Kim Ahram, Morato Nicolás M, Saha Prabir, Eyimegwu Pascal N, Niyaz Aqeel A, Huang Rui, Cooks R Graham, Phelan Ryan M, Lewis Jared C
Department of Chemistry, Indiana University, Bloomington, Indiana, 47405, USA.
Department of Chemistry, Bindley Bioscience Center, and Purdue Institute for Cancer Research, Purdue University, West Lafayette, Indiana, 47907, USA.
Angew Chem Int Ed Engl. 2025 Jun 17:e202503538. doi: 10.1002/anie.202503538.
Phosphorylation plays important roles in biology by modulating the structure, reactivity, and biological function of a broad range of molecules. Biocatalytic phosphorylation has attracted attention from synthetic chemists due to its selectivity and mild reaction conditions using ATP as a phosphate donor. Given the potential synthetic utility of kinases with activity on small molecule substrates, we explored the activity of PsiK, the enzyme responsible for selective 4-O-phosphorylation of 4-hydroxytryptamine or psilocin in psylocybin biosynthesis by Psilocybe cubensis. We find that PsiK has good activity on a range of substituted phenols and benzenediols beyond its native substrate, enabling preparative phosphorylation of different substrates, and substantially expands the substrate scope of biocatalytic phosphorylation. We also show that active site mutations can further expand substrate scope and improve site-selectivity. This engineering effort was greatly expedited using DESI-MS screening, which enabled analysis of 2688 reactions in only 40 min. Finally, gram-scale phosphorylation of a representative substrate was achieved with a turnover number over 10 000. Together, these results highlight the biocatalytic utility of PsiK and derivatives thereof for selective phosphorylation of phenols and benzenediols under mild conditions.
磷酸化通过调节多种分子的结构、反应活性和生物学功能在生物学中发挥重要作用。生物催化磷酸化因其选择性以及使用ATP作为磷酸盐供体的温和反应条件而受到合成化学家的关注。鉴于激酶对小分子底物具有活性的潜在合成效用，我们探索了PsiK的活性，PsiK是负责在古巴裸盖菇的裸盖菇素生物合成中对4-羟色胺或脱磷酸裸盖菇素进行选择性4-O-磷酸化的酶。我们发现PsiK对一系列超出其天然底物的取代酚和苯二酚具有良好的活性，能够对不同底物进行制备性磷酸化，并大大扩展了生物催化磷酸化的底物范围。我们还表明，活性位点突变可以进一步扩大底物范围并提高位点选择性。使用DESI-MS筛选极大地加快了这项工程工作，该筛选仅需40分钟就能分析2688个反应。最后，实现了代表性底物的克级磷酸化，周转数超过10000。总之，这些结果突出了PsiK及其衍生物在温和条件下对酚类和苯二酚进行选择性磷酸化的生物催化效用。
