RtcB enzymes comprise a widely distributed family of manganese- and GTP-dependent RNA repair enzymes that join 2′,3′-cyclic phosphate ends to 5′-OH ends via RtcB-(histidinyl-N)–GMP, RNA 3′-phosphate, and RNA_3′pp_5′G intermediates. RtcB can ligate either 5′-OH RNA or 5′-OH DNA strands in vitro. The nucleic acid contacts of RtcB are uncharted. Here we report a 2.7 Å crystal structure of Pyrococcus horikoshii RtcB in complex with a 6-mer 5′-OH DNA oligonucleotide _HOA¹pT²pG³pT⁴pC⁵pC⁶, which reveals enzymic contacts of Asn202 to the terminal 5′-OH nucleophile; Arg238 to the A¹pT² and T²pG³ phosphates; Arg190 and Gln194 to the T²pG³ phosphate; and an Arg190 π-cation interaction with the G³ nucleobase. The structural insights affirm functional studies of E. coli RtcB that implicated the conserved counterpart of Arg238 in engagement of the 5′-OH strand for ligation. The essential active site Cys98 that coordinates two manganese ions is oxidized to cysteine sulfonic acid in our structure, raising the prospect that RtcB activity might be sensitive to modulation during oxidative stress.

中文翻译：

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3'-PO4/5'-OH RNA 连接酶 RtcB 与 5'-OH 寡核苷酸复合物的结构


RtcB 酶包含广泛分布的锰和 GTP 依赖性 RNA 修复酶家族，这些酶通过 RtcB-(组氨酸基-N)–GMP、RNA 3'-磷酸将 2',3'-环状磷酸末端连接到 5'-OH 末端，和 RNA _3' pp _5' G 中间体。 RtcB 可以在体外连接 5'-OH RNA 或 5'-OH DNA 链。 RtcB 的核酸接触是未知的。在这里，我们报道了Pyrococcus horikoshii RtcB 与 6 聚体 5'-OH DNA 寡核苷酸_HO A ¹ pT ² pG ³ pT ⁴ pC ⁵ pC ⁶复合物的 2.7 Å 晶体结构，揭示了 Asn202 与末端 5' 的酶接触-OH亲核体； Arg238 至 A ¹ pT ²和 T ² pG ³磷酸盐； Arg190 和 Gln194 至 T ² pG ³磷酸盐；以及Arg190 π-阳离子与G ³核碱基的相互作用。结构见解证实了大肠杆菌RtcB 的功能研究，表明 Arg238 的保守对应物参与了 5'-OH 链的连接。在我们的结构中，协调两个锰离子的重要活性位点 Cys98 被氧化为半胱氨酸磺酸，这提出了 RtcB 活性可能对氧化应激期间的调节敏感的前景。