Specificity and catalysis hardwired at the RNA–protein interface in a translational proofreading enzyme

Ahmad, Sadeem; Muthukumar, Sowndarya; Kuncha, Santosh Kumar; Routh, Satya Brata; Yerabham, Antony S.K.; Hussain, Tanweer; Kamarthapu, Venu; Kruparani, Shobha P; Sankaranarayanan, Rajan

doi:10.1038/ncomms8552

Cited by 24 publications

(27 citation statements)

References 58 publications

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“…We could generate 3'-end modifications of tRNA Gly with A76 being replaced by either 2′-deoxyadenosine (2′-dA76) or 2′-fluoro-2′-deoxyadenosine (2′-FdA76) and could also charge the modified tRNAs with glycine using glycyl-tRNA synthetase. Employing this strategy to test our hypothesis on NTDs, we have very recently been successful in proving that NTDs can catalyse deacylation of their substrates only with the assistance of 2′-OH of tRNA [ 41 ]. We used the same strategy to substantiate our hypothesis on DTD.…”

Section: Resultsmentioning

confidence: 99%

Elongation Factor Tu Prevents Misediting of Gly-tRNA(Gly) Caused by the Design Behind the Chiral Proofreading Site of D-Aminoacyl-tRNA Deacylase

et al. 2016

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D-aminoacyl-tRNA deacylase (DTD) removes D-amino acids mischarged on tRNAs and is thus implicated in enforcing homochirality in proteins. Previously, we proposed that selective capture of D-aminoacyl-tRNA by DTD’s invariant, cross-subunit Gly-cisPro motif forms the mechanistic basis for its enantioselectivity. We now show, using nuclear magnetic resonance (NMR) spectroscopy-based binding studies followed by biochemical assays with both bacterial and eukaryotic systems, that DTD effectively misedits Gly-tRNAGly. High-resolution crystal structure reveals that the architecture of DTD’s chiral proofreading site is completely porous to achiral glycine. Hence, L-chiral rejection is the only design principle on which DTD functions, unlike other chiral-specific enzymes such as D-amino acid oxidases, which are specific for D-enantiomers. Competition assays with elongation factor thermo unstable (EF-Tu) and DTD demonstrate that EF-Tu precludes Gly-tRNAGly misediting at normal cellular concentrations. However, even slightly higher DTD levels overcome this protection conferred by EF-Tu, thus resulting in significant depletion of Gly-tRNAGly. Our in vitro observations are substantiated by cell-based studies in Escherichia coli that show that overexpression of DTD causes cellular toxicity, which is largely rescued upon glycine supplementation. Furthermore, we provide direct evidence that DTD is an RNA-based catalyst, since it uses only the terminal 2′-OH of tRNA for catalysis without the involvement of protein side chains. The study therefore provides a unique paradigm of enzyme action for substrate selection/specificity by DTD, and thus explains the underlying cause of DTD’s activity on Gly-tRNAGly. It also gives a molecular and functional basis for the necessity and the observed tight regulation of DTD levels, thereby preventing cellular toxicity due to misediting.

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Section: Resultsmentioning

confidence: 99%

Elongation Factor Tu Prevents Misediting of Gly-tRNA(Gly) Caused by the Design Behind the Chiral Proofreading Site of D-Aminoacyl-tRNA Deacylase

et al. 2016

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“…With respect to catalysis, mutations in the conserved activesite residues of DTD do not affect its deacylation activity. Similar to NTD, the side chains have no role in either substrate specificity or catalysis; instead, the 2Ј-OH of A76 (of tRNA) was shown to be indispensable for its activity (108,109). Taking cues from NTD and on the basis of structure, a similar RNAbased substrate-assisted catalysis mechanism was proposed for DTD.…”

Section: D-aminoacyl-trna Deacylasementioning

confidence: 99%

Chiral checkpoints during protein biosynthesis

Kuncha

Kruparani

Sankaranarayanan

2019

Journal of Biological Chemistry

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Edited by Karin Musier-Forsyth Protein chains contain only L-amino acids, with the exception of the achiral glycine, making the chains homochiral. This homochirality is a prerequisite for proper protein folding and, hence, normal cellular function. The importance of D-amino acids as a component of the bacterial cell wall and their roles in neurotransmission in higher eukaryotes are well-established. However, the wider presence and the corresponding physiological roles of these specific amino acid stereoisomers have been appreciated only recently. Therefore, it is expected that enantiomeric fidelity has to be a key component of all of the steps in translation. Cells employ various molecular mechanisms for keeping D-amino acids away from the synthesis of nascent polypeptide chains. The major factors involved in this exclusion are aminoacyl-tRNA synthetases (aaRSs), elongation factor thermo-unstable (EF-Tu), the ribosome, and D-aminoacyl-tRNA deacylase (DTD). aaRS, EF-Tu, and the ribosome act as "chiral checkpoints" by preferentially binding to L-amino acids or L-aminoacyl-tRNAs, thereby excluding D-amino acids. Interestingly, DTD, which is conserved across all life forms, performs "chiral proofreading," as it removes D-amino acids erroneously added to tRNA. Here, we comprehensively review D-amino acids with respect to their occurrence and physiological roles, implications for chiral checkpoints required for translation fidelity, and potential use in synthetic biology. The authors declare that they have no conflicts of interest with the contents of this article.

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“…The probable active site cavity consisting of “SQFT” motif was also found to be well conserved amidst all structures (Bhatt et al, 2010 ). In addition to stand-alone DTD proteins, the DTD-like domain (Pab-NTD) was also found appended to threonyl-tRNA synthetase (ThrRS) of Pyrococcus abyssi with the similar hydrolytic activity of DTD (Hussain et al, 2006 ; Ahmad et al, 2015 ). The presence of a free standing DTD probably would have evolved from ThrRS under extensive pressure of D-amino acid toxicity.…”

Section: Structure Of Dtdmentioning

confidence: 99%

“…However, the mechanism of discrimination between D and L amino acids by DTD enzyme is yet to be elucidated. The enantio-selectivity of the DTD explained by the presence of Gly-Cys-Pro dipeptide in PfDTD which is responsible for maintaining homochirality by selecting only D-amino acids and rejecting L-amino acids (Ahmad et al, 2015 ). Although, most of the studies related to the enzymatic mechanism have been conducted for Plasmodium DTD, but it is assumed that it will be valid for all DTD proteins owing to highly conserved sequences.…”

Section: Enzymatic Mechanism Of Dtdmentioning

confidence: 99%

Recent Updates on DTD (D-Tyr-tRNATyr Deacylase): An Enzyme Essential for Fidelity and Quality of Protein Synthesis

Bhatt

Soni

Sharma

2016

Front. Cell Dev. Biol.

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During protein synthesis, there are several checkpoints in the cell to ensure that the information encoded within genetic material is decoded correctly. Charging of tRNA with its cognate amino acid is one of the important steps in protein synthesis and is carried out by aminoacyl-tRNA synthetase (aaRS) with great accuracy. However, due to presence of D-amino acids in the cell, sometimes aaRS charges tRNA with D-amino acids resulting in the hampering of protein translational process, which is lethal to the cell. Every species has some mechanism in order to prevent the formation of D-amino acid-tRNA complex, for instance DTD (D-Tyr-tRNA deacylase) is an enzyme responsible for the cleavage of ester bond formed between D-amino acid and tRNA leading to error free translation process. In this review, structure, function, and enzymatic mechanism of DTD are discussed. The role of DTD as a drug target is also considered.

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Specificity and catalysis hardwired at the RNA–protein interface in a translational proofreading enzyme

Cited by 24 publications

References 58 publications

Elongation Factor Tu Prevents Misediting of Gly-tRNA(Gly) Caused by the Design Behind the Chiral Proofreading Site of D-Aminoacyl-tRNA Deacylase

Elongation Factor Tu Prevents Misediting of Gly-tRNA(Gly) Caused by the Design Behind the Chiral Proofreading Site of D-Aminoacyl-tRNA Deacylase

Chiral checkpoints during protein biosynthesis

Recent Updates on DTD (D-Tyr-tRNATyr Deacylase): An Enzyme Essential for Fidelity and Quality of Protein Synthesis

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