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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]
+ Show Author Affiliations
  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.
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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. https://doi.org/10.1038/s41467-017-01060-y
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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. https://doi.org/10.1038/s41467-017-01060-y. https://www.osti.gov/servlets/purl/1407771.
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@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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Journal Article:
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Publisher's Version of Record
https://doi.org/10.1038/s41467-017-01060-y
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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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