot
journal, March 2010

  • Emsley, P.; Lohkamp, B.; Scott, W. G.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
  • DOI: 10.1107/S0907444910007493

Universal Solvation Model Based on Solute Electron Density and on a Continuum Model of the Solvent Defined by the Bulk Dielectric Constant and Atomic Surface Tensions
journal, May 2009

  • Marenich, Aleksandr V.; Cramer, Christopher J.; Truhlar, Donald G.
  • The Journal of Physical Chemistry B, Vol. 113, Issue 18, p. 6378-6396
  • DOI: 10.1021/jp810292n

Pyridoxal 5′-phosphate: electrophilic catalyst extraordinaire
journal, October 2009

  • Richard, John P.; Amyes, Tina L.; Crugeiras, Juan
  • Current Opinion in Chemical Biology, Vol. 13, Issue 4
  • DOI: 10.1016/j.cbpa.2009.06.023

A genomic overview of pyridoxal-phosphate-dependent enzymes
journal, September 2003


NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity
journal, November 2016

  • Caulkins, Bethany G.; Young, Robert P.; Kudla, Ryan A.
  • Journal of the American Chemical Society, Vol. 138, Issue 46
  • DOI: 10.1021/jacs.6b08937

15 N Nuclear Magnetic Resonance Studies of Acid−Base Properties of Pyridoxal-5‘-Phosphate Aldimines in Aqueous Solution
journal, April 2007

  • Sharif, Shasad; Huot, Monique Chan; Tolstoy, Peter M.
  • The Journal of Physical Chemistry B, Vol. 111, Issue 15
  • DOI: 10.1021/jp067334g

Hyperconjugation in hydrocarbons: Not just a “mild sort of conjugation”
journal, April 2013


Glycine Enolates:  The Large Effect of Iminium Ion Formation on α-Amino Carbon Acidity
journal, August 2001

  • Rios, Ana; Crugeiras, Juan; Amyes, Tina L.
  • Journal of the American Chemical Society, Vol. 123, Issue 32
  • DOI: 10.1021/ja016250c

Theoretical studies of strong hydrogen bonds
journal, January 2006

  • Grabowski, S?awomir J.
  • Annual Reports Section "C" (Physical Chemistry), Vol. 102
  • DOI: 10.1039/b417200k

Protonation States of the Tryptophan Synthase Internal Aldimine Active Site from Solid-State NMR Spectroscopy: Direct Observation of the Protonated Schiff Base Linkage to Pyridoxal-5′-Phosphate
journal, September 2014

  • Caulkins, Bethany G.; Bastin, Baback; Yang, Chen
  • Journal of the American Chemical Society, Vol. 136, Issue 37
  • DOI: 10.1021/ja506267d

Low-barrier hydrogen bonds and enzymic catalysis
journal, June 1994


LAUEGEN , an X-windows-based program for the processing of Laue diffraction data
journal, April 1995


NBO 6.0 : Natural bond orbital analysis program
journal, March 2013

  • Glendening, Eric D.; Landis, Clark R.; Weinhold, Frank
  • Journal of Computational Chemistry, Vol. 34, Issue 16
  • DOI: 10.1002/jcc.23266

LSCALE – the new normalization, scaling and absorption correction program in the Daresbury Laue software suite
journal, June 1999

  • Arzt, Steffi; Campbell, John W.; Harding, Marjorie M.
  • Journal of Applied Crystallography, Vol. 32, Issue 3
  • DOI: 10.1107/S0021889898015350

NMR Studies of Protonation and Hydrogen Bond States of Internal Aldimines of Pyridoxal 5′-Phosphate Acid–Base in Alanine Racemase, Aspartate Aminotransferase, and Poly- l -lysine
journal, November 2013

  • Chan-Huot, Monique; Dos, Alexandra; Zander, Reinhard
  • Journal of the American Chemical Society, Vol. 135, Issue 48
  • DOI: 10.1021/ja408988z

Accurate Coulomb-fitting basis sets for H to Rn
journal, January 2006

  • Weigend, Florian
  • Physical Chemistry Chemical Physics, Vol. 8, Issue 9
  • DOI: 10.1039/b515623h

Reaction specificity in pyridoxal phosphate enzymes
journal, January 2005


Long-Range Electrostatics-Induced Two-Proton Transfer Captured by Neutron Crystallography in an Enzyme Catalytic Site
journal, March 2016

  • Gerlits, Oksana; Wymore, Troy; Das, Amit
  • Angewandte Chemie International Edition, Vol. 55, Issue 16
  • DOI: 10.1002/anie.201509989

Global indicators of X-ray data quality
journal, April 2001


Generalized X-ray and neutron crystallographic analysis: more accurate and complete structures for biological macromolecules
journal, May 2009

  • Adams, Paul D.; Mustyakimov, Marat; Afonine, Pavel V.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 65, Issue 6
  • DOI: 10.1107/S0907444909011548

Visualizing the Bohr effect in hemoglobin: neutron structure of equine cyanomethemoglobin in the R state and comparison with human deoxyhemoglobin in the T state
journal, June 2016

  • Dajnowicz, Steven; Seaver, Sean; Hanson, B. Leif
  • Acta Crystallographica Section D Structural Biology, Vol. 72, Issue 7
  • DOI: 10.1107/S2059798316009049

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