Crystal structure at 1.8 A resolution and identification of active site residues of Sulfolobus solfataricus peptidyl-tRNA hydrolase.
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.
Fromant
Michel
M
Laboratoire de Biochimie, Unité Mixte de Recherche 7654 CNRS-Ecole Polytechnique, 91128 Palaiseau Cedex, France.
Schmitt
Emmanuelle
E
Mechulam
Yves
Y
Lazennec
Christine
C
Plateau
Pierre
P
Blanquet
Sylvain
S
D000595:Amino Acid Sequence; D000818:Animals; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D003460:Crystallization; D018360:Crystallography, X-Ray; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013379:Substrate Specificity / Q000235:genetics; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics; D013912:Threonine / Q000235:genetics / Q000378:metabolism
D019843:Archaeal Proteins; D013912:Threonine; D001224:Aspartic Acid; D002265:Carboxylic Ester Hydrolases; C019674:aminoacyl-tRNA hydrolase; D008239:Lysine