Laboratory of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Berlin, Germany.
FEBS J. 2013 Mar;280(6):1475-90. doi: 10.1111/febs.12143. Epub 2013 Feb 21.
Thermostable nucleoside phosphorylases are attractive biocatalysts for the synthesis of modified nucleosides. Hence we report on the recombinant expression of three 'high molecular mass' purine nucleoside phosphorylases (PNPs) derived from the thermophilic bacteria Deinococcus geothermalis, Geobacillus thermoglucosidasius and from the hyperthermophilic archaeon Aeropyrum pernix (5'-methythioadenosine phosphorylase; ApMTAP). Thermostability studies, kinetic analysis and substrate specificities are reported. The PNPs were stable at their optimal temperatures (DgPNP, 55 °C; GtPNP, 70 °C; ApMTAP, activity rising to 99 °C). Substrate properties were investigated for natural purine nucleosides [adenosine, inosine and their C2'-deoxy counterparts (activity within 50-500 U·mg(-1))], analogues with 2'-amino modified 2'-deoxy-adenosine and -inosine (within 0.1-3 U·mg(-1)) as well as 2'-deoxy-2'-fluoroadenosine (9) and its C2'-arabino diastereomer (10, within 0.01-0.03 U·mg(-1)). Our results reveal that the structure of the heterocyclic base (e.g. adenine or hypoxanthine) can play a critical role in the phosphorolysis reaction. The implications of this finding may be helpful for reaction mechanism studies or optimization of reaction conditions. Unexpectedly, the diastereomeric 2'-deoxyfluoro adenine ribo- and arabino-nucleosides displayed similar substrate properties. Moreover, cytidine and 2'-deoxycytidine were found to be moderate substrates of the prepared PNPs, with substrate activities in a range similar to those determined for 2'-deoxyfluoro adenine nucleosides 9 and 10. C2'-modified nucleosides are accepted as substrates by all recombinant enzymes studied, making these enzymes promising biocatalysts for the synthesis of modified nucleosides. Indeed, the prepared PNPs performed well in preliminary transglycosylation reactions resulting in the synthesis of 2'-deoxyfluoro adenine ribo- and arabino- nucleosides in moderate yield (24%).
耐热核苷磷酸化酶是合成修饰核苷的有吸引力的生物催化剂。因此，我们报告了三种来源于嗜热细菌 Deinococcus geothermalis、Geobacillus thermoglucosidasius 和超嗜热古菌 Aeropyrum pernix 的“高分子量”嘌呤核苷磷酸化酶（PNP）的重组表达（5'-甲硫腺苷磷酸化酶；ApMTAP）。报告了热稳定性研究、动力学分析和底物特异性。PNP 在其最适温度下稳定（DgPNP，55°C；GtPNP，70°C；ApMTAP，活性升高至 99°C）。研究了天然嘌呤核苷[腺苷、肌苷及其 C2'-脱氧对应物（活性在 50-500 U·mg(-1)）]、2'-氨基修饰的 2'-脱氧-腺苷和 -肌苷类似物（活性在 0.1-3 U·mg(-1)）以及 2'-脱氧-2'-氟腺苷（9）及其 C2'-阿拉伯糖差向异构体（10，活性在 0.01-0.03 U·mg(-1)）的底物特性。我们的结果表明，杂环碱基（例如腺嘌呤或次黄嘌呤）的结构在磷酸解反应中起着关键作用。这一发现的意义可能有助于反应机制研究或反应条件的优化。出乎意料的是，非对映异构的 2'-脱氧氟腺苷核糖和阿拉伯糖核苷显示出相似的底物特性。此外，发现胞苷和 2'-脱氧胞苷是制备的 PNP 的中等底物，其底物活性与确定的 2'-脱氧氟腺苷核苷 9 和 10 的活性相似。所有研究的重组酶都接受 C2'-修饰的核苷作为底物，这使得这些酶成为修饰核苷合成的有前途的生物催化剂。事实上，制备的 PNP 在初步的糖基转移反应中表现良好，导致 2'-脱氧氟腺苷核糖和阿拉伯糖核苷以中等产率（24%）合成。
