Functional characterization of leucine-specific domain 1 from eukaryal and archaeal leucyl-tRNA synthetases

2010 ◽  
Vol 429 (3) ◽  
pp. 505-513 ◽  
Author(s):  
Xiao-Long Zhou ◽  
Meng Wang ◽  
Min Tan ◽  
Qian Huang ◽  
Gilbert Eriani ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Trna Synthetase ◽  
Acid Activation ◽  
Reaction Step ◽  
Specific Domain ◽  
Binding Strength ◽  
Trna Synthetases ◽  
Amino Acid Activation ◽  
Common Domain

LeuRS (leucyl-tRNA synthetase) catalyses the esterification of tRNAsLeu with leucine. This family of enzymes is divided into prokaryotic and eukaryal/archaeal groups according to the presence and position of specific insertions and extensions. In the present study, we investigated the function of LSD1 (leucine-specific domain 1), which is naturally present in eukaryal/archaeal LeuRSs, but absent from prokaryotic LeuRSs. When mutated in their common domain, the eukaryal and archaeal LeuRSs exhibited defects in the first reaction step of amino acid activation with variations of leucine or ATP-binding strength, whereas the tRNA aminoacylation was moderately affected. When the eukaryal extension was mutated, severe tRNA charging defects were observed, suggesting that eukaryotes evolved this LSD1 extension in order to improve the aminoacylation reaction step. The results also showed that the LSD1s from organisms of both groups are dispensable for post-transfer editing. Together, the data provide us with a further understanding of the organization and structure of LeuRS domains.

scholarly journals Virus-Encoded Aminoacyl-tRNA Synthetases: Structural and Functional Characterization of Mimivirus TyrRS and MetRS

2007 ◽  
Vol 81 (22) ◽  
pp. 12406-12417 ◽  
Author(s):  
Chantal Abergel ◽  
Joëlle Rudinger-Thirion ◽  
Richard Giegé ◽  
Jean-Michel Claverie
Keyword(s):  
Active Site ◽  
Functional Characterization ◽  
Evolutionary Relationship ◽  
Trna Synthetase ◽  
Dna Viruses ◽  
Trna Synthetases ◽  
Functional Studies ◽  
Aminoacyl Trna Synthetases ◽  
Viral Enzyme

ABSTRACT Aminoacyl-tRNA synthetases are pivotal in determining how the genetic code is translated in amino acids and in providing the substrate for protein synthesis. As such, they fulfill a key role in a process universally conserved in all cellular organisms from their most complex to their most reduced parasitic forms. In contrast, even complex viruses were not found to encode much translation machinery, with the exception of isolated components such as tRNAs. In this context, the discovery of four aminoacyl-tRNA synthetases encoded in the genome of mimivirus together with a full set of translation initiation, elongation, and termination factors appeared to blur what was once a clear frontier between the cellular and viral world. Functional studies of two mimivirus tRNA synthetases confirmed the MetRS specificity for methionine and the TyrRS specificity for tyrosine and conformity with the identity rules for tRNATyr for archea/eukarya. The atomic structure of the mimivirus tyrosyl-tRNA synthetase in complex with tyrosinol exhibits the typical fold and active-site organization of archaeal-type TyrRS. However, the viral enzyme presents a unique dimeric conformation and significant differences in its anticodon binding site. The present work suggests that mimivirus aminoacyl-tRNA synthetases function as regular translation enzymes in infected amoebas. Their phylogenetic classification does not suggest that they have been acquired recently by horizontal gene transfer from a cellular host but rather militates in favor of an intricate evolutionary relationship between large DNA viruses and ancestral eukaryotes.

scholarly journals Streptogramin B biosynthesis in Streptomyces pristinaespiralis and Streptomyces virginiae: molecular characterization of the last structural peptide synthetase gene.

1997 ◽  
Vol 41 (9) ◽  
pp. 1904-1909 ◽  
Author(s):  
V de Crécy-Lagard ◽  
W Saurin ◽  
D Thibaut ◽  
P Gil ◽  
L Naudin ◽  
...  
Keyword(s):  
Amino Acid ◽  
Acid Activation ◽  
Activation Domains ◽  
Amino Acid Activation ◽  
Streptomyces Virginiae ◽  
Streptomyces Pristinaespiralis ◽  
Degenerate Oligonucleotide ◽  
Peptide Synthetase ◽  
Different Origins

Streptomyces pristinaespiralis and S. virginiae both produce closely related hexadepsipeptide antibiotics of the streptogramin B family. Pristinamycins I and virginiamycins S differ only in the fifth incorporated precursor, di(mono)methylated amine and phenylalanine, respectively. By using degenerate oligonucleotide probes derived from internal sequences of the purified S. pristinaespiralis SnbD and SnbE proteins, the genes from two streptogramin B producers, S. pristinaespiralis and S. virginiae, encoding the peptide synthetase involved in the activation and incorporation of the last four precursors (proline, 4-dimethylparaaminophenylalanine [for pristinamycin I(A)] or phenylalanine [for virginiamycin S], pipecolic acid, and phenylglycine) were cloned. Analysis of the sequence revealed that SnbD and SnbE are encoded by a unique snbDE gene. SnbDE (4,849 amino acids [aa]) contains four amino acid activation domains, four condensation domains, an N-methylation domain, and a C-terminal thioesterase domain. Comparison of the sequences of 55 amino acid-activating modules from different origins confirmed that these sequences contain enough information for the performance of legitimate predictions of their substrate specificity. Partial sequencing (1,993 aa) of the SnbDE protein of S. virginiae allowed comparison of the proline and aromatic acid activation domains of the two species and the identification of coupled frameshift mutations.

The stereochemical course of amino acid activation by methionyl- and tyrosyl-tRNA synthetases

Nature ◽  
1979 ◽  
Vol 281 (5729) ◽  
pp. 320-321 ◽  
Author(s):  
Simon P. Langdon ◽  
Gordon Lowe
Keyword(s):  
Amino Acid ◽  
Acid Activation ◽  
Trna Synthetases ◽  
Amino Acid Activation

scholarly journals RNA-assisted catalysis in a protein enzyme: The 2′-hydroxyl of tRNAThr A76 promotes aminoacylation by threonyl-tRNA synthetase

2008 ◽  
Vol 105 (46) ◽  
pp. 17748-17753 ◽  
Author(s):  
Anand Minajigi ◽  
Christopher S. Francklyn
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Class Ii ◽  
Trna Synthetase ◽  
Hydroxyl Groups ◽  
Acid Activation ◽  
Mutant Proteins ◽  
Amino Acid Activation ◽  
Rate Determining Step ◽  
Aminoacyl Transfer

Aminoacyl-tRNA synthetases (aaRSs) join amino acids to 1 of 2 terminal hydroxyl groups of their cognate tRNAs, thereby contributing to the overall fidelity of protein synthesis. In class II histidyl-tRNA synthetase (HisRS) the nonbridging Sp-oxygen of the adenylate is a potential general base for aminoacyl transfer. To test for conservation of this mechanism in other aaRSs and the role of terminal hydroxyls of tRNA in aminoacyl transfer, we investigated the class II Escherichia coli threonyl-tRNA synthetase (ThrRS). As with other class II aaRSs, the rate-determining step for ThrRS is amino acid activation. In ThrRS, however, the 2′-OH of A76 of tRNAThr and a conserved active-site histidine (His-309) collaborate to catalyze aminoacyl transfer by a mechanism distinct from HisRS. Conserved residues in the ThrRS active site were replaced with alanine, and then the resulting mutant proteins were analyzed by steady-state and rapid kinetics. Nearly all mutants preferentially affected the amino acid activation step, with only a modest effect on aminoacyl transfer. By contrast, H309A ThrRS decreased transfer 242-fold and imposed a kinetic block to CCA accommodation. His-309 hydrogen bonds to the 2′-OH of A76, and substitution of the latter by hydrogen or fluorine decreased aminoacyl transfer by 763- and 94-fold, respectively. The proton relay mechanism suggested by these data to promote aminoacylation is reminiscent of the NAD+-dependent mechanisms of alcohol dehydrogenases and sirtuins and the RNA-mediated catalysis of the ribosomal peptidyl transferase center.

scholarly journals Mimivirus TyrRS: preliminary structural and functional characterization of the first amino-acyl tRNA synthetase found in a virus

2005 ◽  
Vol 61 (2) ◽  
pp. 212-215 ◽  
Author(s):  
Chantal Abergel ◽  
Sabine Chenivesse ◽  
Deborah Byrne ◽  
Karsten Suhre ◽  
Vincent Arondel ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Trna Synthetase
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