Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Sauletekio Av. 7, LT-10257 Vilnius, Lithuania.
Molecules. 2024 Aug 9;29(16):3767. doi: 10.3390/molecules29163767.
Nucleosides, nucleotides, and their analogues are an important class of molecules that are used as substrates in research of enzymes and nucleic acid, or as antiviral and antineoplastic agents. Nucleoside phosphorylation is usually achieved with chemical methods; however, enzymatic phosphorylation is a viable alternative. Here, we present a chemoenzymatic synthesis of modified cytidine monophosphates, where a chemical synthesis of novel -modified cytidines is followed by an enzymatic phosphorylation of the nucleosides by nucleoside kinases. To enlarge the substrate scope, multiple mutant variants of deoxynucleoside kinase (dNK) (EC:2.7.1.145) and deoxycytidine kinase (dCK) (EC:2.7.1.74) have been created and tested. It has been determined that certain point mutations in the active sites of the kinases alter their substrate specificities noticeably and allow phosphorylation of compounds that had been otherwise not phosphorylated by the wild-type dNK or dCK.
核苷、核苷酸及其类似物是一类重要的分子，可用作酶和核酸研究的底物，也可用作抗病毒和抗肿瘤药物。核苷的磷酸化通常采用化学方法，但酶磷酸化是一种可行的替代方法。在这里，我们展示了一种化学酶法合成修饰的胞苷单磷酸酯的方法，其中通过化学合成新型修饰的胞苷，然后由核苷激酶对核苷进行酶磷酸化。为了扩大底物范围，我们创建并测试了脱氧核苷激酶 (dNK) (EC:2.7.1.145) 和脱氧胞苷激酶 (dCK) (EC:2.7.1.74) 的多种突变变体。已经确定，激酶活性位点的某些点突变明显改变了它们的底物特异性，并允许对原本不能被野生型 dNK 或 dCK 磷酸化的化合物进行磷酸化。
