Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02903, USA.
Department of Chemistry, McGill University, Montreal, Quebec H3A 0B8, Canada.
RNA. 2022 Jul;28(7):927-936. doi: 10.1261/rna.079159.122. Epub 2022 Apr 22.
In eukaryotic cells, intron lariats produced by the spliceosome contain a 2'5' phosphodiester linkage. The RNA lariat debranching enzyme, Dbr1, is the only enzyme known to hydrolyze this bond. Dbr1 is a member of the metallophosphoesterase (MPE) family of enzymes, and recent X-ray crystal structures and biochemistry data demonstrate that Dbr1 from uses combinations of Mn, Zn, and Fe as enzymatic cofactors. Here, we examine the kinetic properties and metal dependence of the Dbr1 homolog from (yDbr1). Elemental analysis measured stoichiometric quantities of Fe and Zn in yDbr1 purified following heterologous expression We analyzed the ability of Fe, Zn, and Mn to reconstitute activity in metal-free apoenzyme. Purified yDbr1 was highly active, turning over substrate at 5.6 sec, and apo-yDbr1 reconstituted with Fe was the most active species, turning over at 9.2 sec We treated human lymphoblastoid cells with the iron-chelator deferoxamine and measured a twofold increase in cellular lariats. These data suggest that Fe is an important biological cofactor for Dbr1 enzymes.
在真核细胞中，由剪接体产生的内含子套索含有 2'-5' 磷酸二酯键。RNA 套索去分支酶 Dbr1 是已知唯一能够水解该键的酶。Dbr1 是金属磷酸酯酶（MPE）酶家族的成员，最近的 X 射线晶体结构和生物化学数据表明， 来自 的 Dbr1 使用 Mn、Zn 和 Fe 的组合作为酶辅因子。在这里，我们研究了 （yDbr1）的 Dbr1 同源物的动力学特性和金属依赖性。通过异源表达纯化后，元素分析测量了 yDbr1 中化学计量的 Fe 和 Zn 量。我们分析了 Fe、Zn 和 Mn 在无金属 apo 酶中重新构成活性的能力。纯化的 yDbr1 具有很高的活性，以 5.6 秒的速度转化底物，并且用 Fe 重组的 apo-yDbr1 是最活跃的物种，以 9.2 秒的速度转化。我们用铁螯合剂去铁胺处理人淋巴母细胞系，并测量细胞套索增加了两倍。这些数据表明 Fe 是 Dbr1 酶的重要生物辅因子。
