Molecular, kinetic and thermodynamic characterization of Mycobacterium tuberculosis orotate phosphoribosyltransferase
Molecular, kinetic and thermodynamic characterization of Mycobacterium tuberculosis orotate phosphoribosyltransferase
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DOI：
10.1039/c1mb05402c
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发表时间：
2012-01-01
影响因子：
--
通讯作者：
Santos, Diogenes Santiago
中科院分区：
文献类型：
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作者：
Breda, Ardala;Rosado, Leonardo Astolfi;Santos, Diogenes Santiago
结核病（TB）是由结核分枝杆菌（Mycobacterium tuberculosis）引起的慢性传染病。耐药菌株在世界范围内的出现、免疫受损人群中感染患者数量的增加以及大量潜伏感染个体是该疾病的储存库，这些都强调了迫切需要新的策略来治疗结核病。核苷酸代谢途径为开发抗活动性结核病的新药提供了有希望的分子靶点，并且有希望对潜伏形式的病原体也有效。从头嘧啶合成途径的乳清酸磷酸核糖转移酶（OPRT）催化从5 '-磷酸-α-D-核糖1'-二磷酸（PRPP）到乳清酸（OA）的可逆磷酸核糖转移，形成焦磷酸和乳清酸苷5 '-单磷酸（OMP）。在这里，我们描述的克隆和pyrE编码的蛋白质的M。结核病H37 Rv菌株作为同源二聚体功能性OPRT酶。分枝结核杆菌OPRT正向反应的真实动力学常数和产物抑制结果表明单-异有序双-双动力学机制，这在以前没有描述过。没有检测到半反应和等温滴定量热法（ITC）的数据支持所提出的机制。ITC数据还提供了底物/产物与M之间非共价相互作用的热力学特征。结核病OPRT。这些数据为基于靶点的抗结核药物的合理设计提供了坚实的基础，并应告知我们如何更好地设计M的抑制剂。结核病OPRT。
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-alpha-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.