Streptomyces coelicolor Polynucleotide Phosphorylase Can Polymerize Nucleoside Diphosphates under Phosphorolysis Conditions, with Implications for the Degradation of Structured RNAs
Streptomyces coelicolor Polynucleotide Phosphorylase Can Polymerize Nucleoside Diphosphates under Phosphorolysis Conditions, with Implications for the Degradation of Structured RNAs
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DOI：
10.1128/jb.00936-13
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发表时间：
2013-11-01
影响因子：
3.2
通讯作者：
Mackie, George A.
中科院分区：
文献类型：
--
作者：
Jones, George H.;Mackie, George A.
我们研究了天蓝色链霉菌（Streptomycescoelicolor）的野生型多核苷酸磷酸化酶（PNIPs）及其两种突变体N459 D和C468 A在ADP聚合和S.天蓝色rpsO-pnp操纵子。野生型酶的聚合活性是N459 D的两倍，是C468 A的四倍。N459 D对结构化RNA底物的磷酸解的k（cat）/K-m值与野生型酶观察到的基本相同，而C468 A对该底物的活性为50%。所有四种常见的核苷二磷酸的混合物增加了由野生型酶的结构化底物的磷酸解的k（cat）/K-m的1.7倍，但不影响由N459 D或C468 A催化的磷酸解。我们在核苷二磷酸存在下进行结构化底物的磷酸解，并使用[P-32]pCp标记这些反应产物的3'末端。末端标记RNA的消化和产物在测序凝胶上的展示显示野生型S。coelicolorPN 10能在磷酸解条件下合成RNA 3'尾，而N459 D和C468 A突变体不能。野生型酶不向已经具有非结构化3'尾的底物添加3'尾。我们提出了一个模型，在该模型中，3'尾巴的瞬时合成促进了结构底物的磷酸解链霉菌PNDY。
We have examined the ability of wild-type polynucleotide phosphorylase (PNPase) from Streptomyces coelicolor and two mutant forms of the enzyme, N459D and C468A, to function in the polymerization of ADP and in the phosphorolysis of RNA substrates derived from the S. coelicolor rpsO-pnp operon. The wild-type enzyme was twice as active in polymerization as N459D and four times as active as C468A. The k(cat)/K-m value for phosphorolysis of a structured RNA substrate by N459D was essentially the same as that observed for the wild-type enzyme, while C468A was 50% as active with this substrate. A mixture of all four common nucleoside diphosphates increased the k(cat)/K-m for phosphorolysis of the structured substrate by the wild-type enzyme by a factor of 1.7 but did not affect phosphorolysis catalyzed by N459D or C468A. We conducted phosphorolysis of the structured substrate in the presence of nucleoside diphosphates and labeled the 3' ends of the products of those reactions using [P-32]pCp. Digestion of the end-labeled RNAs and display of the products on a sequencing gel revealed that wild-type S. coelicolor PNPase was able to synthesize RNA 3' tails under phosphorolysis conditions while the N459D and C468A mutants could not. The wild-type enzyme did not add 3' tails to a substrate that already possessed an unstructured 3' tail. We propose a model in which the transient synthesis of 3' tails facilitates the phosphorolysis of structured substrates by Streptomyces PNPase.