Crystal structure at 1.2Aresolution and active site mapping of Escherichia coli peptidyl-tRNA hydrolase

Schmitt, Emmanuelle; Mechulam, Yves; Fromant, Michel; Plateau, Pierre; Blanquet, Sylvain

doi:10.1093/emboj/16.15.4760

Cited by 91 publications

(206 citation statements)

References 46 publications

Supporting

Mentioning

198

Contrasting

Order By: Relevance

“…Rate data were best fit to the theoretical Michaelis-Menten equation with k cat ϭ 1.3 Ϯ 0.1 s Ϫ1 and K m ϭ 0.22 Ϯ 0.06 M. Thus, E. coli-derived diacetyl-lysyl-tRNA Lys represents an efficient substrate for human Pth2. The catalytic efficiency (k cat /K m ϭ 5.9 M Ϫ1 s Ϫ1 ) for the human enzyme is also comparable with that of the archaea S. solfataricus Pth2 (9), although both are higher than that reported for E. coli Pth (Table II) (6). Most likely Pth2s recognize more extensively the tRNA structure than Pths accounting for the lower K m of Pth2s.…”

Section: Resultssupporting

confidence: 56%

“…The Pth2 fold differs from that of Pth. E. coli Pth (Protein Data Bank code 2PTH) is 193 residues long and shows a hydrolase-like fold with a mixed ␤-sheet of five strands (order 2-1-3-5-4) in its core with strand 4 antiparallel to the rest (6).…”

Section: Resultsmentioning

confidence: 99%

“…E. coli Pth crystal structure has been solved and exhibits an ␣/␤ fold formed by three layers with a mixed five-strand ␤-sheet at the core. Its catalytic parameters have been measured, and several residues affecting the 5Ј-phosphate and 3Ј-end recognition of the peptidyl-tRNA substrate have been mapped (6,7).…”

mentioning

confidence: 99%

“…Pth2 lacks any detectable homology to solved three-dimensional structures. S. solfataricus Pth2 displays a greater catalytic efficiency toward E. coli diacetyl-lysyl-tRNA Lys than E. coli Pth (9,6). In addition, both enzymes differ in their mode of recognition of several tRNA features, like the 1-72-base pair mismatch in E. coli tRNA fMet or the presence of a phosphorylated 5Ј-end.…”

mentioning

confidence: 99%

See 3 more Smart Citations

Crystal Structure of a Human Peptidyl-tRNA Hydrolase Reveals a New Fold and Suggests Basis for a Bifunctional Activity

Pereda

Waas

Jan

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Peptidyl-tRNA hydrolase (Pth) activity releases tRNA from the premature translation termination product peptidyl-tRNA. Two different enzymes have been reported to encode such activity, Pth present in bacteria and eukaryotes and Pth2 present in archaea and eukaryotes. Here we report the crystallographic structure of the Homo sapiens Pth2 at a 2.0-Å resolution as well as its catalytic properties. In contrast to the structure of Escherichia coli Pth, H. sapiens Pth2 has an ␣/␤ fold with a four-stranded antiparallel ␤-sheet in its core surrounded by two ␣-helices on each side. This arrangement of secondary structure elements generates a fold not previously reported. Its catalytic efficiency is comparable with that reported for the archaeal Sulfolobus solfataricus Pth2 and higher than that of the bacterial E. coli Pth. Several lines of evidence target the active site to two close loops with highly conserved residues. This active site architecture is unrelated to that of E. coli Pth. In addition, intermolecular contacts in the crystal asymmetric unit cell suggest a likely surface for protein-protein interactions related to the Pth2-mediated apoptosis.During the protein translation process, a significant proportion of the ribosomes that initiate mRNA readout do not reach the stop codon. Peptidyl-tRNA molecules may dissociate from the mRNA template causing a premature end of the process (1, 2). Accumulation of peptidyl-tRNAs reduces the efficiency of translation by sequestering tRNAs and impairing the initiation (3). Prokaryotic and eukaryotic cells show an enzymatic activity that releases the peptidyl moiety from the tRNA, called peptidyl-tRNA hydrolase (Pth), 1 allowing the free tRNA and peptide to be reused in protein synthesis (4, 5). First identified in Escherichia coli, genes encoding Pth have been found in bacteria and eukaryotes but not in archaea. E. coli Pth crystal structure has been solved and exhibits an ␣/␤ fold formed by three layers with a mixed five-strand ␤-sheet at the core. Its catalytic parameters have been measured, and several residues affecting the 5Ј-phosphate and 3Ј-end recognition of the peptidyl-tRNA substrate have been mapped (6, 7).Recently, a Pth-like activity has been identified in Methanocaldococcus jannaschii and characterized in Sulfolobus solfataricus, both archaebacteria (8, 9). The new enzyme called Pth2 is shorter in length and lacks any significant sequence homology to Pth. It coincides with the Pfam data base entry "Uncharacterized Protein Family 0099" (UPF0099) found in archaea and eukaryotes but not in bacterial genomes. Pth2 lacks any detectable homology to solved three-dimensional structures. S. solfataricus Pth2 displays a greater catalytic efficiency toward E. coli diacetyl-lysyl-tRNA Lys than E. coli Pth (9, 6). In addition, both enzymes differ in their mode of recognition of several tRNA features, like the 1-72-base pair mismatch in E. coli tRNA fMet or the presence of a phosphorylated 5Ј-end.Human Pth2, also called Bcl-2 inhibitor of transcription 1 (Bit1), possesses an ...

show abstract

Section: Resultssupporting

confidence: 56%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Crystal Structure of a Human Peptidyl-tRNA Hydrolase Reveals a New Fold and Suggests Basis for a Bifunctional Activity

Pereda

Waas

Jan

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Therefore, complementation was brought about by the M. jannaschii pth2 gene. This was further confirmed by the ability of the M. jannaschii pth2 gene (in pGR518) to complement an E. coli pth deletion (27) strain (data not shown). Thus, the archaeal pth2 gene is functionally equivalent to the E. coli pth gene.…”

Section: Jannaschii Pth2 Complements the Temperature-sensitive Pthmentioning

confidence: 66%

Orthologs of a novel archaeal and of the bacterial peptidyl–tRNA hydrolase are nonessential in yeast

Rosas-Sandoval

Ambrogelly

Rinehart

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Peptidyl-tRNA hydrolase (encoded by pth) is an essential enzyme in all bacteria, where it releases tRNA from the premature translation termination product peptidyl-tRNA. Archaeal genomes lack a recognizable peptidyl-tRNA hydrolase (Pth) ortholog, although it is present in most eukaryotes. However, we detected Pth-like activity in extracts of the archaeon Methanocaldococcus jannaschii. The uncharacterized MJ0051 ORF was shown to correspond to a protein with Pth activity. Heterologously expressed MJ0051 enzyme catalyzed in vitro the cleavage of the Pth substrates diacetyl-[ 14 C]lysyl-tRNA and acetyl-[ 14 C]phenylalanyl-tRNA. On transformation of an Escherichia coli pth ts mutant, the MJ0051 gene (named pth2) rescued the temperature-sensitive phenotype of the strain. Analysis of known genomes revealed the presence of highly conserved orthologs of the archaeal pth2 gene in all archaea and eukaryotes but not in bacteria. The phylogeny of pth2 homologs suggests that the gene has been vertically inherited throughout the archaeal and eukaryal domains. Deletions in Saccharomyces cerevisiae of the pth2 (YBL057c) or pth (YHR189w) orthologs were viable, as was the double deletion strain, implying that the canonical Pth and Pth2 enzymes are not essential for yeast viability. P eptidyl-tRNA hydrolase (Pth) is an essential enzyme in bacteria (1-3). It cleaves peptidyl-tRNAs, premature protein synthesis products that dissociate from the ribosome, and allows the discharged tRNAs to reengage in protein synthesis. In mutants with limited Pth activity, peptidyl-tRNAs accumulate in the cell and reduce the availability of essential acylatable tRNAs below the limit compatible with protein synthesis; thus cell growth is impaired. However, the possibility that peptidyl-tRNA per se is toxic has not been ruled out. A significant proportion of the ribosomes that initiate mRNA translation interrupt protein synthesis before reaching the stop codon (4). These events of abortive translation may result in peptidyl-tRNA dissociation from the ribosome (5), forming the natural substrates for Pth. There are at least two documented instances of peptidyl-tRNA accumulation and growth inhibition in Escherichia coli mutants partly defective in Pth activity: minigene expression and overproduction of a nonfunctional protein. In each case, accumulation of peptidyl-tRNA occurs corresponding to the last sense codon, implying defects in translation termination (6-8).Pth activity is ubiquitous. Orthologs of the E. coli pth gene have been identified in all known bacterial and some eukaryotic genomes (e.g., yeast, maize, mice, and human), and Pth activities were demonstrated in yeast long ago (9-11). Furthermore, the bacterial pth ortholog YHR189w of Saccharomyces cerevisiae complements a temperature-sensitive E. coli strain harboring a pth ts mutant, although YHR189w is not essential for yeast viability (3). Biochemical studies with S. cerevisiae extracts revealed the presence of at least two types of Pth activities, distinguished by the different efficiency o...

show abstract

All in the family: Structural and evolutionary relationships among three modular proteins with diverse functions and variable assembly

Bergdoll¹,

Eltis

Cameron

et al. 1998

Protein Science

View full text Add to dashboard Cite

The crystal structures of three proteins of diverse function and low sequence similarity were analyzed to evaluate structural and evolutionary relationships. The proteins include a bacterial bleomycin resistance protein, a bacterial extradiol dioxygenase, and human glyoxalase I. Structural comparisons, as well as phylogenetic analyses, strongly indicate that the modem family of proteins represented by these structures arose through a rich evolutionary history that includes multiple gene duplication and fusion events. These events appear to be historically shared in some cases, but parallel and historically independent in others. A significant early event is proposed to be the establishment of metalbinding in an oligomeric ancestor prior to the first gene fusion. Variations in the spatial arrangements of homologous modules are observed that are consistent with the structural principles of three-dimensional domain swapping, but in the unusual context of the formation of larger monomers from smaller dimers or tetramers. The comparisons support a general mechanism for metalloprotein evolution that exploits the symmetry of a homooligomeric protein to originate a metal binding site and relies upon the relaxation of symmetry, as enabled by gene duplication, to establish and refine specific functions.Keywords: bleomycin resistance protein; extradiol dioxygenase; glyoxalase I; metalloproteins; modular proteins; protein evolution; three-dimensional domain swapping Gene duplication is recognized as a primary mechanism for the origination of new functions during the course of evolution. Indeed, numerous analyses of the sequences and tertiary structures of proteins have revealed the duplication, and, in some cases the subsequent fusion, of genes that code for protein modules. Recognized families of proteins derived from duplicated genes tend to have shared or closely related functions because identification by sequence analysis is limited to cases where the primary structures

show abstract

Crystal structure at 1.2Aresolution and active site mapping of Escherichia coli peptidyl-tRNA hydrolase

Cited by 91 publications

References 46 publications

Crystal Structure of a Human Peptidyl-tRNA Hydrolase Reveals a New Fold and Suggests Basis for a Bifunctional Activity

Crystal Structure of a Human Peptidyl-tRNA Hydrolase Reveals a New Fold and Suggests Basis for a Bifunctional Activity

Orthologs of a novel archaeal and of the bacterial peptidyl–tRNA hydrolase are nonessential in yeast

All in the family: Structural and evolutionary relationships among three modular proteins with diverse functions and variable assembly

Contact Info

Product

Resources

About