Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
Nucleic Acids Res. 2013 Jan 7;41(1):355-65. doi: 10.1093/nar/gks977. Epub 2012 Oct 30.
T4 polynucleotide kinase-phosphatase (Pnkp) exemplifies a family of enzymes with 5'-kinase and 3'-phosphatase activities that function in nucleic acid repair. The polynucleotide 3'-phosphatase reaction is executed by the Pnkp C-terminal domain, which belongs to the DxDxT acylphosphatase superfamily. The 3'-phosphatase reaction entails formation and hydrolysis of a covalent enzyme-(Asp165)-phosphate intermediate, driven by general acid-base catalyst Asp167. We report that Pnkp also has RNA 2'-phosphatase activity that requires Asp165 and Asp167. The physiological substrate for Pnkp phosphatase is an RNA 2',3'-cyclic phosphate end (RNA > p), but the pathway of cyclic phosphate removal and its enzymic requirements are undefined. Here we find that Pnkp reactivity with RNA > p requires Asp165, but not Asp167. Whereas wild-type Pnkp transforms RNA > p to RNA(OH), mutant D167N converts RNA > p to RNA 3'-phosphate, which it sequesters in the phosphatase active site. In support of the intermediacy of an RNA phosphomonoester, the reaction of mutant S211A with RNA > p results in transient accumulation of RNAp en route to RNA(OH). Our results suggest that healing of 2',3'-cyclic phosphate ends is a four-step processive reaction: RNA > p + Pnkp → RNA-(3'-phosphoaspartyl)-Pnkp → RNA(3')p + Pnkp → RNA(OH) + phosphoaspartyl-Pnkp → P(i) + Pnkp.
T4 多核苷酸激酶-磷酸酶 (Pnkp) 是一类具有 5'-激酶和 3'-磷酸酶活性的酶的典范，它们在核酸修复中发挥作用。多核苷酸 3'-磷酸酶反应由 Pnkp C 末端结构域执行，该结构域属于 DxDxT 酰基磷酸酶超家族。3'-磷酸酶反应需要形成和水解一个共价酶-(Asp165)-磷酸中间物，由广义酸碱催化剂 Asp167 驱动。我们报告 Pnkp 还具有 RNA 2'-磷酸酶活性，需要 Asp165 和 Asp167。Pnkp 磷酸酶的生理底物是 RNA 2',3'-环磷酸末端 (RNA > p)，但环磷酸去除途径及其酶学要求尚不清楚。在这里，我们发现 Pnkp 与 RNA > p 的反应需要 Asp165，但不需要 Asp167。野生型 Pnkp 将 RNA > p 转化为 RNA(OH)，而突变体 D167N 将 RNA > p 转化为 RNA 3'-磷酸，其将其隔离在磷酸酶活性位点中。为了支持 RNA 磷酸单酯的中间物的存在，突变体 S211A 与 RNA > p 的反应导致 RNAp 中间体在转化为 RNA(OH)的过程中暂时积累。我们的结果表明，2',3'-环磷酸末端的修复是一个四步连续反应过程：RNA > p + Pnkp → RNA-(3'-磷酸天冬酰胺酰)-Pnkp → RNA(3')p + Pnkp → RNA(OH) + 磷酸天冬酰胺酰-Pnkp → P(i) + Pnkp。
