No abstract

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.

Strict L-chiral rejection through Gly-cisPro motif during chiral proofreading underlies the inability of D-aminoacyl-tRNA deacylase (DTD) to discriminate between D-amino acids and achiral glycine. The consequent Gly-tRNAGly ‘misediting paradox’ is resolved by EF-Tu in the cell. Here, we show that DTD’s active site architecture can efficiently edit mischarged Gly-tRNAAla species four orders of magnitude more efficiently than even AlaRS, the only ubiquitous cellular checkpoint known for clearing the error. Also, DTD knockout in AlaRS editing-defective background causes pronounced toxicity in Escherichia coli even at low-glycine levels which is alleviated by alanine supplementation. We further demonstrate that DTD positively selects the universally invariant tRNAAla-specific G3•U70. Moreover, DTD’s activity on non-cognate Gly-tRNAAla is conserved across all bacteria and eukaryotes, suggesting DTD’s key cellular role as a glycine deacylator. Our study thus reveals a hitherto unknown function of DTD in cracking the universal mechanistic dilemma encountered by AlaRS, and its physiological importance.DOI: http://dx.doi.org/10.7554/eLife.24001.001

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.