title: Molecular, kinetic and thermodynamic characterization of Mycobacterium tuberculosis orotate 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 novo pyrimidine 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. tuberculosis H37Rv strain as a homodimeric functional OPRT enzyme. The M. tuberculosis OPRT 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. tuberculosis OPRT. 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. tuberculosis OPRT.
authors.affiliation: Instituto Nacional de Ciência e Tecnologia em Tuberculose, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Ardala
authors.initials: A
authors.lastname: Breda
authors.forename: Leonardo Astolfi
authors.initials: LA
authors.lastname: Rosado
authors.forename: Daniel Macedo
authors.initials: DM
authors.lastname: Lorenzini
authors.forename: Luiz Augusto
authors.initials: LA
authors.lastname: Basso
authors.forename: Diógenes Santiago
authors.initials: DS
authors.lastname: Santos
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000493:pharmacokinetics; D003001:Cloning, Molecular; D057075:Enzyme Assays; D015870:Gene Expression; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000378:metabolism; D009962:Orotate Phosphoribosyltransferase / Q000235:genetics / Q000493:pharmacokinetics*; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D009962:Orotate Phosphoribosyltransferase