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Elucidating the catalytic reaction mechanism of orotate phosphoribosyltransferase by means of X-ray crystallography and computational simulations
- Source :
- Digital.CSIC. Repositorio Institucional del CSIC, instname
- Publication Year :
- 2020
- Publisher :
- American Chemical Society, 2020.
-
Abstract
- 15 p.-8 fig.-2 tab.1 graph. abst.+ 8 fig. supl.-1 tab. supl.<br />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.<br />This work was supported by the Spanish Ministerio de Economı́a y Competitividad (2015) and Ministerio de Ciencia,Innovación y Universidades (2018) through grants cofinanced with European Union ERDF funds PGC2018-094852-B-C22 to IT and CTQ2015-66206-C2-2-R and RTI2018-1018-102242-B-I00 to M.C.V., by Generalitat Valenciana AICO/2018/238 project and by Universitat Jaume I UJI-B2016-28project to IT and M.R., respectively, and CSIC intramuralgrant 2016E064 to M.C.V. M.R. thanks Ministerio deEconomía y Competitividad for a“Ramón y Cajal”contract(RYC-2014-16592)
- Subjects :
- Pyrimidine
Stereochemistry
010402 general chemistry
01 natural sciences
Catalysis
Enzyme catalysis
chemistry.chemical_compound
Orotidine
Nucleotide
Reaction mechanism
chemistry.chemical_classification
MD simulations
010405 organic chemistry
Hydrogen bond
General Chemistry
Tautomer
0104 chemical sciences
Pyrimidine metabolism
Orotate phosphoribosyltransferase
chemistry
QM/MM methods
X-ray structure
Subjects
Details
- Database :
- OpenAIRE
- Journal :
- Digital.CSIC. Repositorio Institucional del CSIC, instname
- Accession number :
- edsair.doi.dedup.....4b05ead028a457f0d7840c55a8f4d872