Crystal Structure at 1.8 Å Resolution and Identification of Active Site Residues of Sulfolobus solfataricus Peptidyl-tRNA Hydrolase

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Abstract
The 3-D structure of the peptidyl-tRNA hydrolase from the archaea Sulfolobus solfataricus has been solved at 1.8 Å 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 β-sheet surrounded by two groups of two α-helices. The dimer interface is mainly formed by van der Waals interactions between hydrophobic residues belonging to the two N-terminal α1 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 α1 and in the β3−β4 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.