Department of Chemistry & Biochemistry, The University of Wisconsin- Milwaukee, Milwaukee, WI, 53217, USA.
Department of Chemistry & Biochemistry, The University of Wisconsin- Milwaukee, Milwaukee, WI, 53217, USA.
Anal Biochem. 2020 Apr 15;595:113622. doi: 10.1016/j.ab.2020.113622. Epub 2020 Feb 12.
Nudix proteins are members of a large family of homologous enzymes that hydrolyze nucleoside diphosphates linked to other compounds. The substrates for a subset of Nudix enzymes are all nucleotides linked to RNA, like the mG mRNA caps and the more recently discovered NAD(H) RNA caps. However, the RNA affinity and nucleic acid specificity of Nudix proteins has not yet been explored in depth. In this study we designed new fluorescence-based assays to examine the interaction of purified recombinant E. coli NudC and human Nudt1 (aka MTH1) Nudt3, Nudt12, Nudt16, and Nudt20 (aka Dcp2). All Nudix proteins except Nudt1 and Nudt12 bound both RNA and DNA stoichiometrically with high affinity (dissociation constants in the nanomolar range) and no clear sequence specificity. In stark contrast, Nudt12 binds RNA but not similar DNA oligonucleotides. Nudt12 also bound RNAs with 5' NAD caps more tightly than those with NADH or mG cap. NudC was similarly selective against mG caps but did not differentiate between NAD and NADH capped RNA. Nudt3, Nudt16, and Nudt20 bound mG capped RNA more tightly than RNA with NADH caps.
Nudix 蛋白是一大类同源酶的成员，这些酶能水解与其他化合物相连的核苷二磷酸。一组 Nudix 酶的底物都是与 RNA 相连的核苷酸，如 mG mRNA 帽和最近发现的 NAD(H)RNA 帽。然而，Nudix 蛋白对 RNA 的亲和力和核酸特异性尚未被深入研究。在这项研究中，我们设计了新的基于荧光的测定法来研究纯化的重组大肠杆菌 NudC 和人 Nudt1（又名 MTH1）、Nudt3、Nudt12、Nudt16 和 Nudt20（又名 Dcp2）之间的相互作用。除了 Nudt1 和 Nudt12 之外，所有的 Nudix 蛋白都以高亲和力（纳摩尔范围内的解离常数）和没有明显的序列特异性，与 RNA 和 DNA 均以化学计量比结合。与此形成鲜明对比的是，Nudt12 结合 RNA，但不结合类似的 DNA 寡核苷酸。Nudt12 也更紧密地结合带有 5'NAD 帽的 RNA，而不是带有 NADH 或 mG 帽的 RNA。NudC 对 mG 帽也有类似的选择性，但不能区分带有 NAD 和 NADH 帽的 RNA。Nudt3、Nudt16 和 Nudt20 与 mG 帽 RNA 的结合比与 NADH 帽 RNA 的结合更紧密。
