Applied Biotechnology Group, Universidad Europea de Madrid, Urbanización El Bosque, Villaviciosa de Odón, 28670 Madrid, Spain.
Molecular Enzymology Group, University of Groningen, Feringa Building, 9747 AG Groningen, The Netherlands.
Biomolecules. 2024 Sep 23;14(9):1196. doi: 10.3390/biom14091196.
Nucleoside phosphorylases (NPs) are pivotal enzymes in the salvage pathway, catalyzing the reversible phosphorolysis of nucleosides to produce nucleobases and α-D-ribose 1-phosphate. Due to their efficiency in catalyzing nucleoside synthesis from purine or pyrimidine bases, these enzymes hold significant industrial importance in the production of nucleoside-based drugs. Given that the thermodynamic equilibrium for purine NPs (PNPs) is favorable for nucleoside synthesis-unlike pyrimidine NPs (PyNPs, UP, and TP)-multi-enzymatic systems combining PNPs with PyNPs, UPs, or TPs are commonly employed in the synthesis of nucleoside analogs. In this study, we report the first development of two engineered bifunctional fusion enzymes, created through the genetic fusion of purine nucleoside phosphorylase I (PNP I) and thymidine phosphorylase (TP) from . These fusion constructs, PNP I/TP-His and TP/PNP I-His, provide an innovative one-pot, single-step alternative to traditional multi-enzymatic synthesis approaches. Interestingly, both fusion enzymes retain phosphorolytic activity for both purine and pyrimidine nucleosides, demonstrating significant activity at elevated temperatures (60-90 °C) and within a pH range of 6-8. Additionally, both enzymes exhibit high thermal stability, maintaining approximately 80-100% of their activity when incubated at 60-80 °C over extended periods. Furthermore, the transglycosylation capabilities of the fusion enzymes were explored, demonstrating successful catalysis between purine (2'-deoxy)ribonucleosides and pyrimidine bases, and vice versa. To optimize reaction conditions, the effects of pH and temperature on transglycosylation activity were systematically examined. Finally, as a proof of concept, these fusion enzymes were successfully employed in the synthesis of various purine and pyrimidine ribonucleoside and 2'-deoxyribonucleoside analogs, underscoring their potential as versatile biocatalysts in nucleoside-based drug synthesis.
核苷磷酸化酶（NPs）是补救途径中的关键酶，可催化核苷的可逆磷酸解，产生碱基和α-D-核糖 1-磷酸。由于它们在催化嘌呤或嘧啶碱基的核苷合成方面的效率很高，这些酶在基于核苷的药物生产中具有重要的工业意义。鉴于嘌呤 NPs（PNPs）的热力学平衡有利于核苷合成-与嘧啶 NPs（PyNPs、UP 和 TP 不同）-因此，通常将多酶系统将 PNPs 与 PyNPs、UP 或 TP 结合在一起，用于核苷类似物的合成。在这项研究中，我们报告了通过遗传融合嘌呤核苷磷酸化酶 I（PNP I）和胸苷磷酸化酶（TP）来自. 首次开发两种工程化双功能融合酶的研究结果。这些融合构建体，PNP I/TP-His 和 TP/PNP I-His，为传统多酶合成方法提供了一种创新的一锅法、单步替代方案。有趣的是，两种融合酶均保留对嘌呤和嘧啶核苷的磷酸解活性，在 60-90°C 的高温和 6-8 的 pH 范围内表现出显著的活性。此外，两种酶都表现出很高的热稳定性，在 60-80°C 下孵育时，其活性保持在 80-100%左右。此外，还探索了融合酶的转糖基化能力，证明了嘌呤（2'-脱氧）核糖核苷和嘧啶碱基之间以及反之亦然的成功催化。为了优化反应条件，系统研究了 pH 和温度对转糖基化活性的影响。最后，作为概念验证，这些融合酶成功地用于各种嘌呤和嘧啶核糖核苷和 2'-脱氧核糖核苷类似物的合成，突出了它们作为基于核苷的药物合成中多功能生物催化剂的潜力。
