Center for Computational and Integrative Biology, Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA.
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Science. 2023 Oct 27;382(6669):423-429. doi: 10.1126/science.adh5339. Epub 2023 Oct 26.
A DNA polymerase with a single mutation and a divalent calcium cofactor catalyzes the synthesis of unnatural N3'→P5' phosphoramidate (NP) bonds to form NP-DNA. However, this template-directed phosphoryl transfer activity remains orders of magnitude slower than native phosphodiester synthesis. Here, we used time-resolved x-ray crystallography to show that NP-DNA synthesis proceeds with a single detectable calcium ion in the active site. Using insights from isotopic and elemental effects, we propose that one-metal-ion electrophilic substrate activation is inferior to the native two-metal-ion mechanism. We found that this deficiency in divalent activation could be ameliorated by trivalent rare earth and post-transition metal cations, substantially enhancing NP-DNA synthesis. Scandium(III), in particular, confers highly specific NP activity with kinetics enhanced by more than 100-fold over calcium(II), yielding NP-DNA strands up to 100 nucleotides in length.
一种具有单个突变和二价钙离子辅助因子的 DNA 聚合酶可以催化非天然的 N3'→P5' 膦酸酰胺（NP）键的合成，从而形成 NP-DNA。然而，这种模板指导的磷酸转移活性仍然比天然的磷酸二酯合成慢几个数量级。在这里，我们使用时间分辨 X 射线晶体学表明，NP-DNA 的合成是在活性位点中单个可检测的钙离子进行的。利用同位素和元素效应的见解，我们提出单金属离子亲电底物活化不如天然的双金属离子机制有效。我们发现，这种二价离子活化的缺陷可以通过三价稀土和后过渡金属阳离子得到改善，从而大大提高 NP-DNA 的合成。特别是钪(III)，赋予了高度特异性的 NP 活性，其动力学比钙(II)增强了 100 多倍，生成了长达 100 个核苷酸的 NP-DNA 链。
