Department of Biomedical Chemistry, University of Tokyo, Tokyo 113-0033, Japan.
J Mol Biol. 2011 Jan 7;405(1):92-104. doi: 10.1016/j.jmb.2010.10.050. Epub 2010 Nov 2.
In Archaea, splicing endonuclease (EndA) recognizes and cleaves precursor RNAs to remove introns. Currently, EndAs are classified into three families according to their subunit structures: homotetramer, homodimer, and heterotetramer. The crenarchaeal heterotetrameric EndAs can be further classified into two subfamilies based on the size of the structural subunit. Subfamily A possesses a structural subunit similar in size to the catalytic subunit, whereas subfamily B possesses a structural subunit significantly smaller than the catalytic subunit. Previously, we solved the crystal structure of an EndA from Pyrobaculum aerophilum. The endonuclease was classified into subfamily B, and the structure revealed that the enzyme lacks an N-terminal subdomain in the structural subunit. However, no structural information is available for crenarchaeal heterotetrameric EndAs that are predicted to belong to subfamily A. Here, we report the crystal structure of the EndA from Aeropyrum pernix, which is predicted to belong to subfamily A. The enzyme possesses the N-terminal subdomain in the structural subunit, revealing that the two subfamilies of heterotetrameric EndAs are structurally distinct. EndA from A. pernix also possesses an extra loop region that is characteristic of crenarchaeal EndAs. Our mutational study revealed that the conserved lysine residue in the loop is important for endonuclease activity. Furthermore, the sequence characteristics of the loops and the positions towards the substrate RNA according to a docking model prompted us to propose that crenarchaea-specific loops and an extra amino acid sequence at the catalytic loop of nanoarchaeal EndA are derived by independent convergent evolution and function for recognizing noncanonical bulge-helix-bulge motif RNAs as substrates.
在古菌中，剪接内切酶（EndA）识别并切割前体 RNA 以去除内含子。目前，根据亚基结构，EndA 分为三类：同源四聚体、同源二聚体和异源四聚体。古菌异源四聚体 EndA 可以根据结构亚基的大小进一步分为两个亚科。亚科 A 的结构亚基大小与催化亚基相似，而亚科 B 的结构亚基明显小于催化亚基。此前，我们解析了嗜热焦硫酸盐古菌 Pyrobaculum aerophilum 的 EndA 晶体结构。内切酶被归类为亚科 B，结构显示该酶在结构亚基中缺乏 N 端亚结构域。然而，对于预测属于亚科 A 的古菌异源四聚体 EndA，尚无结构信息。在此，我们报告了 Aeropyrum pernix 的 EndA 晶体结构，该酶预测属于亚科 A。该酶在结构亚基中具有 N 端亚结构域，表明异源四聚体 EndA 的两个亚科在结构上是不同的。A. pernix 的 EndA 还具有一个特征性的额外环区。我们的突变研究表明，环中的保守赖氨酸残基对于内切酶活性很重要。此外，根据对接模型，环的序列特征和朝向底物 RNA 的位置促使我们提出，古菌特异性环和纳米古菌 EndA 催化环上的额外氨基酸序列是通过独立的趋同进化而来，并作为底物识别非典型的突环-螺旋-突环 motif RNA。
