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A flap motif in human serine hydroxymethyltransferase is important for structural stabilization, ligand binding, and control of product release.
J Biol Chem. 2019 07 05; 294(27):10490-10502.JB

Abstract

Human cytosolic 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 the WT but had similar activity in THF-independent aldol cleavage of β-hydroxyamino acid. hcSHMT/Δflap was less sensitive to THF inhibition than the WT (Ki of 0.65 and 0.27 mm 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.

Authors+Show Affiliations

From the Department of Biochemistry and 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), and.Synchrotron Light Research Institute (Public Organization), Nakhon Ratchasima 30000, Thailand.Center of Excellence in Computational Chemistry (CECC), Department of Chemistry, and.Bioinformatics and Computational Biology Program, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand, and.Bioinformatics and Computational Biology Program, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand, and. Biocatalyst and Environmental Biotechnology Research Unit, Department of Biochemistry, Faculty of Science, and.National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency, Pathumthani 12120, Thailand.Biomolecular Analysis and Application Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), and.Biomolecular Analysis and Application Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), and.From the Department of Biochemistry and 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.Department of Biochemistry, Faculty of Science, Burapha University, Chonburi 20131, Thailand somchart@go.buu.ac.th.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

31118236

Citation

Ubonprasert, Sakunrat, et al. "A Flap Motif in Human Serine Hydroxymethyltransferase Is Important for Structural Stabilization, Ligand Binding, and Control of Product Release." The Journal of Biological Chemistry, vol. 294, no. 27, 2019, pp. 10490-10502.
Ubonprasert S, Jaroensuk J, Pornthanakasem W, et al. A flap motif in human serine hydroxymethyltransferase is important for structural stabilization, ligand binding, and control of product release. J Biol Chem. 2019;294(27):10490-10502.
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. (2019). A flap motif in human serine hydroxymethyltransferase is important for structural stabilization, ligand binding, and control of product release. The Journal of Biological Chemistry, 294(27), 10490-10502. https://doi.org/10.1074/jbc.RA119.007454
Ubonprasert S, et al. A Flap Motif in Human Serine Hydroxymethyltransferase Is Important for Structural Stabilization, Ligand Binding, and Control of Product Release. J Biol Chem. 2019 07 5;294(27):10490-10502. PubMed PMID: 31118236.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A flap motif in human serine hydroxymethyltransferase is important for structural stabilization, ligand binding, and control of product release. AU - Ubonprasert,Sakunrat, AU - Jaroensuk,Juthamas, AU - Pornthanakasem,Wichai, AU - Kamonsutthipaijit,Nuntaporn, AU - Wongpituk,Peerapong, AU - Mee-Udorn,Pitchayathida, AU - Rungrotmongkol,Thanyada, AU - Ketchart,Onuma, AU - Chitnumsub,Penchit, AU - Leartsakulpanich,Ubolsree, AU - Chaiyen,Pimchai, AU - Maenpuen,Somchart, Y1 - 2019/05/22/ PY - 2019/04/20/received PY - 2019/05/21/revised PY - 2020/07/05/pmc-release PY - 2019/5/24/pubmed PY - 2020/3/11/medline PY - 2019/5/24/entrez KW - PLP-dependent enzymes KW - analytical ultracentrifugation KW - dTMP synthesis cycle KW - enzyme kinetics KW - flap motif/flexible loop KW - folate KW - molecular dynamics KW - nucleoside/nucleotide biosynthesis KW - oligomerization KW - protein structure KW - pyridoxal phosphate KW - serine hydroxymethyl transferase KW - small-angle X-ray scattering (SAXS) KW - structure-function KW - tetrahydrofolate-dependent reactions SP - 10490 EP - 10502 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 294 IS - 27 N2 - Human cytosolic 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 the WT but had similar activity in THF-independent aldol cleavage of β-hydroxyamino acid. hcSHMT/Δflap was less sensitive to THF inhibition than the WT (Ki of 0.65 and 0.27 mm 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. SN - 1083-351X UR - https://www.unboundmedicine.com/medline/citation/31118236/A_flap_motif_in_human_serine_hydroxymethyltransferase_is_important_for_structural_stabilization_ligand_binding_and_control_of_product_release_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=31118236 DB - PRIME DP - Unbound Medicine ER -