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In this study, interactions of the catalytically active binuclear form of glycerophosphodiesterase (GpdQ) with four chemically diverse substrates, i.e. NPP (a phosphomonoester), BNPP and GPE (both phosphodiesters), and paraoxon (a phosphotriester) have been investigated using all-atom molecular dynamics (MD) simulations. The roles of metal ions and key amino acid residues, coordination flexibility, and dynamic transformations in all enzyme–substrate complexes have been elucidated. The roles of important first and second coordination shell residues in substrate binding and coordination flexibility of the enzyme suggested by simulations are supported by experimental data. The chemical nature of the substrate is found to influence the mode of binding, electrostatic surface potential, metal–metal distance, and reorganization of the active site. The experimentally proposed association between the substrate binding and coordination flexibility is analyzed using principal component analysis (PCA), movements of loops, and root-mean-square-fluctuations (RMSF) as parameters. The PCA of these substrates provides different energy basins, i.e. one, three, two and five for NPP, BNPP, GPE, and paraoxon, respectively. Additionally, the area of an irregular hexagon (268.3, 288.9, 350.8, and 362.5 Å 2 ) formed by the residues on these loops illustrates their distinct motions. The substrate binding free energies of NPP, BNPP, and GPE are quite close (22.4–24.3 kcal mol −1 ), but paraoxon interacts with the smallest binding free energy (14.1 kcal mol −1 ). The metal binding energies in the presence of these substrates are substantially different, i.e. the lowest for NPP and the highest for paraoxon. These results thus provide deeper insight into the chemical promiscuity and coordination flexibility of this important enzyme.
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The structure of a Lactobacillus helveticus chlorogenic acid esterase and the dynamics of its insertion domain provide insights into substrate binding
Chlorogenic acid esterases (ChlEs) are a useful class of enzymes that hydrolyze chlorogenic acid (CGA) into caffeic and quinic acids. ChlEs can break down CGA in foods to improve their sensory properties and release caffeic acid in the digestive system to improve the absorption of bioactive compounds. This work presents the structure, molecular dynamics, and biochemical characterization of a ChlE fromLactobacillus helveticus(Lh). Molecular dynamics simulations suggest that substrate access to the active site ofLhChlE is modulated by two hairpin loops above the active site. Docking simulations and mutational analysis suggest that two residues within the loops, Gln145and Lys164, are important for CGA binding. Lys164provides a slight substrate preference for CGA, whereas Gln145is required for efficient turnover. This work is the first to examine the dynamics of a bacterial ChlE and provides insights on substrate binding preference and turnover in this type of enzyme.
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- Award ID(s):
- 1905399
- PAR ID:
- 10467569
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- FEBS Letters
- ISSN:
- 0014-5793
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1002/1873-3468.14731
