Identification of Rv0535 as methylthioadenosine phosphorylase from Mycobacterium tuberculosis.
Identification of Rv0535 as methylthioadenosine phosphorylase from Mycobacterium tuberculosis.
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DOI：
10.1016/j.tube.2011.11.010
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发表时间：
2012-03
期刊：
影响因子：
--
通讯作者：
Parker WB
中科院分区：
文献类型：
--
作者：
Buckoreelall K;Sun Y;Hobrath JV;Wilson L;Parker WB
5'-甲硫腺苷 (MTA) 是一种天然嘌呤，在真核生物和古细菌中由甲硫腺苷磷酸化酶 (MTAP, E.C 2.4.2.28) 代谢，但在细菌中通常不代谢。在这项工作中，Rv0535（已被注释为结核分枝杆菌中可能的 MTAP）在大肠杆菌 BL21 (DE3) 中表达并纯化。纯化的蛋白质显示出磷酸化酶的特性，MTA 是优选的底物。腺苷和S-腺苷-L-高半胱氨酸是不良底物，并且未检测到5'-甲硫基肌苷、其他天然嘌呤或天然嘧啶的活性。结核分枝杆菌MTAP的动力学分析表明MTA的Km值为9.1 μM。 Rv0535 在变性 SDS-PAGE 凝胶上估计为 30 kDa 蛋白质，这与其基因序列预测的分子量一致。使用凝胶过滤色谱法，确定酶的天然分子量为 60 ± 4 kDa，因此表明结核分枝杆菌 MTAP 是二聚体。通过基于人类酶晶体的同源模型，鉴定了分枝杆菌和人类 MTAP 活性位点的差异。完整的结构活性关系分析可以识别两种酶之间底物特异性的差异，以帮助开发基于嘌呤的抗结核药物。
5′-methylthioadenosine (MTA) is a natural purine that is metabolized by methylthioadenosine phosphorylase (MTAP, E.C 2.4.2.28) in Eukarya and Archaea but generally not in bacteria. In this work, Rv0535, which has been annotated as a probable MTAP in M. tuberculosis, was expressed in and purified from E. coli BL21 (DE3). The purified protein displayed properties of a phosphorylase and MTA was the preferred substrate. Adenosine and S-adenosyl-L-homocysteine were poor substrates and no activity was detected with 5′-methylthioinosine, the other natural purines or the natural pyrimidines. Kinetic analysis of M. tuberculosis MTAP showed that the Km value for MTA was 9.1 μM. Rv0535 was estimated as a 30 kDa protein on a denaturing SDS-PAGE gel, which agreed with the molecular mass predicted by its gene sequence. Using gel filtration chromatography, the native molecular mass of the enzyme was determined to be 60 ± 4 kDa, and thus indicates that M. tuberculosis MTAP is a dimer. Differences in active site between mycobacterial and human MTAPs were identified by homology modeling based on the crystal of the human enzyme. A complete structure activity relationship analysis could identify differences in substrate specificity between the two enzymes to aid in the development of purine-based, anti-tuberculosis drugs.
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影响因子：
2.9
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通讯作者：
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