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A flap motif in human serine hydroxymethyltransferase is important for structural stabilization, ligand binding, and control of product release
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Metadata
Document Title
A flap motif in human serine hydroxymethyltransferase is important for structural stabilization, ligand binding, and control of product release
Author
Ubonprasert S.,Jaroensuk J.,Pornthanakasem W.,Kamonsutthipaijit N.,Wongpituk P.,Mee-Udorn P.,Rungrotmongkol T.,Ketchart O.,Chitnumsub P.,Leartsakulpanich U.,Chaiyen P.,Maenpuen S.
Name from Authors Collection
Scopus Author ID
57208316065
Affiliations
Department of Biochemistry, Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand; School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong, 21210, Thailand; Biomolecular Analysis and Application Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani, 12120, Thailand; National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency, Pathumthani, 12120, Thailand; Synchrotron Light Research Institute (Public Organization), Nakhon Ratchasima, 30000, Thailand; Center of Excellence in Computational Chemistry (CECC), Department of Chemistry, Chulalongkorn University, Bangkok, 10330, Thailand; Biocatalyst and Environmental Biotechnology Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand; Bioinformatics and Computational Biology Program, Graduate School, Chulalongkorn University, Bangkok, 10330, Thailand; Department of Biochemistry, Faculty of Science, Burapha University, 169 Long-Hard Bangsaen Rd., Chonburi, 20131, Thailand
Type
Article
Source Title
Journal of Biological Chemistry
ISSN
00219258
Year
2019
Volume
294
Issue
27
Page
10490-10502
Open Access
All Open Access, Hybrid Gold, Green
Publisher
American Society for Biochemistry and Molecular Biology Inc.
DOI
10.1074/jbc.RA119.007454
Abstract
Humancytosolic serine hydroxymethyltransferase (hcSHMT) is a promising target for anticancer chemotherapy and contains a flexible "flap motif" whose function is yet unknown. Here, using size-exclusion chromatography, analytical ultracentrifugation, small-angle X-ray scattering (SAXS), molecular dynamics (MD) simulations, and ligand-binding and enzyme-kinetic analyses, we studied the functional roles of the flap motif by comparing WT hcSHMT with a flap-deleted variant (hcSHMT/Δflap). We found that deletion of the flap results in a mixture of apo-dimers and holo-tetramers, whereas the WT was mostly in the tetrameric form. MD simulations indicated that the flap stabilizes structural compactness and thereby enhances oligomerization. The hcSHMT/Δflap variant exhibited different catalytic properties in (6S)-tetrahydrofolate (THF)-dependent reactions compared with theWTbut had similar activity in THF-independent aldol cleavage of β-hydroxyamino acid. hcSHMT/Δflap was less sensitive to THF inhibition than theWT(Ki of 0.65 and 0.27mM THF at pH 7.5, respectively), and the THF dissociation constant of the WT was also 3-fold lower than that of hcSHMT/Δflap, indicating that the flap is important for THF binding. hcSHMT/ Δflap did not display the burst kinetics observed in the WT. These results indicate that, upon removal of the flap, product release is no longer the rate-limiting step, implying that the flap is important for controlling product release. The findings reported here improve our understanding of the functional roles of the flap motif in hcSHMT and provide fundamental insight into how a flexible loop can be involved in controlling the enzymatic reactions of hcSHMT and other enzymes. © 2019 Ubonprasert et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
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License
CC BY or a CC BY-NC-ND
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Publication Source
Scopus