Elucidating the Catalytic Reaction Mechanism of Orotate Phosphoribosyltransferase by Means of X‑ray Crystallography and Computational Simulations
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https://doi.org/10.1021/acscatal.9b05294 |
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Títol
Elucidating the Catalytic Reaction Mechanism of Orotate Phosphoribosyltransferase by Means of X‑ray Crystallography and Computational SimulationsAutoria
Data de publicació
2020-01-02Editor
American Chemical SocietyISSN
2155-5435Cita bibliogràfica
Maite Roca, Sergio Navas-Yuste, Kirill Zinovjev, Miguel López-Estepa, Sara Gómez, Francisco J. Fernández, M. Cristina Vega, and Iñaki Tuñón. Elucidating the Catalytic Reaction Mechanism of Orotate Phosphoribosyltransferase by Means of X-ray Crystallography and Computational Simulations. ACS Catalysis 2020 10 (3), 1871-1885 doi: 10.1021/acscatal.9b05294Tipus de document
info:eu-repo/semantics/articleVersió de l'editorial
https://pubs.acs.org/doi/10.1021/acscatal.9b05294Versió
info:eu-repo/semantics/publishedVersionParaules clau / Matèries
Resum
Orotate phosphoribosyltransferase (OPRTase) catalyzes the reaction between the ribose donor α-d-5-phosphoribosyl-1-pyrophosphate (PRPP) and orotate (OA) in the presence of Mg2+ ion to obtain pyrophosphate and pyrimidine ... [+]
Orotate phosphoribosyltransferase (OPRTase) catalyzes the reaction between the ribose donor α-d-5-phosphoribosyl-1-pyrophosphate (PRPP) and orotate (OA) in the presence of Mg2+ ion to obtain pyrophosphate and pyrimidine nucleotide orotidine 5′-monophosphate (OMP), a key precursor in de novo biosynthesis of pyrimidine nucleotides. In this work, several structures of the dimeric Escherichia coli OPRTase (EcOPRTase) have been determined at high resolution, and kinetic measurements have been carried out to obtain the catalytic rate and Michaelis constants. Molecular dynamics (MD) simulations have been carried out, and structural analysis from the X-ray and MD simulation structures reveals conformational changes related to the flexible catalytic loop that establishes hydrogen bond interactions with the pyrophosphoryl group of PRPP. It is proposed that the OA substrate can be in equilibrium in its tautomeric forms. Starting from the most stable tautomeric form, all the plausible mechanisms have been explored by means of quantum mechanics/molecular mechanics (QM/MM) MD simulations using the adaptive string method. The most feasible mechanism consists of the proton transfer from the N1 atom of OA to a water molecule and from the water molecule to the α-phosphate O2A atom of PRPP. After that, the nucleophilic attack of the N1 atom of OA to the C1 atom of PRPP proceeds to yield OMP and pyrophosphate. The free energy barrier obtained is in very good agreement with the experimental data reported. Analysis of some relevant distances between key residues and the substrates (OA and PRPP) at the reactant state and transition state (TS) of the rate-limiting step allows us to understand the role of some conserved residues (Lys73, Asp125, Lys103*, Arg99*, and Mg2+ ion) electrostatically stabilizing the TS and preserving the flexible catalytic loop in a closed conformation during the enzymatic reaction. [-]
Publicat a
ACS Catalysis, 2020, vol. 10 no 3Proyecto de investigación
Spanish Ministerio de Economia y Competitividad (2015); Ministerio de Ciencia, Innovacion y Universidades (2018); European Union (EU): PGC2018-094852-B-C22, CTQ201S-66206-C2-2-R, RTI2018-1018-102242-B-I00; Generalitat Valenciana: AICO/2018/238; Universitat Jaume I: UJI-B2016-28; Consejo Superior de Investigaciones Cientificas (CSIC): 2016E064; Spanish Government: RYC-2014-16592; official Ph.D. program in Biochemistry, Molecular Biology and Biomedicine of the Universidad Complutense de Madrid (UCM)Drets d'accés
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