Human Deoxycytidine Kinase Is a Valuable Biocatalyst for the Synthesis of Nucleotide Analogues
Human Deoxycytidine Kinase Is a Valuable Biocatalyst for the Synthesis of Nucleotide Analogues
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DOI：
10.3390/catal9120997
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发表时间：
2019-12-01
期刊：
影响因子：
3.9
通讯作者：
Wagner, Anke
中科院分区：
文献类型：
--
作者：
Hellendahl, Katja E.;Kamel, Sarah;Wagner, Anke
天然核糖核苷-5 '-单磷酸是核酸的结构单元，用于许多目的，包括食品添加剂。此外，它们的类似物还用于制药应用。例如，氟达拉滨-5 '-单磷酸盐在治疗血液恶性肿瘤中是有效的。迄今为止，核糖核苷-5 '-单磷酸主要通过化学合成来生产，但这种方法的固有缺点导致了酶促合成途径的发展。在这项研究中，我们评估了人脱氧胞苷激酶（HsdCK）作为合适的生物催化剂的潜力，从相应的核苷合成天然和修饰的核糖核苷-5 '-单磷酸。人dCK在E.大肠杆菌，并固定在镍-氮基三乙酸（Ni-NTA）superflow。可溶性和固定化生物催化剂的底物光谱的筛选显示，HsdCK接受广泛的天然和修饰的核苷，除了胸苷和尿苷衍生物。在优化反应条件后，使用增加的底物浓度，将HsdCK用于氟达拉滨-5 '-单磷酸的合成。虽然可溶性生物催化剂揭示了最高的产物形成与最低的底物浓度为0.3 mM，在固定化HsdCK的存在下，产物产率随着底物浓度的增加而增加。因此，固定化HsdCK的应用在使用与工业应用相关的高底物浓度时是有利的。
Natural ribonucleoside-5'-monophosphates are building blocks for nucleic acids which are used for a number of purposes, including food additives. Their analogues, additionally, are used in pharmaceutical applications. Fludarabine-5'-monophosphate, for example, is effective in treating hematological malignancies. To date, ribonucleoside-5'-monophosphates are mainly produced by chemical synthesis, but the inherent drawbacks of this approach have led to the development of enzymatic synthesis routes. In this study, we evaluated the potential of human deoxycytidine kinase (HsdCK) as suitable biocatalyst for the synthesis of natural and modified ribonucleoside-5'-monophosphates from their corresponding nucleosides. Human dCK was heterologously expressed in E. coli and immobilized onto Nickel-nitrilotriacetic acid (Ni-NTA) superflow. A screening of the substrate spectrum of soluble and immobilized biocatalyst revealed that HsdCK accepts a wide range of natural and modified nucleosides, except for thymidine and uridine derivatives. Upon optimization of the reaction conditions, HsdCK was used for the synthesis of fludarabine-5'-monophosphate using increasing substrate concentrations. While the soluble biocatalyst revealed highest product formation with the lowest substrate concentration of 0.3 mM, the product yield increased with increasing substrate concentrations in the presence of the immobilized HsdCK. Hence, the application of immobilized HsdCK is advantageous upon using high substrate concentration which is relevant in industrial applications.