Ponchon Luc, Boulanger Pascale, Labesse Gilles, Letellier Lucienne
Institut de Biochimie et Biophysique Moléculaire et Cellulaire, Unite Mixte de Recherche CNRS 8619, Bātiment 430, Université de Paris-Sud, 91405 Orsay, France.
J Biol Chem. 2006 Mar 3;281(9):5829-36. doi: 10.1074/jbc.M511817200. Epub 2005 Dec 22.
Bacteriophage terminases are essential molecular motors involved in the encapsidation of viral DNA. They are hetero-multimers whose large subunit encodes both ATPase and endonuclease activities. Although the ATPase domain is well characterized from sequence and functional analysis, the C-terminal region remains poorly defined. We describe sequence-structure comparisons of the endonuclease region of various bacteriophages that revealed new sequence similarities shared by this region and the Holliday junction resolvase RuvC and to a lesser extent the HIV integrase and the ribonuclease H. Extensive sequence comparison and motif refinement led to a common signature of terminases and resolvases with three conserved acidic residues engaged in catalytic activity. Sequence analyses were validated by in vivo and in vitro functional assays showing that the nuclease activity of the endonuclease domain of bacteriophage T5 terminase was abolished by mutation of any of the three predicted catalytic aspartates. Overall, these data suggest that the endonuclease domains of terminases operate autonomously and that they adopt a fold similar to that of resolvases and share the same divalent cation-dependent enzymatic mechanism.
噬菌体末端酶是参与病毒DNA包装的必需分子马达。它们是异源多聚体，其大亚基编码ATP酶和内切核酸酶活性。尽管通过序列和功能分析对ATP酶结构域已有充分了解，但C末端区域仍定义不清。我们描述了各种噬菌体内切核酸酶区域的序列-结构比较，结果揭示了该区域与霍利迪连接体解离酶RuvC共享的新序列相似性，在较小程度上还与HIV整合酶和核糖核酸酶H相似。广泛的序列比较和基序优化产生了末端酶和解离酶的共同特征，即有三个保守的酸性残基参与催化活性。体内和体外功能试验验证了序列分析结果，结果表明，噬菌体T5末端酶内切核酸酶结构域的核酸酶活性会因三个预测的催化天冬氨酸中任何一个发生突变而丧失。总体而言，这些数据表明，末端酶的内切核酸酶结构域可自主发挥作用，并且它们采用与解离酶相似的折叠方式，共享相同的二价阳离子依赖性酶促机制。
