Revealing protonation states and tracking substrate in serine hydroxymethyltransferase with room-temperature X-ray and neutron crystallography

Victoria N. Drago; Claudia Campos; Mattea Hooper; Aliyah Collins; Oksana Gerlits; Kevin L. Weiss; Matthew P. Blakeley; Robert S. Phillips; Andrey Kovalevsky

doi:10.1038/s42004-023-00964-9

Revealing protonation states and tracking substrate in serine hydroxymethyltransferase with room-temperature X-ray and neutron crystallography

Victoria N. Drago, Claudia Campos, Mattea Hooper, Aliyah Collins, Oksana Gerlits, Kevin L. Weiss, Matthew P. Blakeley, Robert S. Phillips, Andrey Kovalevsky

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

2 Scopus citations

Abstract

Pyridoxal 5’-phosphate (PLP)-dependent enzymes utilize a vitamin B₆-derived cofactor to perform a myriad of chemical transformations on amino acids and other small molecules. Some PLP-dependent enzymes, such as serine hydroxymethyltransferase (SHMT), are promising drug targets for the design of small-molecule antimicrobials and anticancer therapeutics, while others have been used to synthesize pharmaceutical building blocks. Understanding PLP-dependent catalysis and the reaction specificity is crucial to advance structure-assisted drug design and enzyme engineering. Here we report the direct determination of the protonation states in the active site of Thermus thermophilus SHMT (TthSHMT) in the internal aldimine state using room-temperature joint X-ray/neutron crystallography. Conserved active site architecture of the model enzyme TthSHMT and of human mitochondrial SHMT (hSHMT2) were compared by obtaining a room-temperature X-ray structure of hSHMT2, suggesting identical protonation states in the human enzyme. The amino acid substrate serine pathway through the TthSHMT active site cavity was tracked, revealing the peripheral and cationic binding sites that correspond to the pre-Michaelis and pseudo-Michaelis complexes, respectively. At the peripheral binding site, the substrate is bound in the zwitterionic form. By analyzing the observed protonation states, Glu53, but not His residues, is proposed as the general base catalyst, orchestrating the retro-aldol transformation of L-serine into glycine.

Original language	English
Article number	162
Journal	Communications Chemistry
Volume	6
Issue number	1
DOIs	https://doi.org/10.1038/s42004-023-00964-9
State	Published - Dec 2023

Funding

This research at ORNL’s High Flux Isotope Reactor (IMAGINE beamline) and at ORNL’s Spallation Neutron Source (MaNDi beamline) was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. The Office of Biological and Environmental Research supported research at ORNL’s Center for Structural Molecular Biology (CSMB), a DOE Office of Science User Facility. ORNL is managed by UT-Battelle LLC for DOE’s Office of Science, the single largest supporter of basic research in the physical sciences in the United States. X-ray crystallographic data were in part collected at Argonne National Laboratory using Structural Biology Center (SBC) beamline ID19 at the Advanced Photon Source. Use of the Advanced Photon Source, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357. The authors thank the Institut Laue Langevin (beamline LADI-DALI) for awarded additional neutron beamtime. This research was supported by a grant from NIH-GMS (R01GM137008) to R.S.P. and A.K. This research at ORNL’s High Flux Isotope Reactor (IMAGINE beamline) and at ORNL’s Spallation Neutron Source (MaNDi beamline) was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. The Office of Biological and Environmental Research supported research at ORNL’s Center for Structural Molecular Biology (CSMB), a DOE Office of Science User Facility. ORNL is managed by UT-Battelle LLC for DOE’s Office of Science, the single largest supporter of basic research in the physical sciences in the United States. X-ray crystallographic data were in part collected at Argonne National Laboratory using Structural Biology Center (SBC) beamline ID19 at the Advanced Photon Source. Use of the Advanced Photon Source, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357. The authors thank the Institut Laue Langevin (beamline LADI-DALI) for awarded additional neutron beamtime. This research was supported by a grant from NIH-GMS (R01GM137008) to R.S.P. and A.K.

Funders	Funder number
NIH-GMS	R01GM137008
ORNL’s Center for Structural Molecular Biology
Scientific User Facilities Division
U.S. Department of Energy	DE-AC02-06CH11357
Office of Science
Basic Energy Sciences
Biological and Environmental Research
Argonne National Laboratory
Oak Ridge National Laboratory
Canadian Society for Molecular Biosciences
UT-Battelle
Institut Laue-Langevin

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title = "Revealing protonation states and tracking substrate in serine hydroxymethyltransferase with room-temperature X-ray and neutron crystallography",

abstract = "Pyridoxal 5{\textquoteright}-phosphate (PLP)-dependent enzymes utilize a vitamin B6-derived cofactor to perform a myriad of chemical transformations on amino acids and other small molecules. Some PLP-dependent enzymes, such as serine hydroxymethyltransferase (SHMT), are promising drug targets for the design of small-molecule antimicrobials and anticancer therapeutics, while others have been used to synthesize pharmaceutical building blocks. Understanding PLP-dependent catalysis and the reaction specificity is crucial to advance structure-assisted drug design and enzyme engineering. Here we report the direct determination of the protonation states in the active site of Thermus thermophilus SHMT (TthSHMT) in the internal aldimine state using room-temperature joint X-ray/neutron crystallography. Conserved active site architecture of the model enzyme TthSHMT and of human mitochondrial SHMT (hSHMT2) were compared by obtaining a room-temperature X-ray structure of hSHMT2, suggesting identical protonation states in the human enzyme. The amino acid substrate serine pathway through the TthSHMT active site cavity was tracked, revealing the peripheral and cationic binding sites that correspond to the pre-Michaelis and pseudo-Michaelis complexes, respectively. At the peripheral binding site, the substrate is bound in the zwitterionic form. By analyzing the observed protonation states, Glu53, but not His residues, is proposed as the general base catalyst, orchestrating the retro-aldol transformation of L-serine into glycine.",

author = "Drago, {Victoria N.} and Claudia Campos and Mattea Hooper and Aliyah Collins and Oksana Gerlits and Weiss, {Kevin L.} and Blakeley, {Matthew P.} and Phillips, {Robert S.} and Andrey Kovalevsky",

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AU - Drago, Victoria N.

AU - Campos, Claudia

AU - Hooper, Mattea

AU - Collins, Aliyah

AU - Gerlits, Oksana

AU - Weiss, Kevin L.

AU - Blakeley, Matthew P.

AU - Phillips, Robert S.

AU - Kovalevsky, Andrey

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Revealing protonation states and tracking substrate in serine hydroxymethyltransferase with room-temperature X-ray and neutron crystallography

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