TY - JOUR
T1 - Elucidating the Catalytic Reaction Mechanism of Orotate Phosphoribosyltransferase by Means of X-ray Crystallography and Computational Simulations
AU - Roca, Maite
AU - Navas-Yuste, Sergio
AU - Zinovjev, Kirill
AU - López-Estepa, Miguel
AU - Gómez, Sara
AU - Fernández, Francisco J.
AU - Vega, M. Cristina
AU - Tuñón, Iñaki
PY - 2020/2/7
Y1 - 2020/2/7
N2 - 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.
AB - 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.
KW - enzyme catalysis
KW - MD simulations
KW - orotate phosphoribosyltransferase
KW - pyrimidine metabolism
KW - QM/MM methods
KW - reaction mechanism
KW - X-ray structure
UR - http://www.scopus.com/inward/record.url?scp=85078676759&partnerID=8YFLogxK
U2 - 10.1021/acscatal.9b05294
DO - 10.1021/acscatal.9b05294
M3 - Article (Academic Journal)
AN - SCOPUS:85078676759
SN - 2155-5435
VL - 10
SP - 1871
EP - 1885
JO - ACS Catalysis
JF - ACS Catalysis
M1 - 3
ER -