- Email: [email protected]
d -Tyrosyl-tRNA Deacylase: A New Function
TIBS 1380 No. of Pages 2
Spotlight
D-Tyrosyl-tRNA
Deacylase: A New Function Richard Calendar1,* D-Aminoacyl-tRNA
deacylase (DTD) hydrolyzes [58_TD$IF]D-amino acids mistakenly attached to tRNAs and, thus, has been implicated in perpetuating protein homochirality. Fifty years after the discovery of DTD, it has now been shown that its function extends beyond ‘chiral proofreading’ because it also eliminates glycine that has been erroneously coupled to tRNAAla[57_TD$IF]. Fifty years ago, as a graduate student in Paul Berg’s laboratory at Stanford University, I purified tyrosyl-tRNA synthetase from Escherichia coli to homogeneity [1]. My colleagues and I found that this enzyme could attach [59_TD$IF]D-tyrosine, as well as [60_TD$IF]L-tyrosine, to tRNATyr [2]. D-TyrosyltRNATyr[80_TD$IF] was degraded in extracts of E. coli, yeast, and rat liver [3]. We called the enzyme that degrades D[62_TD$IF] -tyrosyl-tRNATyr ‘D-tyrosyl-tRNA deacylase’ (DTD). Using the same methods I had used to purify tyrosyl-tRNA synthetase to homogeneity, I was able to purify DTD only 25-fold at the time. More than 30 years later, Soutourina et al. [4] extended my purification and obtained homogeneous DTD from E. coli. They obtained the amino acid sequence of DTD, as well as the DNA sequence of its gene. The authors deleted the gene encoding DTD from E. coli and found the deletion strain to be sensitive to D-tyrosine, D-tryptophan, D-aspartate, D-serine, and D-glutamine [5]. Biochemically, DTD was shown to act on multiple D[5_TD$IF] -aminoacyl-tRNAs besides [61_TD$IF]D-tyrosyl-tRNATyr[63_TD$IF], such as [64_TD$IF]D-phenylalanyl-tRNAPhe, D-aspartyl-tRNAAsp[65_TD$IF], and D[6_TD$IF] -tryptophanyl-tRNATrp [3–5]. Therefore, the enzyme also came to be known as ‘D-aminoacyl-tRNA
deacylase’, although the acronym DTD phenomenon is precluded by the translational elongation factor thermo unstable was retained. (EF-Tu). The latter is responsible for the Despite biochemical characterization of delivery of all aminoacylated tRNAs to DTD and in vivo studies in E. coli and ribosomes for protein synthesis. EF-Tu yeast [3–5], two key aspects of this homo- binds and protects glycyl-tRNAGly, thus dimeric translational proofreading avoiding misediting of the achiral enzyme remained elusive: the mecha- substrate (Figure 1A). nisms of enantioselectivity (i.e., how DTD discriminates between [68_TD$IF]L- and D- Fifty years after the discovery of DTD, the amino acids) and the mechanism of cat- physiological relevance of its activity on alytic removal of the aminoacyl moiety achiral glycine has now been elucidated from the tRNA. Research over the past by Pawar et al. [8]. In addition to charging decade has revealed that DTD is an abso- L-alanine, alanyl-tRNA synthetase (AlaRS) lute configuration-specific enzyme that occasionally mischarges glycine or L[74_TD$IF] -serdoes not cross-react with [69_TD$IF]L-aminoacyl- ine on tRNAAla[73_TD$IF]. Hence, dedicated prooftRNAs, despite the large excess of the reading domains, both as part of the latter in the cellular pool. Thus, the activity enzyme itself (called the cis-editing of DTD has been referred to as ‘chiral domain) or as freestanding modules proofreading’. An invariant Gly-cisPro (called trans-editing domains), have dipeptide motif in DTD is crucial for the evolved to take care of the errors comchiral specificity of the enzyme. The Gly- mitted by AlaRS. However, most of the cisPro motif of one monomer is inserted AlaRS trans-editing domains were previinto the active site of its dimeric counter- ously shown to act on L[75_TD$IF] -seryl-tRNAAla and part. Hence, the two active sites of DTD only poorly on glycyl-tRNAAla. Defects in are present at the dimeric interface [6]. proofreading by AlaRS were demonMutational and biochemical analyses strated to cause severe pathological conhave also demonstrated that DTD does ditions, including neurodegeneration in not require active site protein side chains the mouse [9]. The recent study by Pawar to perform catalysis. The enzyme instead et al. [8] revealed a hitherto unknown uses only the 20 -OH of adenosine-76 at function of DTD, namely, to clear glycylthe 30 terminus of any tRNA to hydrolyze tRNAAla. Biochemical probing has shown its substrates. Thus, DTD is an RNA- that the activity of DTD on glycyl-tRNAAla based catalyst [6,7]. is significantly higher than that of the AlaRS cis-editing domain. The activity of Interestingly, Routh et al. [7] showed that DTD is so high on glycyl-tRNAAla that it the architecture of the chiral proofreading can eliminate the noncognate achiral subsite of DTD is such that it can bind the strate even in the presence of EF-Tu achiral amino acid, glycine, in the active (Figure 1A). This function of DTD has been site pocket. This implies that DTD uses further validated in in vivo assays, wherein strict L[70_TD$IF] -chiral rejection by the Gly-cisPro a knockout of DTD in a AlaRS editingmotif rather than D[71_TD$IF] -chiral selection as the defective background caused marked mechanism for enantioselectivity. Such glycine toxicity in E. coli. This shows that an unprecedented chirality-based exclu- the physiological significance of DTD sionary mechanism allows DTD to avoid extends beyond chiral proofreading durcross-reacting with the abundant L[69_TD$IF] -ami- ing translation of the genetic code. noacyl-tRNA pool. However, the lack of discrimination between D[58_TD$IF] -amino acid and In a nutshell, the recent work by Pawar glycine leads to an untenable scenario in et al. [8] has expanded the horizons of the the cell, since DTD can act on and deplete physiological role of DTD as a proofread[72_TD$IF]glycyl-tRNAGly[67_TD$IF], thereby adversely affect- ing factor, adding a totally unanticipated ing protein synthesis. Such a deleterious dimension to its cellular function. Yet,
Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy
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TIBS 1380 No. of Pages 2
study. Furthermore, a DTD knockout in bacteria or yeast is not lethal per se under laboratory growth conditions [5]. The specific conditions (such as stress) under which DTD knockout would be lethal, as is the case for other proofreading activities, remain to be discovered [10]. Moreover, the physiological relevance of DTD in higher systems, such as the mouse, needs additional probing. This is especially important in the neuronal context, where a correlation exists between higher DTD levels and higher amounts of certain amino acids (e.g., D-serine, D-aspartate, and glycine) that act as neurotransmitters. Nevertheless, it would not be an exaggeration to say that future investigations into the unexplored territories of DTD could bring unforeseen surprises. 1 Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202, USA
*Correspondence: [email protected] (R. Calendar). http://dx.doi.org/10.1016/j.tibs.2017.06.012 References 1. Calendar, R. and Berg, P. (1966) Purification and physical characterization of tyrosyl ribonucleic acid synthetases from Escherichia coli and Bacillus subtilis. Biochemistry 5, 1681–1690 2. Calendar, R. and Berg, P. (1966) The catalytic properties of tyrosyl ribonucleic acid synthetases from Escherichia coli and Bacillus subtilis. Biochemistry 5, 1690–1695 3. Calendar, R. and Berg, P. (1967) D-Tyrosyl RNA: formation, hydrolysis and utilization for protein synthesis. J. Mol. Biol. 26, 39–54
Figure 1. The role of D[5_TD$IF] -aminoacyl-tRNA deacylase (DTD) extends beyond ‘chiral proofreading’. (A) Gly
On the one hand, misediting of Gly-tRNA by DTD is prevented through protection of the cognate achiral substrate by elongation factor thermo unstable (EF-Tu). On the other hand, DTD is able to edit the noncognate Gly-tRNAAla species even in the presence of EF-Tu, thereby bolstering translational fidelity. (B) DTD has higher activity on glycine charged on tRNAAla than on tRNAGly and a single base-pair change to G3U70 (present in tRNAAla) enhances the activity of DTD on tRNAGly, thus indicating the role of tRNA elements in modulating the activity of this translational proofreading enzyme.
4. Soutourina, J. et al. (1999) Functional characterization of the D-Tyr-tRNATyr deacylase from Escherichia coli. J. Biol. Chem. 274, 19109–19114 5. Soutourina, J. et al. (2000) Metabolism of D-aminoacyltRNAs in Escherichia coli and Saccharomyces cerevisiae cells. J. Biol. Chem. 275, 32535–32542 6. Ahmad, S. et al. (2013) Mechanism of chiral proofreading during translation of the genetic code. Elife 2, e01519 7. Routh, S.B. et al. (2016) Elongation Factor Tu prevents misediting of Gly-tRNA(Gly) caused by the design behind the chiral proofreading site of ᴅ-aminoacyl-tRNA deacylase. PLoS Biol. 14, e1002465
several aspects of this chiral proofreading enzyme need to be addressed and deciphered. For instance, there is a significantly higher activity of DTD on glycyltRNAAla compared with that on glycyltRNAGly [8]. This clearly indicates, for
2
the first time, that the tRNA moiety has a major role in modulating the activity of DTD (Figure 1B). Identification of tRNA elements that modulate its activity and the magnitude of their contribution to the process are areas that deserve further
Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy
8. Pawar, K.I. et al. (2017) Role of D-aminoacyl-tRNA deacylase beyond chiral proofreading as a cellular defense against glycine mischarging by AlaRS. Elife 6, e24001 9. Lee, J.W. et al. (2006) Editing-defective tRNA synthetase causes protein misfolding and neurodegeneration. Nature 443, 50–55 10. Kermgard, E. et al. (2017) Quality control by isoleucyl-tRNA synthetase of Bacillus subtilis Is required for efficient sporulation. Sci. Rep. 7, 41763
Spotlight
D-Tyrosyl-tRNA
Deacylase: A New Function Richard Calendar1,* D-Aminoacyl-tRNA
deacylase (DTD) hydrolyzes [58_TD$IF]D-amino acids mistakenly attached to tRNAs and, thus, has been implicated in perpetuating protein homochirality. Fifty years after the discovery of DTD, it has now been shown that its function extends beyond ‘chiral proofreading’ because it also eliminates glycine that has been erroneously coupled to tRNAAla[57_TD$IF]. Fifty years ago, as a graduate student in Paul Berg’s laboratory at Stanford University, I purified tyrosyl-tRNA synthetase from Escherichia coli to homogeneity [1]. My colleagues and I found that this enzyme could attach [59_TD$IF]D-tyrosine, as well as [60_TD$IF]L-tyrosine, to tRNATyr [2]. D-TyrosyltRNATyr[80_TD$IF] was degraded in extracts of E. coli, yeast, and rat liver [3]. We called the enzyme that degrades D[62_TD$IF] -tyrosyl-tRNATyr ‘D-tyrosyl-tRNA deacylase’ (DTD). Using the same methods I had used to purify tyrosyl-tRNA synthetase to homogeneity, I was able to purify DTD only 25-fold at the time. More than 30 years later, Soutourina et al. [4] extended my purification and obtained homogeneous DTD from E. coli. They obtained the amino acid sequence of DTD, as well as the DNA sequence of its gene. The authors deleted the gene encoding DTD from E. coli and found the deletion strain to be sensitive to D-tyrosine, D-tryptophan, D-aspartate, D-serine, and D-glutamine [5]. Biochemically, DTD was shown to act on multiple D[5_TD$IF] -aminoacyl-tRNAs besides [61_TD$IF]D-tyrosyl-tRNATyr[63_TD$IF], such as [64_TD$IF]D-phenylalanyl-tRNAPhe, D-aspartyl-tRNAAsp[65_TD$IF], and D[6_TD$IF] -tryptophanyl-tRNATrp [3–5]. Therefore, the enzyme also came to be known as ‘D-aminoacyl-tRNA
deacylase’, although the acronym DTD phenomenon is precluded by the translational elongation factor thermo unstable was retained. (EF-Tu). The latter is responsible for the Despite biochemical characterization of delivery of all aminoacylated tRNAs to DTD and in vivo studies in E. coli and ribosomes for protein synthesis. EF-Tu yeast [3–5], two key aspects of this homo- binds and protects glycyl-tRNAGly, thus dimeric translational proofreading avoiding misediting of the achiral enzyme remained elusive: the mecha- substrate (Figure 1A). nisms of enantioselectivity (i.e., how DTD discriminates between [68_TD$IF]L- and D- Fifty years after the discovery of DTD, the amino acids) and the mechanism of cat- physiological relevance of its activity on alytic removal of the aminoacyl moiety achiral glycine has now been elucidated from the tRNA. Research over the past by Pawar et al. [8]. In addition to charging decade has revealed that DTD is an abso- L-alanine, alanyl-tRNA synthetase (AlaRS) lute configuration-specific enzyme that occasionally mischarges glycine or L[74_TD$IF] -serdoes not cross-react with [69_TD$IF]L-aminoacyl- ine on tRNAAla[73_TD$IF]. Hence, dedicated prooftRNAs, despite the large excess of the reading domains, both as part of the latter in the cellular pool. Thus, the activity enzyme itself (called the cis-editing of DTD has been referred to as ‘chiral domain) or as freestanding modules proofreading’. An invariant Gly-cisPro (called trans-editing domains), have dipeptide motif in DTD is crucial for the evolved to take care of the errors comchiral specificity of the enzyme. The Gly- mitted by AlaRS. However, most of the cisPro motif of one monomer is inserted AlaRS trans-editing domains were previinto the active site of its dimeric counter- ously shown to act on L[75_TD$IF] -seryl-tRNAAla and part. Hence, the two active sites of DTD only poorly on glycyl-tRNAAla. Defects in are present at the dimeric interface [6]. proofreading by AlaRS were demonMutational and biochemical analyses strated to cause severe pathological conhave also demonstrated that DTD does ditions, including neurodegeneration in not require active site protein side chains the mouse [9]. The recent study by Pawar to perform catalysis. The enzyme instead et al. [8] revealed a hitherto unknown uses only the 20 -OH of adenosine-76 at function of DTD, namely, to clear glycylthe 30 terminus of any tRNA to hydrolyze tRNAAla. Biochemical probing has shown its substrates. Thus, DTD is an RNA- that the activity of DTD on glycyl-tRNAAla based catalyst [6,7]. is significantly higher than that of the AlaRS cis-editing domain. The activity of Interestingly, Routh et al. [7] showed that DTD is so high on glycyl-tRNAAla that it the architecture of the chiral proofreading can eliminate the noncognate achiral subsite of DTD is such that it can bind the strate even in the presence of EF-Tu achiral amino acid, glycine, in the active (Figure 1A). This function of DTD has been site pocket. This implies that DTD uses further validated in in vivo assays, wherein strict L[70_TD$IF] -chiral rejection by the Gly-cisPro a knockout of DTD in a AlaRS editingmotif rather than D[71_TD$IF] -chiral selection as the defective background caused marked mechanism for enantioselectivity. Such glycine toxicity in E. coli. This shows that an unprecedented chirality-based exclu- the physiological significance of DTD sionary mechanism allows DTD to avoid extends beyond chiral proofreading durcross-reacting with the abundant L[69_TD$IF] -ami- ing translation of the genetic code. noacyl-tRNA pool. However, the lack of discrimination between D[58_TD$IF] -amino acid and In a nutshell, the recent work by Pawar glycine leads to an untenable scenario in et al. [8] has expanded the horizons of the the cell, since DTD can act on and deplete physiological role of DTD as a proofread[72_TD$IF]glycyl-tRNAGly[67_TD$IF], thereby adversely affect- ing factor, adding a totally unanticipated ing protein synthesis. Such a deleterious dimension to its cellular function. Yet,
Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy
1
TIBS 1380 No. of Pages 2
study. Furthermore, a DTD knockout in bacteria or yeast is not lethal per se under laboratory growth conditions [5]. The specific conditions (such as stress) under which DTD knockout would be lethal, as is the case for other proofreading activities, remain to be discovered [10]. Moreover, the physiological relevance of DTD in higher systems, such as the mouse, needs additional probing. This is especially important in the neuronal context, where a correlation exists between higher DTD levels and higher amounts of certain amino acids (e.g., D-serine, D-aspartate, and glycine) that act as neurotransmitters. Nevertheless, it would not be an exaggeration to say that future investigations into the unexplored territories of DTD could bring unforeseen surprises. 1 Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202, USA
*Correspondence: [email protected] (R. Calendar). http://dx.doi.org/10.1016/j.tibs.2017.06.012 References 1. Calendar, R. and Berg, P. (1966) Purification and physical characterization of tyrosyl ribonucleic acid synthetases from Escherichia coli and Bacillus subtilis. Biochemistry 5, 1681–1690 2. Calendar, R. and Berg, P. (1966) The catalytic properties of tyrosyl ribonucleic acid synthetases from Escherichia coli and Bacillus subtilis. Biochemistry 5, 1690–1695 3. Calendar, R. and Berg, P. (1967) D-Tyrosyl RNA: formation, hydrolysis and utilization for protein synthesis. J. Mol. Biol. 26, 39–54
Figure 1. The role of D[5_TD$IF] -aminoacyl-tRNA deacylase (DTD) extends beyond ‘chiral proofreading’. (A) Gly
On the one hand, misediting of Gly-tRNA by DTD is prevented through protection of the cognate achiral substrate by elongation factor thermo unstable (EF-Tu). On the other hand, DTD is able to edit the noncognate Gly-tRNAAla species even in the presence of EF-Tu, thereby bolstering translational fidelity. (B) DTD has higher activity on glycine charged on tRNAAla than on tRNAGly and a single base-pair change to G3U70 (present in tRNAAla) enhances the activity of DTD on tRNAGly, thus indicating the role of tRNA elements in modulating the activity of this translational proofreading enzyme.
4. Soutourina, J. et al. (1999) Functional characterization of the D-Tyr-tRNATyr deacylase from Escherichia coli. J. Biol. Chem. 274, 19109–19114 5. Soutourina, J. et al. (2000) Metabolism of D-aminoacyltRNAs in Escherichia coli and Saccharomyces cerevisiae cells. J. Biol. Chem. 275, 32535–32542 6. Ahmad, S. et al. (2013) Mechanism of chiral proofreading during translation of the genetic code. Elife 2, e01519 7. Routh, S.B. et al. (2016) Elongation Factor Tu prevents misediting of Gly-tRNA(Gly) caused by the design behind the chiral proofreading site of ᴅ-aminoacyl-tRNA deacylase. PLoS Biol. 14, e1002465
several aspects of this chiral proofreading enzyme need to be addressed and deciphered. For instance, there is a significantly higher activity of DTD on glycyltRNAAla compared with that on glycyltRNAGly [8]. This clearly indicates, for
2
the first time, that the tRNA moiety has a major role in modulating the activity of DTD (Figure 1B). Identification of tRNA elements that modulate its activity and the magnitude of their contribution to the process are areas that deserve further
Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy
8. Pawar, K.I. et al. (2017) Role of D-aminoacyl-tRNA deacylase beyond chiral proofreading as a cellular defense against glycine mischarging by AlaRS. Elife 6, e24001 9. Lee, J.W. et al. (2006) Editing-defective tRNA synthetase causes protein misfolding and neurodegeneration. Nature 443, 50–55 10. Kermgard, E. et al. (2017) Quality control by isoleucyl-tRNA synthetase of Bacillus subtilis Is required for efficient sporulation. Sci. Rep. 7, 41763