Direct visualization of critical hydrogen atoms in a pyridoxal 5′-phosphate enzyme

Steven Dajnowicz; Ryne C. Johnston; Jerry M. Parks; Matthew P. Blakeley; David A. Keen; Kevin L. Weiss; Oksana Gerlits; Andrey Kovalevsky; Timothy C. Mueser

doi:10.1038/s41467-017-01060-y

Direct visualization of critical hydrogen atoms in a pyridoxal 5′-phosphate enzyme

Steven Dajnowicz, Ryne C. Johnston, Jerry M. Parks, Matthew P. Blakeley, David A. Keen, Kevin L. Weiss, Oksana Gerlits, Andrey Kovalevsky, Timothy C. Mueser

Research output: Contribution to journal › Article › peer-review

49 Scopus citations

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. 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.

Original language	English
Article number	955
Journal	Nature Communications
Volume	8
Issue number	1
DOIs	https://doi.org/10.1038/s41467-017-01060-y
State	Published - Dec 1 2017

Funding

The Office of Biological and Environmental Research supported research at ORNL’s Center for Structural Molecular Biology (CSMB) using facilities supported by the Scientific User Facilities Division, Office of Basic Energy Sciences, US Department of Energy. This work used resources of the Compute and Data Environment for Science (CADES) at ORNL, which is managed by UT-Battelle, LLC for the U.S. Department of Energy under Contract No. DE-AC05-00OR22725. This research at ORNL’s High Flux Isotope Reactor (IMAGINE beamline) was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. The authors thank Institut Laue Langevin (beamline LADI-III) for neutron beam time. This research was supported in part by a grant from The Center for the Advancement of Science in Space, Inc. (CASIS NASA Award No. GA-2013-117, T.C.M Co-I and GA-2017-251, T.C.M. P.I.). We thank Prof. Michael Toney for insightful conversations during the manuscript preparation. S.D. was supported by CASIS, the University of Toledo, and the ORNL GO! program (Award No. N-125688-01, T.C.M. PI, A.K. ORNL PI).

Funders	Funder number
CADES
Data Environment for Science
Scientific User Facilities Division
U.S. Department of Energy	DE-AC05-00OR22725
Basic Energy Sciences	GA-2017-251, GA-2013-117
Biological and Environmental Research
Oak Ridge National Laboratory
Center for the Advancement of Science in Space
University of Toledo

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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. 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.",

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Direct visualization of critical hydrogen atoms in a pyridoxal 5′-phosphate enzyme

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