Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, Russia.
Int J Mol Sci. 2022 Oct 19;23(20):12540. doi: 10.3390/ijms232012540.
Development of efficient approaches for the production of medically important nucleosides is a highly relevant challenge for biotechnology. In particular, cascade synthesis of arabinosides would allow relatively easy production of various cytostatic and antiviral drugs. However, the biocatalyst necessary for this approach, ribokinase from (RK), has a very low activity towards D-arabinose, making the synthesis using the state-of-art native enzyme technologically unfeasible. Here, we report the results of our enzyme design project, dedicated to engineering a mutant form of RK with elevated activity towards arabinose. Analysis of the active site structure has allowed us to hypothesize the reasons behind the low RK activity towards arabinose and select feasible mutations. Enzyme assay and kinetic studies have shown that the A98G mutation has caused a large 15-fold increase in k and 1.5-fold decrease in K for arabinose phosphorylation. As a proof of concept, we have performed the cascade synthesis of 2-chloroadenine arabinoside utilizing the A98G mutant with 10-fold lower amount of enzyme compared to the wild type without any loss of synthesis efficiency. Our results are valuable both for the development of new technologies of synthesis of modified nucleosides and providing insight into the structural reasons behind RK substrate specificity.
开发高效的方法来生产具有医学重要性的核苷是生物技术的一个极具挑战性的课题。特别是，阿拉伯糖苷的级联合成将允许相对容易地生产各种细胞抑制剂和抗病毒药物。然而，这种方法所需的生物催化剂，即来自 (RK) 的核酮激酶，对 D-阿拉伯糖的活性非常低，使得使用最先进的天然酶技术合成在技术上不可行。在这里，我们报告了我们的酶设计项目的结果，该项目致力于设计一种突变形式的 RK，使其对阿拉伯糖具有更高的活性。对活性位点结构的分析使我们能够假设 RK 对阿拉伯糖活性低的原因，并选择可行的突变。酶测定和动力学研究表明，A98G 突变导致阿拉伯糖磷酸化的 k 值增加了 15 倍，而 K 值降低了 1.5 倍。作为概念验证，我们利用 A98G 突变体进行了 2-氯腺嘌呤阿拉伯糖苷的级联合成，与野生型相比，酶的用量降低了 10 倍，而合成效率没有任何损失。我们的结果对于开发新型修饰核苷的合成技术以及深入了解 RK 底物特异性的结构原因都具有重要价值。
