Glutamyl-queuosine-tRNAAsp synthetase (Glu-Q-RS) is a paralog of glutamyl-tRNA synthetase (GluRS) and is found in more than forty species of proteobacteria, cyanobacteria, and actinobacteria. Glu-Q-RS shows striking structural similarity with N-terminal catalytic domain of GluRS (NGluRS) but it lacks the C-terminal anticodon binding domain (CGluRS). In spite of structural similarities, Glu-Q-RS and NGluRS differ in their functional properties. Glu-Q-RS glutamylates the Q34 nucleotide of the anticodon of tRNAAsp whereas NGluRS constitutes the catalytic domain of GluRS catalyzing the transfer of Glu on the acceptor end of tRNAGlu. Since NGluRS is able to catalyze aminoacylation of only tRNAGlu the glutamylation capacity of tRNAAsp by Glu-Q-RS is surprising. To understand the substrate specificity of Glu-Q-RS we undertook a systemic approach by investigating the biophysical and biochemical properties of the NGluRS (1-301), CGluRS (314-471) and Glu-Q-RS-CGluRS, (1-298 of Glu-Q-RS fused to 314-471 from GluRS). Circular dichroism, fluorescence spectroscopy and differential scanning calorimetry analyses revealed absence of N-terminal domain (1-298 of Glu-Q-RS) and C-terminal domain (314-471 from GluRS) communication in chimera, in contrast to the native full length GluRS. The chimeric Glu-Q-RS is still able to aminoacylate tRNAAsp but has also the capacity to bind tRNAGlu. However the chimeric protein is unable to aminoacylate tRNAGlu probably as a consequence of the lack of domain-domain communication. © Springer Science+Business Media New York 2013.

RAJAT BANERJEE

Biotechnology and Dr. B. C Guha Centre for Genetic Engineering and Biotechnology

S RAY

M BLAISE

B ROY

S GHOSH

University of Calcutta (informally known as Calcutta University or CU) is a collegiate public state university located in Kolkata, West Bengal, India.&nbsp;Within India it is recognized as a &quot;Five-Star University&quot; and accredited &quot;A&quot; Grade by National Assessment and Accreditation Council.

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The University of Calcutta has secured the Fifth position among the Universities of India in the prestigious &quot;Indian University Ranking 2019&quot; list, released by the NIRF of the Ministry of Human Resource Development, Government of India.

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It was established on 24 January 1857, and was one of the first institutions in Asia to be established as a multidisciplinary and Western-style university. Its alumni and faculty include several heads of state, heads of government, social reformers, prominent artists, only academy award winner and Dirac medal winner in India, many Fellows of the Royal Society and five Nobel laureates as of 2019 which is highest in South Asia.

University Of Calcutta

Protein Journal

Molten globule and other disordered states of proteins are now known to play important roles in many cellular processes. From equilibrium unfolding studies of two paralogous proteins and their variants, glutaminyl-tRNA synthetase (GlnRS) and two of its variants [glutamyl-tRNA synthetase (GluRS) and its isolated domains, and a GluRS-GlnRS chimera], we demonstrate that only GlnRS forms a molten globule-like intermediate at low urea concentrations. We demonstrated that a loop in the GlnRS C-terminal anticodon binding domain that promotes communication with the N-terminal domain and indirectly modulates amino acid binding is also responsible for stabilization of the molten globule state. This loop was inserted into GluRS in the eukaryotic branch after the archaea-eukarya split, right around the time when GlnRS evolved. Because of the structural and functional importance of the loop, it is proposed that the insertion of the loop into a putative ancestral GluRS in eukaryotes produced a catalytically active molten globule state. Because of their enhanced dynamic nature, catalytically active molten globules are likely to possess broad substrate specificity. It is further proposed that the putative broader substrate specificity allowed the catalytically active molten globule to accept glutamine in addition to glutamic acid, leading to the evolution of GlnRS. (Figure Presented). © 2012 American Chemical Society.

Biochemistry

A functional loop spanning distant domains of glutaminyl-tRNA synthetase also stabilizes a molten globule state

Discrimination of tRNAGln is an integral function of several bacterial glutamyl-tRNA synthetases (GluRS). The origin of the discrimination is thought to arise from unfavorable interactions between tRNAGln and the anticodon-binding domain of GluRS. From experiments on an anticodon-binding domain truncated Escherichia coli (E. coli) GluRS (catalytic domain) and a chimeric protein, constructed from the catalytic domain of E. coli GluRS and the anticodon-binding domain of E. coli glutaminyl-tRNA synthetase (GlnRS), we show that both proteins discriminate against E. coli tRNAGln. Our results demonstrate that in addition to the anticodon-binding domain, tRNAGln discriminatory elements may be present in the catalytic domain in E. coli GluRS as well. © 2009 Federation of European Biochemical Societies.

Fulltext

FEBS Letters

The role of the catalytic domain of E. coli GluRS in tRNAGln discrimination

aaRSs (aminoacyl-tRNA synthetases) are multi-domain proteins that have evolved by domain acquisition. The anti-codon binding domain was added to the more ancient catalytic domain during aaRS evolution. Unlike in eukaryotes, the anti-codon binding domains of GluRS (glutamyl-tRNA synthetase) and GlnRS (glutaminyl-tRNA synthetase) in bacteria are structurally distinct. This originates from the unique evolutionary history of GlnRSs. Starting from the catalytic domain, eukaryotic GluRS evolved by acquiring the archaea/eukaryote-specific anti-codon binding domain after branching away from the eubacteria family. Subsequently, eukaryotic GlnRS evolved from GluRS by gene duplication and horizontally transferred to bacteria. In order to study the properties of the putative ancestral GluRS in eukaryotes, formed immediately after acquiring the anti-codon binding domain, we have designed and constructed a chimaeric protein, cGluGlnRS, consisting of the catalytic domain, Ec GluRS (Escherichia coli GluRS), and the anti-codon binding domain of EcGlnRS (E. coli GlnRS). In contrast to the isolated EcN-GluRS, cGluGlnRS showed detectable activity of glutamylation of E. coli tRNAglu and was capable of complementing an E. coli ts (temperature-sensitive)-GluRS strain at non-permissive temperatures. Both cGluGlnRS and EcN-GluRS were found to bind E. coli tRNAglu with native EcGluRS-like affinity, suggesting that the anticodon-binding domain in cGluGlnRS enhances kcat for glutamylation. This was further confirmed from similar experiments with a chimaera between EcN-GluRS and the substrate-binding domain of EcDnaK (E. coli DnaK). We also show that an extended loop, present in the anticodon-binding domains of GlnRSs, is absent in archaeal GluRS, suggesting that the loop was a later addition, generating additional anti-codon discrimination capability in GlnRS as it evolved from GluRS in eukaryotes. © The Authors Journal compilation.

Biochemical Journal

A chimaeric glutamyl:glutaminyl-tRNA synthetase: Implications for evolution

Glutamyl-queuosine tRNAAsp synthetase (Glu-Q-RS) from Escherichia coli is a paralog of the catalytic core of glutamyl-tRNA synthetase (GluRS) that catalyzes glutamylation of queuosine in the wobble position of tRNAAsp. Despite important structural similarities, Glu-Q-RS and GluRS diverge strongly by their functional properties. The only feature common to both enzymes consists in the activation of Glu to form Glu-AMP, the intermediate of transfer RNA (tRNA) aminoacylation. However, both enzymes differ by the mechanism of selection of the cognate amino acid and by the mechanism of its activation. Whereas GluRS selects l-Glu and activates it only in the presence of the cognate tRNAGlu, Glu-Q-RS forms Glu-AMP in the absence of tRNA. Moreover, while GluRS transfers the activated Glu to the 3′ accepting end of the cognate tRNAGlu, Glu-Q-RS transfers the activated Glu to Q34 located in the anticodon loop of the noncognate tRNAAsp. In order to gain insight into the structural elements leading to distinct mechanisms of amino acid activation, we solved the three-dimensional structure of Glu-Q-RS complexed to Glu and compared it to the structure of the GluRS·Glu complex. Comparison of the catalytic site of Glu-Q-RS with that of GluRS, combined with binding experiments of amino acids, shows that a restricted number of residues determine distinct catalytic properties of amino acid recognition and activation by the two enzymes. Furthermore, to explore the structural basis of the distinct aminoacylation properties of the two enzymes and to understand why Glu-Q-RS glutamylates only tRNAAsp among the tRNAs possessing queuosine in position 34, we performed a tRNA mutational analysis to search for the elements of tRNAAsp that determine recognition by Glu-Q-RS. The analyses made on tRNAAsp and tRNAAsn show that the presence of a C in position 38 is crucial for glutamylation of Q34. The results are discussed in the context of the evolution and adaptation of the tRNA glutamylation system. © 2008 Elsevier Ltd. All rights reserved.

Journal of Molecular Biology

Crystal Structure of Glutamyl-Queuosine tRNAAsp Synthetase Complexed with l-Glutamate: Structural Elements Mediating tRNA-Independent Activation of Glutamate and Glutamylation of tRNAAsp Anticodon

Journal	Data powered by TypesetProtein Journal
Publisher	Data powered by TypesetSpringer New York LLC
ISSN	1572-3887