Molecular, kinetic and thermodynamic characterization of Mycobacterium tuberculosisorotate phosphoribosyltransferase

Abstract

Tuberculosis (TB) is a chronic infectious disease caused mainly by Mycobacterium tuberculosis. The worldwide emergence of drug-resistant strains, the increasing number of infected patients among immune compromised populations, and the large number of latent infected individuals that are reservoir to the disease have underscored the urgent need of new strategies to treat TB. The nucleotide metabolism pathways provide promising molecular targets for the development of novel drugs against active TB and may, hopefully, also be effective against latent forms of the pathogen. The orotate phosphoribosyltransferase (OPRT) enzyme of the de novopyrimidine synthesis pathway catalyzes the reversible phosphoribosyl transfer from 5′-phospho-α-D-ribose 1′-diphosphate (PRPP) to orotic acid (OA), forming pyrophosphate and orotidine 5′-monophosphate (OMP). Here we describe cloning and characterization of pyrE-encoded protein of M. tuberculosisH37Rv strain as a homodimeric functional OPRT enzyme. The M. tuberculosisOPRT true kinetic constants for forward reaction and product inhibition results suggest a Mono-Iso Ordered Bi–Bi kinetic mechanism, which has not been previously described for this enzyme family. Absence of detection of half reaction and isothermal titration calorimetry (ITC) data support the proposed mechanism. ITC data also provided thermodynamic signatures of non-covalent interactions between substrate/product and M. tuberculosisOPRT. These data provide a solid foundation on which to base target-based rational design of anti-TB agents and should inform us how to better design inhibitors of M. tuberculosisOPRT.