Dissection of a class II tRNA synthetase: Determinants for minihelix recognition are tightly associated with domain for amino acid activation

Biochemistry ◽  
1993 ◽  
Vol 32 (19) ◽  
pp. 5267-5272 ◽  
Author(s):  
Douglas D. Buechter ◽  
Paul Schimmel
Keyword(s):  
Amino Acid ◽  
Class Ii ◽  
Trna Synthetase ◽  
Acid Activation ◽  
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 Asymmetric Amino Acid Activation by Class II Histidyl-tRNA Synthetase from Escherichia coli

2009 ◽  
Vol 284 (31) ◽  
pp. 20753-20762 ◽  
Author(s):  
Ethan Guth ◽  
Mindy Farris ◽  
Michael Bovee ◽  
Christopher S. Francklyn
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Class Ii ◽  
Trna Synthetase ◽  
Acid Activation ◽  
Amino Acid Activation

scholarly journals The mechanism of β-N-methylamino-l-alanine inhibition of tRNA aminoacylation and its impact on misincorporation

2019 ◽  
Vol 295 (5) ◽  
pp. 1402-1410 ◽  
Author(s):  
Nien-Ching Han ◽  
Tammy J. Bullwinkle ◽  
Kaeli F. Loeb ◽  
Kym F. Faull ◽  
Kyle Mohler ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Direct Evidence ◽  
Trna Synthetase ◽  
Acid Activation ◽  
Aminoacyl Trna Synthetase ◽  
Amino Acid Activation ◽  
Biochemical Assays ◽  
Serine Codons ◽  
Lateral Sclerosis

β-N-methylamino-l-alanine (BMAA) is a nonproteinogenic amino acid that has been associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). BMAA has been found in human protein extracts; however, the mechanism by which it enters the proteome is still unclear. It has been suggested that BMAA is misincorporated at serine codons during protein synthesis, but direct evidence of its cotranslational incorporation is currently lacking. Here, using LC-MS–purified BMAA and several biochemical assays, we sought to determine whether any aminoacyl-tRNA synthetase (aaRS) utilizes BMAA as a substrate for aminoacylation. Despite BMAA's previously predicted misincorporation at serine codons, following a screen for amino acid activation in ATP/PPi exchange assays, we observed that BMAA is not a substrate for human seryl-tRNA synthetase (SerRS). Instead, we observed that BMAA is a substrate for human alanyl-tRNA synthetase (AlaRS) and can form BMAA-tRNAAla by escaping from the intrinsic AlaRS proofreading activity. Furthermore, we found that BMAA inhibits both the cognate amino acid activation and the editing functions of AlaRS. Our results reveal that, in addition to being misincorporated during translation, BMAA may be able to disrupt the integrity of protein synthesis through multiple different mechanisms.

Corrigendum to “Structural Snapshots of the KMSKS Loop Rearrangement for Amino Acid Activation by Bacterial Tyrosyl-tRNA Synthetase” [J. Mol. Biol. (2005) 346, 105–117]

2005 ◽  
Vol 354 (3) ◽  
pp. 739 ◽  
Author(s):  
Takatsugu Kobayashi ◽  
Tetsuo Takimura ◽  
Ryo Sekine ◽  
Vincent P. Kelly ◽  
Kenji Kamata ◽  
...  
Keyword(s):  
Amino Acid ◽  
Trna Synthetase ◽  
Acid Activation ◽  
Amino Acid Activation

Amino acid activation of a dual-specificity tRNA synthetase is independent of tRNA

2002 ◽  
Vol 316 (3) ◽  
pp. 421-427 ◽  
Author(s):  
Richard S.A Lipman ◽  
Penny J Beuning ◽  
Karin Musier-Forsyth ◽  
Ya-Ming Hou
Keyword(s):  
Amino Acid ◽  
Trna Synthetase ◽  
Acid Activation ◽  
Amino Acid Activation ◽  
Dual Specificity
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