Thermus thermophilus Nucleoside Phosphorylases Active in the Synthesis of Nucleoside Analogues
Thermus thermophilus Nucleoside Phosphorylases Active in the Synthesis of Nucleoside Analogues
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DOI：
10.1128/aem.07605-11
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发表时间：
2012-05-01
影响因子：
4.4
通讯作者：
Sinisterra, Jose-Vicente
中科院分区：
文献类型：
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作者：
Almendros, Marcos;Berenguer, Jose;Sinisterra, Jose-Vicente
嗜热栖热菌 HB27 的细胞提取物对嘌呤和嘧啶核苷表达磷酸解活性。克隆并在大肠杆菌中表达了五个假定的编码基因，并纯化和研究了相应的重组蛋白。其中两种在体外显示出针对嘌呤核苷的磷酸解活性，第三种在体外显示出针对嘧啶核苷的磷酸解活性，这三种分别被命名为TtPNPI、TtPNPII和TtPyNP。三种酶活性的最适温度超出了水沸点，无法准确测量，而它们都表现出较宽的最适 pH 值，范围为 5.0 至 7.0。分析超速离心实验表明，TtPNPI 是同源六聚体，TtPNPII 是单体，TtPyNP 是同源二聚体。测定了每种酶的天然底物的磷酸解的动力学常数。核苷类似物的反应测试揭示了核苷中识别的关键位置。检测到与合成核碱基类似物（如5-碘尿嘧啶或2,6-二氨基嘌呤）和阿拉伯糖苷的活性，支持这些酶可用于合成具有药理活性的新核苷类似物。
Cells extracts from Thermus thermophilus HB27 express phosphorolytic activities on purines and pyrimidine nucleosides. Five putative encoding genes were cloned and expressed in Escherichia coli, and the corresponding recombinant proteins were purified and studied. Two of these showed phosphorolytic activities against purine nucleosides, and third one showed phosphorolytic activity against pyrimidine nucleosides in vitro, and the three were named TtPNPI, TtPNPII, and TtPyNP, respectively. The optimal temperature for the activity of the three enzymes was beyond the water boiling point and could not be measured accurately, whereas all of them exhibited a wide plateau of optimal pHs that ranged from 5.0 to 7.0. Analytical ultracentrifugation experiments revealed that TtPNPI was a homohexamer, TtPNPII was a monomer, and TtPyNP was a homodimer. Kinetic constants were determined for the phosphorolysis of the natural substrates of each enzyme. Reaction tests with nucleoside analogues revealed critical positions in the nucleoside for its recognition. Activities with synthetic nucleobase analogues, such as 5-iodouracil or 2,6-diaminopurine, and arabinosides were detected, supporting that these enzymes could be applied for the synthesis of new nucleoside analogs with pharmacological activities.