Fromant Michel, Schmitt Emmanuelle, Mechulam Yves, Lazennec Christine, Plateau Pierre, Blanquet Sylvain
Laboratoire de Biochimie, Unité Mixte de Recherche 7654 CNRS-Ecole Polytechnique, 91128 Palaiseau Cedex, France.
Biochemistry. 2005 Mar 22;44(11):4294-301. doi: 10.1021/bi047711k.
The 3-D structure of the peptidyl-tRNA hydrolase from the archaea Sulfolobus solfataricus has been solved at 1.8 A resolution. Homologues of this enzyme are found in archaea and eucarya. Bacteria display a different type of peptidyl-tRNA hydrolase that is also encountered in eucarya. In solution, the S. solfataricus hydrolase behaves as a dimer. In agreement, the crystalline structure of this enzyme indicates the formation of a dimer. Each protomer is made of a mixed five-stranded beta-sheet surrounded by two groups of two alpha-helices. The dimer interface is mainly formed by van der Waals interactions between hydrophobic residues belonging to the two N-terminal alpha1 helices contributed by two protomers. Site-directed mutagenesis experiments were designed for probing the basis of specificity of the archaeal hydrolase. Among the strictly conserved residues within the archaeal/eucaryal peptidyl-tRNA hydrolase family, three residues, K18, D86, and T90, appear of utmost importance for activity. They are located in the N-part of alpha1 and in the beta3-beta4 loop. K18 and D86, which form a salt bridge, might play a role in the catalysis thanks to their acid and basic functions, whereas the OH group of T90 could act as a nucleophile. These observations clearly distinguish the active site of the archaeal/eucaryal hydrolases from that of the bacterial/eucaryal ones, where a histidine is believed to serve as the catalytic base.
嗜热栖热菌（Sulfolobus solfataricus）的肽基 - tRNA水解酶的三维结构已在1.8埃分辨率下解析出来。该酶的同源物存在于古菌和真核生物中。细菌则具有一种不同类型的肽基 - tRNA水解酶，在真核生物中也能发现。在溶液中，嗜热栖热菌水解酶表现为二聚体。与此一致，该酶的晶体结构表明形成了二聚体。每个原体由一个混合的五链β - 折叠组成，周围环绕着两组两个α - 螺旋。二聚体界面主要由两个原体贡献的两个N端α1螺旋上的疏水残基之间的范德华相互作用形成。设计了定点诱变实验来探究古菌水解酶特异性的基础。在古菌/真核生物肽基 - tRNA水解酶家族中严格保守的残基中，三个残基K18、D86和T90对活性似乎最为重要。它们位于α1的N端部分以及β3 - β4环中。形成盐桥的K18和D86，因其酸性和碱性功能可能在催化中起作用，而T90的OH基团可作为亲核试剂。这些观察结果清楚地将古菌/真核生物水解酶的活性位点与细菌/真核生物水解酶的活性位点区分开来，在细菌/真核生物水解酶中，一个组氨酸被认为是催化碱基。
