Dipartimento di Genetica e Microbiologia, Università degli Studi di Pavia, Pavia, Italy.
PLoS One. 2010 Nov 15;5(11):e15494. doi: 10.1371/journal.pone.0015494.
The selection and soaring spread of Mycobacterium tuberculosis multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) is a severe public health problem. Currently, there is an urgent need for new drugs for tuberculosis treatment, with novel mechanisms of action and, moreover, the necessity to identify new drug targets. Mycobacterial phosphoribosylpyrophosphate synthetase (MtbPRPPase) is a crucial enzyme involved in the biosynthesis of decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Moreover, phosphoribosylpyrophosphate, which is the product of the PRPPase catalyzed reaction, is the precursor for the biosynthesis of nucleotides and of some amino acids such as histidine and tryptophan. In this context, the elucidation of the molecular and functional features of MtbPRPPase is mandatory. MtbPRPPase was obtained as a recombinant form, purified to homogeneity and characterized. According to its hexameric form, substrate specificity and requirement of phosphate for activity, the enzyme proved to belong to the class I of PRPPases. Although the sulfate mimicked the phosphate, it was less effective and required higher concentrations for the enzyme activation. MtbPRPPase showed hyperbolic response to ribose 5-phosphate, but sigmoidal behaviour towards Mg-ATP. The enzyme resulted to be allosterically activated by Mg(2+) or Mn(2+) and inhibited by Ca(2+) and Cu(2+) but, differently from other characterized PRPPases, it showed a better affinity for the Mn(2+) and Cu(2+) ions, indicating a different cation binding site geometry. Moreover, the enzyme from M. tuberculosis was allosterically inhibited by ADP, but less sensitive to inhibition by GDP. The characterization of M. tuberculosis PRPPase provides the starting point for the development of inhibitors for antitubercular drug design.
结核分枝杆菌耐多药（MDR-TB）和广泛耐药（XDR-TB）菌株的选择和快速传播是一个严重的公共卫生问题。目前，迫切需要新的结核病治疗药物，这些药物具有新的作用机制，而且有必要确定新的药物靶点。分枝杆菌磷酸核糖焦磷酸合酶（MtbPRPPase）是参与脱磷酸烯醇式丙酮酸阿拉伯糖合成的关键酶，脱磷酸烯醇式丙酮酸阿拉伯糖是分枝杆菌细胞壁生物合成的必需前体。此外，PRPPase 催化反应的产物磷酸核糖焦磷酸是核苷酸和一些氨基酸（如组氨酸和色氨酸）生物合成的前体。在这种情况下，阐明 MtbPRPPase 的分子和功能特征是强制性的。MtbPRPPase 以重组形式获得，纯化至均一性并进行了表征。根据其六聚体形式、底物特异性和对活性的磷酸盐要求，该酶属于 PRPPase 类 I。尽管硫酸盐模拟了磷酸盐，但它的效果较差，并且需要更高的浓度才能激活酶。MtbPRPPase 对核糖 5-磷酸呈双曲线响应，但对 Mg-ATP 呈 S 形行为。该酶被 Mg 2+或 Mn 2+别构激活，并被 Ca 2+和 Cu 2+抑制，但与其他已鉴定的 PRPPase 不同，它对 Mn 2+和 Cu 2+离子具有更好的亲和力，表明其阳离子结合位点的几何形状不同。此外，来自结核分枝杆菌的酶被 ADP 别构抑制，但对 GDP 的抑制不太敏感。结核分枝杆菌 PRPPase 的特性为抗结核药物设计提供了抑制剂开发的起点。
