Elucidating the Catalytic Reaction Mechanism of Orotate Phosphoribosyltransferase by means of X-ray Crystallography and Computational Simulations

Orotate phosphoribosyltransferase (OPRTase) catalyzes the reaction between the ribose donor -D-5-phosphoribosyl-1-pyrophosphate (PRPP) and orotate (OA) in 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 orotate phosphoribosyltransferase (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 h...