Enzyme Structure with Two Catalytic Sites for Double-Sieve Selection of Substrate

Aminoacylation of the minihelix mimicking the amino acid acceptor arm of tRNA has been demonstrated in more than 10 aminoacyl-tRNA synthetase systems. Although Escherichia coli or Homo sapiens cytoplasmic leucyl-tRNA synthetase (LeuRS) is unable to charge the cognate minihelix or microhelix, we show here that minihelix Leu is efficiently charged by Aquifex aeolicus synthetase, the only known heterodimeric LeuRS (␣␤-LeuRS). Aminoacylation of minihelices is strongly dependent on the presence of the A73 identity nucleotide and greatly stimulated by destabilization of the first base pair as reported for the E. coli isoleucyl-tRNA synthetase and methionyl-tRNA synthetase systems. In the E. coli LeuRS system, the anticodon of tRNA Leu is not important for recognition by the synthetase. However, the addition of RNA helices that mimic the anticodon domain stimulates minihelix Leu charging by ␣␤-LeuRS, indicating possible domain-domain communication within ␣␤-LeuRS. The leucine-specific domain of ␣␤-LeuRS is responsible for minihelix recognition. To ensure accurate translation of the genetic code, LeuRS functions to hydrolyze misactivated amino acids (pretransfer editing) and misaminoacylated tRNA (posttransfer editing). In contrast to tRNA Leu , minihelix Leu is unable to induce posttransfer editing even upon the addition of the anticodon domain of tRNA. Therefore, the context of tRNA is crucial for the editing of mischarged products. However, the minihelix Leu cannot be misaminoacylated, perhaps because of the tRNA-independent pretransfer editing activity of ␣␤-LeuRS.Aminoacyl-tRNA synthetases (aaRSs) 1 establish the genetic code by catalyzing the esterification of cognate amino acids to their specific transfer RNAs (tRNA) that bear the corresponding anticodons, which are defined by the genetic code (1). Aminoacylation of tRNA catalyzed by aaRSs is a two-step reaction: (a) activation of amino acids with ATP by formation of aminoacyl adenylate and (b) transfer of the aminoacyl moiety from the aminoacyl adenylate to the cognate tRNA substrate. On the basis of the architecture of the conserved active site domain, aaRSs are divided into two groups (2). Class I enzymes have an active site based on the Rossmann fold, whereas the conserved active core of class II enzymes contains an antiparallel ␤-sheet flanked by ␣-helices.One hypothesis about the assembly of aminoacyl-tRNA synthetases is that the enzymes are assembled in a modular fashion, incorporating new domains around the conserved active site representing ancestral aaRS such as the tRNA-binding domain and editing domain (3). Interestingly, two domains of tRNA (specifically the amino acid-accepting stem (minihelix) and anticodon stem biloop (SBL)), which interact with the aminoacylation active site and tRNA-binding domain of aaRS, respectively, may have arisen independently (4). Chargeable minihelices and smaller helices (e.g. microhelices of 7 bp) mimicking the acceptor stem have been identified in more than 10 aaRS systems (5). Thus, whereas it is recognized that aaRS...

Section: Discussionmentioning

confidence: 99%

Leucyl-tRNA Synthetase from the Hyperthermophilic Bacterium Aquifex aeolicus Recognizes Minihelices

Zhao

Wang

2004

“…Class I enzymes are characterized by a Rossmann nucleotide binding fold (48) that forms the catalytic site for amino acid activation (3, 42, 45, 49, 50 -52). For the closely related class I isoleucyl-, leucyl-, and valyl-tRNA synthetases, an insertion known as CP1 splits the active site (42,(52)(53)(54)(55). This insertion harbors the center for editing that, in the threedimensional structures, is about 30 Å from the site for amino acid activation (15, 52, 54, 56 -58).…”

Section: Discussionmentioning

confidence: 99%

Structure-specific tRNA Determinants for Editing a Mischarged Amino Acid

Beebe¹,

Merriman

Schimmel

2003

Self Cite

Alanyl-tRNA synthetase efficiently aminoacylates tRNA Ala and an RNA minihelix that comprises just one domain of the two-domain L-shaped tRNA structure. It also clears mischarged tRNA Ala using a specialized domain in its C-terminal half. In contrast to full-length tRNA Ala , minihelix Ala was robustly mischarged and could not be edited. Addition in trans of the missing anticodon-containing domain did not activate editing of mischarged minihelix Ala . To understand these differences between minihelix Ala and tRNA Ala , several chimeric full tRNAs were constructed. These had the acceptor stem of a non-cognate tRNA replaced with the stem of tRNA Ala . The chimeric tRNAs collectively introduced multiple sequence changes in all parts but the acceptor stem. However, although the acceptor stem in isolation (as the minihelix) lacked determinants for editing, alanyl-tRNA synthetase effectively cleared a mischarged amino acid from each chimeric tRNA. Thus, a covalently continuous two-domain structure per se, not sequence, is a major determinant for clearance of errors of aminoacylation by alanyl-tRNA synthetase. Because errors of aminoacylation are known to be deleterious to cell growth, structure-specific determinants constitute a powerful selective pressure to retain the format of the two-domain L-shaped tRNA.

“…In contrast, some amino acids are difficult for aminoacyl-tRNA synthetases to distinguish with high accuracy as they can differ by as little as a single methyl group (11). For example, isoleucyl-tRNA synthetase has difficulty distinguishing between the isosteric amino acids isoleucine and valine, whereas the active site of alanyl-tRNA synthetase (AlaRS) is able to accommodate alanine, glycine, and serine (12)(13)(14)(15)(16). * This work was supported by National Science Foundation Grant MCB-Misactivation of noncognate serine and glycine by AlaRS occurs at frequencies of 1/500 and 1/250, respectively.…”

mentioning

confidence: 99%

Lipid II-independent trans Editing of Mischarged tRNAs by the Penicillin Resistance Factor MurM

Shepherd¹,

Ibba

2013

Background: MurM utilizes aminoacyl-tRNAs and Lipid II for peptidoglycan biosynthesis in Streptococcus pneumoniae. Results: MurM deacylates mischarged aminoacyl-tRNAs in the absence of Lipid II. Conclusion:The ability of MurM to function in quality control can compensate for the absence of AlaXp proteins in S. pneumoniae. Significance: MurM can function in translation as a lipid-independent trans editing factor.