Polynucleotide phosphorylase (PNPase) catalyzes stepwise phosphorolysis of the 3′-terminal phosphodiesters of RNA chains to yield nucleoside diphosphate products. In the reverse reaction, PNPase acts as a polymerase, using NDPs as substrates to add NMPs to the 3′-OH terminus of RNA chains while expelling inorganic phosphate. The apparent essentiality of PNPase for growth of M. tuberculosis militates for mycobacterial PNPase as a potential drug target. A cryo-EM structure of Mycobacterium smegmatis PNPase (MsmPNPase) reveals a characteristic ring-shaped homotrimer in which each protomer consists of two RNase PH-like domains and an intervening α-helical module on the inferior surface of the ring. The carboxy-terminal KH and S1 domains, which impart RNA specificity to MsmPNPase, are on the opposite face of the core ring and are conformationally mobile. Single particle reconstructions of MsmPNPase in the act of poly(A) synthesis highlight a 3′-terminal (rA)₄ oligonucleotide and two magnesium ions in the active site and an adenine nucleobase in the central tunnel. We identify amino acids that engage the 3′ segment of the RNA chain (Phe68, Arg105, Arg112, Arg430, Arg431) and the two metal ions (Asp526, Asp532, Gln546, Asp548), and we infer those that bind inorganic phosphate (Thr470, Ser471, His435, Lys534). Alanine mutagenesis pinpointed RNA and phosphate contacts as essential (Arg105, Arg431, Lys534, Thr470 + Ser471), important (Arg112, Arg430), or unimportant (Phe68) for PNPase activity. Severe phosphorolysis and polymerase defects accompanying alanine mutations of the enzymic metal ligands suggest a two-metal mechanism of catalysis by MsmPNPase.

中文翻译：

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耻垢分枝杆菌多核苷酸磷酸化酶的结构和机制


多核苷酸磷酸化酶 (PNPase) 催化 RNA 链 3' 末端磷酸二酯的逐步磷酸解，产生核苷二磷酸产物。在逆反应中，PNPase充当聚合酶，使用NDP作为底物将NMP添加到RNA链的3'-OH末端，同时排出无机磷酸盐。 PNPase 对于结核分枝杆菌生长的明显重要性不利于分枝杆菌 PNPase 作为潜在的药物靶点。耻垢分枝杆菌PNPase (MsmPNPase) 的冷冻电镜结构揭示了一种特征性的环形同源三聚体，其中每个原聚体由两个 RNase PH 样结构域和环下表面上的介入 α 螺旋模块组成。羧基末端 KH 和 S1 结构域赋予 MsmPNPase RNA 特异性，位于核心环的相对面上，并且在构象上可移动。在 Poly(A) 合成过程中，MsmPNPase 的单粒子重建突出显示了活性位点中的 3' 末端 (rA) ₄寡核苷酸和两个镁离子以及中央通道中的腺嘌呤核碱基。我们鉴定了与 RNA 链 3' 片段（Phe68、Arg105、Arg112、Arg430、Arg431）和两种金属离子（Asp526、Asp532、Gln546、Asp548）结合的氨基酸，并推断出那些结合无机磷酸盐的氨基酸（Thr470） 、Ser471、His435、Lys534)。丙氨酸诱变确定 RNA 和磷酸盐接触对于 PNPase 活性是必需的（Arg105、Arg431、Lys534、Thr470 + Ser471）、重要（Arg112、Arg430）或不重要（Phe68）。伴随酶金属配体丙氨酸突变的严重磷酸解和聚合酶缺陷表明了 MsmPNPase 的双金属催化机制。