Retracing the evolution of amino acid specificity in glutaminyl‐tRNA synthetase

Hong, Kwang Won; Ibba, Michael; Söll, Dieter

doi:10.1016/s0014-5793(98)00968-5

Cited by 12 publications

(8 citation statements)

References 39 publications

(39 reference statements)

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“…1D). In the context of WT GlnRS, mutations of Tyr240 to glutamate or glycine decreased K m for glutamine by two to-three fold, with no effect on k cat / K m for aminoacylation (29). The adjacent amino acid, Asp241 of E. coli GlnRS, was also not substituted in the construction of GlnRS S1/L1/L2.…”

Section: Resultsmentioning

confidence: 99%

Synthesis of Glu-tRNA^Gln by Engineered and Natural Aminoacyl-tRNA Synthetases

et al. 2010

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A protein engineering approach is described to delineate which distinct elements of homologous tRNA synthetase architectures are responsible for divergent RNA-amino acid pairing specificities. Previously, we constructed a hybrid enzyme in which 23 amino acids from the catalytic domain of E. coli glutaminyl-tRNA synthetase (GlnRS) were replaced with the corresponding residues of human glutamyl-tRNA synthetase (GluRS). The engineered hybrid (GlnRS S1/L1/L2) synthesizes Glu-tRNAGln over 104-fold more efficiently than GlnRS. Detailed comparison of kinetic parameters between GlnRS S1/L1/L2 and the naturally occurring M. thermautotrophicus GluRSND, which is also capable of Glu-tRNAGln synthesis, now shows that both kcat and Km for glutamate are recapitulated in the engineered enzyme, but that Km for tRNA is 200-fold higher. Thus, the simultaneous optimization of paired amino acid and tRNA binding sites found in a naturally occurring enzyme is not recapitulated in a hybrid that is successfully engineered for amino acid complementarity. We infer that the GlnRS architecture has differentiated to match only cognate amino acid-RNA pairs, and that the substrate selection functions do not operate independently of each other. Design and characterization of four additional hybrids identifies further residues involved in improving complementarity for glutamate, and in communicating between amino acid and tRNA binding sites. The robust catalytic function demonstrated in this engineered system offers a novel platform for exploring the stereochemical origins of coding as a property of the ancient Rossmann fold.

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Section: Resultsmentioning

confidence: 99%

Synthesis of Glu-tRNA^Gln by Engineered and Natural Aminoacyl-tRNA Synthetases

et al. 2010

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“…The same studies indicate that the emergence of glutaminyl-tRNA synthetase is a relatively late event that probably took place within the emerging euykaryotic line of descent, via duplication and mutation of the glutamyl-tRNA synthetase gene. This new ARS was then laterally transferred to some proteobacteria, where it is still functional today [58,[71][72][73][74]. Thus, the analysis of the biochemical and phylogenetic characteristics of GlnRS offers us a window to the early metabolism of LUCA, as well as an indication of the cellular interactions that took place at the onset of the eukaryotic evolutionary branch.…”

Section: Visions and Reflectionsmentioning

confidence: 99%

A view into the origin of life: aminoacyl-tRNA synthetases

Pouplana¹,

Schimmel²

2000

CMLS, Cell. Mol. Life Sci.

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The genetic code converts nucleic acid sequences into protein sequences through the action of aminoacyltransfer RNA (tRNA) synthetases. These enzymes are responsible for the specific attachment of each amino acid to its cognate tRNAs. The aminoacylated tRNAs are then transported to the ribosomal complex by elongation factors, where the amino acid is incorporated into the translated polypeptide chain. The RNA world hypothesis states that modern aminoacyl-tRNA synthetases replaced aminoacylating ribozymes. From this perspective, the establishment of modern aminoacyltRNA synthetases would span the transition from the RNA world to the world of DNA and proteins. Thus, the study of the evolution of aminoacyl-tRNA synthetases could offer insights into that process. Here we review our current understanding of the origin and evolution of aminoacyl-tRNA synthetases, and discuss the implications of these studies on the origin of life. The RNA backgroundThe theory of an RNA world postulates that life evolved through a stage where all functions currently performed by proteins and DNA depended chiefly on RNA [1]. From physical and chemical considerations, this RNA-based metabolism has been proposed to be preceded by a 'chemical world', perhaps based on surface-based catalytic processes [2 -6]. The products of these reactions laid the foundation for the eventual appearance of RNA-based protogenomes and membranes, essential requirements for the establishment of populations subjected to natural selection. The categorical demonstration of the existence of an RNA world is impossible without the discovery of an extant form of RNA-based organisms. The probability of such discovery being made is small because one would expect such organisms to have been eliminated by competing species bearing protein-based physiologies. Thus, the theory of an RNA world springs out of theoretical considerations based on three observations: the central role of RNA and ribonucleoproteins in decoding genetic information [9]; the use of catalytic RNA molecules in limited aspects of modern cell metabolism [10, 11]; the potential of in vitro selected RNA molecules to catalyze important biochemical reactions [12][13][14]. These considerations all point to RNA as a plausible transition molecule, a jack-of-all-trades that was capable of undertaking the necessary functions required for establishment of a primitive cell [7,15]. Eventually, the superior stability of DNA, and the greater catalytic plasticity and capacity of proteins, relegated RNA to limited metabolic chores, as evolution and selection pressed populations into the extant world of DNA and proteins. The modern genetic code may have started as an operational RNA code or a primitive second genetic code. The codon-amino acid relationships were formed through interactions between residues and RNA molecules, perhaps out of aminoacylation reactions, that linked specific amino acids with specific RNAs. These RNAs may have been minihelix-like structures,

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“…Regardless of the research discipline, some common themes or parameters can be observed in the application of directed evolution. For example, directed evolution increasingly appears to be the tool of choice for studying the evolution of and relationship between protein structure and function (2,114,138,192,226,259) and for interpretation of the evolutionary significance of biomolecular systems (122,323). It is also a popular tool for accelerated adaptation of protein functions (e.g., stability, specificity, or affinity) in extreme conditions such as unusual temperatures and organic solvents (198,204,221,222,(327)(328)(329)(330), as well as for improvement of recombinant protein biosynthesis (152,185).…”

Section: Applications Of Directed Evolutionmentioning

confidence: 99%

Laboratory-Directed Protein Evolution

Yuan

Kurek

English

et al. 2005

Microbiol Mol Biol Rev

175

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Systematic approaches to directed evolution of proteins have been documented since the 1970s. The ability to recruit new protein functions arises from the considerable substrate ambiguity of many proteins. The substrate ambiguity of a protein can be interpreted as the evolutionary potential that allows a protein to acquire new specificities through mutation or to regain function via mutations that differ from the original protein sequence. All organisms have evolutionarily exploited this substrate ambiguity. When exploited in a laboratory under controlled mutagenesis and selection, it enables a protein to “evolve” in desired directions. One of the most effective strategies in directed protein evolution is to gradually accumulate mutations, either sequentially or by recombination, while applying selective pressure. This is typically achieved by the generation of libraries of mutants followed by efficient screening of these libraries for targeted functions and subsequent repetition of the process using improved mutants from the previous screening. Here we review some of the successful strategies in creating protein diversity and the more recent progress in directed protein evolution in a wide range of scientific disciplines and its impacts in chemical, pharmaceutical, and agricultural sciences

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Retracing the evolution of amino acid specificity in glutaminyl‐tRNA synthetase

Cited by 12 publications

References 39 publications

Synthesis of Glu-tRNA^Gln by Engineered and Natural Aminoacyl-tRNA Synthetases

Synthesis of Glu-tRNA^Gln by Engineered and Natural Aminoacyl-tRNA Synthetases

A view into the origin of life: aminoacyl-tRNA synthetases

Laboratory-Directed Protein Evolution

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Retracing the evolution of amino acid specificity in glutaminyl‐tRNA synthetase

Cited by 12 publications

References 39 publications

Synthesis of Glu-tRNAGln by Engineered and Natural Aminoacyl-tRNA Synthetases

Synthesis of Glu-tRNAGln by Engineered and Natural Aminoacyl-tRNA Synthetases

A view into the origin of life: aminoacyl-tRNA synthetases

Laboratory-Directed Protein Evolution

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Synthesis of Glu-tRNA^Gln by Engineered and Natural Aminoacyl-tRNA Synthetases

Synthesis of Glu-tRNA^Gln by Engineered and Natural Aminoacyl-tRNA Synthetases