Amino Acid Activation

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Charles W. Carter - One of the best experts on this subject based on the ideXlab platform.

  • The Rodin-Ohno hypothesis that two enzyme superfamilies descended from one ancestral gene: an unlikely scenario for the origins of translation that will not be dismissed
    Biology Direct, 2014
    Co-Authors: Charles W. Carter, Violetta Weinreb, Li Li, Martha Collier, Katiria Gonzalez-rivera, Mariel Jimenez-rodriguez, Ozgün Erdogan, Brian Kuhlman, Xavier Ambroggio, Tishan Williams
    Abstract:

    Background Because Amino Acid Activation is rate-limiting for uncatalyzed protein synthesis, it is a key puzzle in understanding the origin of the genetic code. Two unrelated classes (I and II) of contemporary Aminoacyl-tRNA synthetases (aaRS) now translate the code. Observing that codons for the most highly conserved, Class I catalytic peptides, when read in the reverse direction, are very nearly anticodons for Class II defining catalytic peptides, Rodin and Ohno proposed that the two superfamilies descended from opposite strands of the same ancestral gene. This unusual hypothesis languished for a decade, perhaps because it appeared to be unfalsifiable. Results The proposed sense/antisense alignment makes important predictions. Fragments that align in antiparallel orientations, and contain the respective active sites, should catalyze the same two reactions catalyzed by contemporary synthetases. Recent experiments confirmed that prediction. Invariant cores from both classes, called Urzymes after Ur = primitive, authentic, plus enzyme and representing ~20% of the contemporary structures, can be expressed and exhibit high, proportionate rate accelerations for both Amino-Acid Activation and tRNA acylation. A major fraction (60%) of the catalytic rate acceleration by contemporary synthetases resides in segments that align sense/antisense. Bioinformatic evidence for sense/antisense ancestry extends to codons specifying the invariant secondary and tertiary structures outside the active sites of the two synthetase classes. Peptides from a designed, 46-residue gene constrained by Rosetta to encode Class I and II ATP binding sites with fully complementary sequences both accelerate Amino Acid Activation by ATP ~400 fold. Conclusions Biochemical and bioinformatic results substantially enhance the posterior probability that ancestors of the two synthetase classes arose from opposite strands of the same ancestral gene. The remarkable acceleration by short peptides of the rate-limiting step in uncatalyzed protein synthesis, together with the synergy of synthetase Urzymes and their cognate tRNAs, introduce a new paradigm for the origin of protein catalysts, emphasize the potential relevance of an operational RNA code embedded in the tRNA acceptor stems, and challenge the RNA-World hypothesis. Reviewers This article was reviewed by Dr. Paul Schimmel (nominated by Laura Landweber), Dr. Eugene Koonin and Professor David Ardell.

  • Aminoacylating urzymes challenge the rna world hypothesis
    Journal of Biological Chemistry, 2013
    Co-Authors: Li Li, Christopher S Francklyn, Charles W. Carter
    Abstract:

    Abstract We describe experimental evidence that ancestral peptide catalysts substantially accelerated development of genetic coding. Structurally invariant 120–130-residue Urzymes (Ur = primitive plus enzyme) derived from Class I and Class II Aminoacyl-tRNA synthetases (aaRSs) acylate tRNA far faster than the uncatalyzed rate of nonribosomal peptide bond formation from activated Amino Acids. These new data allow us to demonstrate statistically indistinguishable catalytic profiles for Class I and II aaRSs in both Amino Acid Activation and tRNA acylation, over a time period extending to well before the assembly of full-length enzymes and even further before the Last Universal Common Ancestor. Both Urzymes also exhibit ∼60% of the contemporary catalytic proficiencies. Moreover, they are linked by ancestral sense/antisense genetic coding, and their evident modularities suggest descent from even simpler ancestral pairs also coded by opposite strands of the same gene. Thus, aaRS Urzymes substantially pre-date modern aaRS but are, nevertheless, highly evolved. Their unexpectedly advanced catalytic repertoires, sense/antisense coding, and ancestral modularities imply considerable prior protein-tRNA co-evolution. Further, unlike ribozymes that motivated the RNA World hypothesis, Class I and II Urzyme·tRNA pairs represent consensus ancestral forms sufficient for codon-directed synthesis of nonrandom peptides. By tracing aaRS catalytic activities back to simpler ancestral peptides, we demonstrate key steps for a simpler and hence more probable peptide·RNA development of rapid coding systems matching Amino Acids with anticodon trinucleotides.

  • histidyl trna synthetase urzymes class i and ii Aminoacyl trna synthetase urzymes have comparable catalytic activities for cognate Amino Acid Activation
    Journal of Biological Chemistry, 2011
    Co-Authors: Li Li, Christopher S Francklyn, Violetta Weinreb, Charles W. Carter
    Abstract:

    Abstract Four minimal (119 - 145 residue) active site fragments of Escherichia coli Class II histidyl-tRNA synthetase were constructed, expressed as maltose-binding protein fusions, and assayed for histidine Activation as fusion proteins and after TEV cleavage, using the 32PPi exchange assay. All contain conserved Motifs 1 and 2. Two contain an N-terminal extension of Motif 1 and two contain Motif 3. Five experimental results argue strongly for the authenticity of the observed catalytic activities: (i) active site titration experiments showing high (∼0.1–0.55) fractions of active molecules, (ii) release of cryptic activity by TEV cleavage of the fusion proteins, (iii) reduced activity associated with an active site mutation, (iv) quantitative attribution of increased catalytic activity to the intrinsic effects of Motif 3, the N-terminal extension and their synergistic effect, and (v) significantly altered Km values for both ATP and histidine substrates. It is therefore plausible that neither the insertion domain nor Motif 3 were essential for catalytic activity in the earliest Class II Aminoacyl-tRNA synthetases. The mean rate enhancement of all four cleaved constructs is ∼109 times that of the estimated uncatalyzed rate. As observed for the tryptophanyl-tRNA synthetase (TrpRS) Urzyme, these fragments bind ATP tightly but have reduced affinity for cognate Amino Acids. These fragments thus likely represent Urzymes (Ur = primitive, original, earliest + enzyme) comparable in size and catalytic activity and coded by sequences proposed to be antisense to that coding the previously described Class I TrpRS Urzyme. Their catalytic activities provide metrics for experimental recapitulation of very early evolutionary events.

  • mg2 free bacillus stearothermophilus tryptophanyl trna synthetase retains a major fraction of the overall rate enhancement for tryptophan Activation
    Journal of the American Chemical Society, 2008
    Co-Authors: Violetta Weinreb, Charles W. Carter
    Abstract:

    Few experimental data are available for rates of enzymatic phosphoryl-transfer reactions in the absence of the divalent metal ions associated with such reactions. Such data are of interest for Amino Acid Activation by class Ic Aminoacyl-tRNA synthetases, for which there is substantial evidence that binding energy of ATP may account for a major fraction of the overall rate enhancement, and it is crucial to know if these effects themselves depend on the divalent metal ion. We describe a nested, nonlinear model for the sum of metal-free and metal-catalyzed activities and its use in determining metal-free enzyme activity jointly with transition-state metal binding affinity, by fitting observed values obtained from Mg2+-depleted assays with increasing [EDTA] at known [Mg2+]total. Tryptophan Activation by Bacillus stearothermophilus tryptophanyl-tRNA synthetase falls asymptotically to a plateau value 5 orders of magnitude below that observed for the Mg2+-supplemented enzyme at EDTA concentrations that reduce th...

  • crystal structure of tryptophanyl trna synthetase complexed with adenosine 5 tetraphosphate evidence for distributed use of catalytic binding energy in Amino Acid Activation by class i Aminoacyl trna synthetases
    Journal of Molecular Biology, 2007
    Co-Authors: Pascal Retailleau, Violetta Weinreb, Mei Hu, Charles W. Carter
    Abstract:

    Abstract Tryptophanyl-tRNA synthetase (TrpRS) is a functionally dimeric ligase, which specifically couples hydrolysis of ATP to AMP and pyrophosphate to the formation of an ester bond between tryptophan and the cognate tRNA. TrpRS from Bacillus stearothermophilus binds the ATP analogue, adenosine-5′ tetraphosphate (AQP) competitively with ATP during pyrophosphate exchange. Estimates of binding affinity from this competitive inhibition and from isothermal titration calorimetry show that AQP binds 200 times more tightly than ATP both under conditions of induced-fit, where binding is coupled to an unfavorable conformational change, and under exchange conditions, where there is no conformational change. These binding data provide an indirect experimental measurement of + 3.0 kcal/mol for the conformational free energy change associated with induced-fit assembly of the active site. Thermodynamic parameters derived from the calorimetry reveal very modest enthalpic changes, consistent with binding driven largely by a favorable entropy change. The 2.5 A structure of the TrpRS:AQP complex, determined de novo by X-ray crystallography, resembles that of the previously described, pre-transition state TrpRS:ATP complexes. The anticodon-binding domain untwists relative to the Rossmann-fold domain by 20% of the way toward the orientation observed for the Products complex. An unexpected tetraphosphate conformation allows the γ and δd phosphate groups to occupy positions equivalent to those occupied by the β and γ phosphates of ATP. The β-phosphate effects a 1.11 A extension that relocates the α-phosphate toward the tryptophan carboxylate while the PPi mimic moves deeper into the KMSKS loop. This configuration improves interactions between enzyme and nucleotide significantly and uniformly in the adenosine and PPi binding subsites. A new hydrogen bond forms between S194 from the class I KMSKS signature sequence and the PPi mimic. These complementary thermodynamic and structural data are all consistent with the conclusion that the tetraphosphate mimics a transition-state in which the KMSKS loop develops increasingly tight bonds to the PPi leaving group, weakening linkage to the Pα as it is relocated by an energetically favorable domain movement. Consistent with extensive mutational data on Tyrosyl-tRNA synthetase, this aspect of the mechanism develops high transition-state affinity for the adenosine and pyrophosphate moieties, which move significantly, relative to one another, during the catalytic step.

Violetta Weinreb - One of the best experts on this subject based on the ideXlab platform.

  • The Rodin-Ohno hypothesis that two enzyme superfamilies descended from one ancestral gene: an unlikely scenario for the origins of translation that will not be dismissed
    Biology Direct, 2014
    Co-Authors: Charles W. Carter, Violetta Weinreb, Li Li, Martha Collier, Katiria Gonzalez-rivera, Mariel Jimenez-rodriguez, Ozgün Erdogan, Brian Kuhlman, Xavier Ambroggio, Tishan Williams
    Abstract:

    Background Because Amino Acid Activation is rate-limiting for uncatalyzed protein synthesis, it is a key puzzle in understanding the origin of the genetic code. Two unrelated classes (I and II) of contemporary Aminoacyl-tRNA synthetases (aaRS) now translate the code. Observing that codons for the most highly conserved, Class I catalytic peptides, when read in the reverse direction, are very nearly anticodons for Class II defining catalytic peptides, Rodin and Ohno proposed that the two superfamilies descended from opposite strands of the same ancestral gene. This unusual hypothesis languished for a decade, perhaps because it appeared to be unfalsifiable. Results The proposed sense/antisense alignment makes important predictions. Fragments that align in antiparallel orientations, and contain the respective active sites, should catalyze the same two reactions catalyzed by contemporary synthetases. Recent experiments confirmed that prediction. Invariant cores from both classes, called Urzymes after Ur = primitive, authentic, plus enzyme and representing ~20% of the contemporary structures, can be expressed and exhibit high, proportionate rate accelerations for both Amino-Acid Activation and tRNA acylation. A major fraction (60%) of the catalytic rate acceleration by contemporary synthetases resides in segments that align sense/antisense. Bioinformatic evidence for sense/antisense ancestry extends to codons specifying the invariant secondary and tertiary structures outside the active sites of the two synthetase classes. Peptides from a designed, 46-residue gene constrained by Rosetta to encode Class I and II ATP binding sites with fully complementary sequences both accelerate Amino Acid Activation by ATP ~400 fold. Conclusions Biochemical and bioinformatic results substantially enhance the posterior probability that ancestors of the two synthetase classes arose from opposite strands of the same ancestral gene. The remarkable acceleration by short peptides of the rate-limiting step in uncatalyzed protein synthesis, together with the synergy of synthetase Urzymes and their cognate tRNAs, introduce a new paradigm for the origin of protein catalysts, emphasize the potential relevance of an operational RNA code embedded in the tRNA acceptor stems, and challenge the RNA-World hypothesis. Reviewers This article was reviewed by Dr. Paul Schimmel (nominated by Laura Landweber), Dr. Eugene Koonin and Professor David Ardell.

  • histidyl trna synthetase urzymes class i and ii Aminoacyl trna synthetase urzymes have comparable catalytic activities for cognate Amino Acid Activation
    Journal of Biological Chemistry, 2011
    Co-Authors: Li Li, Christopher S Francklyn, Violetta Weinreb, Charles W. Carter
    Abstract:

    Abstract Four minimal (119 - 145 residue) active site fragments of Escherichia coli Class II histidyl-tRNA synthetase were constructed, expressed as maltose-binding protein fusions, and assayed for histidine Activation as fusion proteins and after TEV cleavage, using the 32PPi exchange assay. All contain conserved Motifs 1 and 2. Two contain an N-terminal extension of Motif 1 and two contain Motif 3. Five experimental results argue strongly for the authenticity of the observed catalytic activities: (i) active site titration experiments showing high (∼0.1–0.55) fractions of active molecules, (ii) release of cryptic activity by TEV cleavage of the fusion proteins, (iii) reduced activity associated with an active site mutation, (iv) quantitative attribution of increased catalytic activity to the intrinsic effects of Motif 3, the N-terminal extension and their synergistic effect, and (v) significantly altered Km values for both ATP and histidine substrates. It is therefore plausible that neither the insertion domain nor Motif 3 were essential for catalytic activity in the earliest Class II Aminoacyl-tRNA synthetases. The mean rate enhancement of all four cleaved constructs is ∼109 times that of the estimated uncatalyzed rate. As observed for the tryptophanyl-tRNA synthetase (TrpRS) Urzyme, these fragments bind ATP tightly but have reduced affinity for cognate Amino Acids. These fragments thus likely represent Urzymes (Ur = primitive, original, earliest + enzyme) comparable in size and catalytic activity and coded by sequences proposed to be antisense to that coding the previously described Class I TrpRS Urzyme. Their catalytic activities provide metrics for experimental recapitulation of very early evolutionary events.

  • mg2 free bacillus stearothermophilus tryptophanyl trna synthetase retains a major fraction of the overall rate enhancement for tryptophan Activation
    Journal of the American Chemical Society, 2008
    Co-Authors: Violetta Weinreb, Charles W. Carter
    Abstract:

    Few experimental data are available for rates of enzymatic phosphoryl-transfer reactions in the absence of the divalent metal ions associated with such reactions. Such data are of interest for Amino Acid Activation by class Ic Aminoacyl-tRNA synthetases, for which there is substantial evidence that binding energy of ATP may account for a major fraction of the overall rate enhancement, and it is crucial to know if these effects themselves depend on the divalent metal ion. We describe a nested, nonlinear model for the sum of metal-free and metal-catalyzed activities and its use in determining metal-free enzyme activity jointly with transition-state metal binding affinity, by fitting observed values obtained from Mg2+-depleted assays with increasing [EDTA] at known [Mg2+]total. Tryptophan Activation by Bacillus stearothermophilus tryptophanyl-tRNA synthetase falls asymptotically to a plateau value 5 orders of magnitude below that observed for the Mg2+-supplemented enzyme at EDTA concentrations that reduce th...

  • crystal structure of tryptophanyl trna synthetase complexed with adenosine 5 tetraphosphate evidence for distributed use of catalytic binding energy in Amino Acid Activation by class i Aminoacyl trna synthetases
    Journal of Molecular Biology, 2007
    Co-Authors: Pascal Retailleau, Violetta Weinreb, Mei Hu, Charles W. Carter
    Abstract:

    Abstract Tryptophanyl-tRNA synthetase (TrpRS) is a functionally dimeric ligase, which specifically couples hydrolysis of ATP to AMP and pyrophosphate to the formation of an ester bond between tryptophan and the cognate tRNA. TrpRS from Bacillus stearothermophilus binds the ATP analogue, adenosine-5′ tetraphosphate (AQP) competitively with ATP during pyrophosphate exchange. Estimates of binding affinity from this competitive inhibition and from isothermal titration calorimetry show that AQP binds 200 times more tightly than ATP both under conditions of induced-fit, where binding is coupled to an unfavorable conformational change, and under exchange conditions, where there is no conformational change. These binding data provide an indirect experimental measurement of + 3.0 kcal/mol for the conformational free energy change associated with induced-fit assembly of the active site. Thermodynamic parameters derived from the calorimetry reveal very modest enthalpic changes, consistent with binding driven largely by a favorable entropy change. The 2.5 A structure of the TrpRS:AQP complex, determined de novo by X-ray crystallography, resembles that of the previously described, pre-transition state TrpRS:ATP complexes. The anticodon-binding domain untwists relative to the Rossmann-fold domain by 20% of the way toward the orientation observed for the Products complex. An unexpected tetraphosphate conformation allows the γ and δd phosphate groups to occupy positions equivalent to those occupied by the β and γ phosphates of ATP. The β-phosphate effects a 1.11 A extension that relocates the α-phosphate toward the tryptophan carboxylate while the PPi mimic moves deeper into the KMSKS loop. This configuration improves interactions between enzyme and nucleotide significantly and uniformly in the adenosine and PPi binding subsites. A new hydrogen bond forms between S194 from the class I KMSKS signature sequence and the PPi mimic. These complementary thermodynamic and structural data are all consistent with the conclusion that the tetraphosphate mimics a transition-state in which the KMSKS loop develops increasingly tight bonds to the PPi leaving group, weakening linkage to the Pα as it is relocated by an energetically favorable domain movement. Consistent with extensive mutational data on Tyrosyl-tRNA synthetase, this aspect of the mechanism develops high transition-state affinity for the adenosine and pyrophosphate moieties, which move significantly, relative to one another, during the catalytic step.

  • Crystal structure of tryptophanyl-tRNA synthetase complexed with adenosine-5' tetraphosphate: evidence for distributed use of catalytic binding energy in Amino Acid Activation by class I Aminoacyl-tRNA synthetases.
    Journal of Molecular Biology, 2007
    Co-Authors: Pascal Retailleau, Violetta Weinreb, Charles W. Carter
    Abstract:

    Tryptophanyl-tRNA synthetase (TrpRS) is a functionally dimeric ligase, which specifically couples hydrolysis of ATP to AMP and pyrophosphate to the formation of an ester bond between tryptophan and the cognate tRNA. TrpRS from Bacillus stearothermophilus binds the ATP analogue, adenosine-5' tetraphosphate (AQP) competitively with ATP during pyrophosphate exchange. Estimates of binding affinity from this competitive inhibition and from isothermal titration calorimetry show that AQP binds 200 times more tightly than ATP both under conditions of induced-fit, where binding is coupled to an unfavorable conformational change, and under exchange conditions, where there is no conformational change. These binding data provide an indirect experimental measurement of +3.0 kcal/mol for the conformational free energy change associated with induced-fit assembly of the active site. Thermodynamic parameters derived from the calorimetry reveal very modest enthalpic changes, consistent with binding driven largely by a favorable entropy change. The 2.5 A structure of the TrpRS:AQP complex, determined de novo by X-ray crystallography, resembles that of the previously described, pre-transition state TrpRS:ATP complexes. The anticodon-binding domain untwists relative to the Rossmann-fold domain by 20% of the way toward the orientation observed for the Products complex. An unexpected tetraphosphate conformation allows the gamma and deltad phosphate groups to occupy positions equivalent to those occupied by the beta and gamma phosphates of ATP. The beta-phosphate effects a 1.11 A extension that relocates the alpha-phosphate toward the tryptophan carboxylate while the PPi mimic moves deeper into the KMSKS loop. This configuration improves interactions between enzyme and nucleotide significantly and uniformly in the adenosine and PPi binding subsites. A new hydrogen bond forms between S194 from the class I KMSKS signature sequence and the PPi mimic. These complementary thermodynamic and structural data are all consistent with the conclusion that the tetraphosphate mimics a transition-state in which the KMSKS loop develops increasingly tight bonds to the PPi leaving group, weakening linkage to the Palpha as it is relocated by an energetically favorable domain movement. Consistent with extensive mutational data on Tyrosyl-tRNA synthetase, this aspect of the mechanism develops high transition-state affinity for the adenosine and pyrophosphate moieties, which move significantly, relative to one another, during the catalytic step.

En-duo Wang - One of the best experts on this subject based on the ideXlab platform.

  • acetylation of lysine ϵ Amino groups regulates Aminoacyl trna synthetase activity in escherichia coli
    Journal of Biological Chemistry, 2017
    Co-Authors: Qing Ye, Quanquan Ji, Fang Yang, En-duo Wang
    Abstract:

    Abstract Previous proteomic analyses have shown that Aminoacyl-tRNA synthetases (aaRSs) in many organisms can be modified by acetylation of lysine (Lys). In this present study, leucyl-tRNA synthetase and arginyl-tRNA synthetase from Escherichia coli (EcLeuRS and EcArgRS) were overexpressed and purified, and found to be acetylated on Lys residues by mass spectrometry (MS). Glutamine (Gln) scanning mutagenesis revealed that Lys619, Lys624 and Lys809 in EcLeuRS and Lys126 and Lys408 in EcArgRS might play important roles in enzyme activity. Furthermore, we utilized a novel protein expression system to obtain enzymes harboring acetylated-Lys (AcK) at specific sites, and investigated their catalytic activity. Acetylation of these Lys residues could affect their Aminoacylation activity by influencing Amino Acid Activation and/or the affinity for tRNA. In vitro assays showed that acetyl-phosphate (AcP) nonenzymatically acetylates EcLeuRS and EcArgRS, and suggested the sirtuin class deacetylase CobB might regulate acetylation of these two enzymes. These findings imply a potential regulatory role for Lys acetylation in controlling the activity of aaRSs and thus protein synthesis.

  • Functional characterization of leucine-specific domain 1 from eukaryal and archaeal leucyl-tRNA synthetases.
    Biochemical Journal, 2010
    Co-Authors: Xiao-long Zhou, Min Tan, Gilbert Eriani, Meng Wang, Qian Huang, En-duo Wang
    Abstract:

    LeuRS (leucyl-tRNA synthetase) catalyses the esterification of tRNAsLeu with leucine. This family of enzymes is divided into prokaryotic and eukaryal/archaeal groups according to the presence and position of specific insertions and extensions. In the present study, we investigated the function of LSD1 (leucine-specific domain 1), which is naturally present in eukaryal/archaeal LeuRSs, but absent from prokaryotic LeuRSs. When mutated in their common domain, the eukaryal and archaeal LeuRSs exhibited defects in the first reaction step of Amino Acid Activation with variations of leucine or ATP-binding strength, whereas the tRNA Aminoacylation was moderately affected. When the eukaryal extension was mutated, severe tRNA charging defects were observed, suggesting that eukaryotes evolved this LSD1 extension in order to improve the Aminoacylation reaction step. The results also showed that the LSD1s from organisms of both groups are dispensable for post-transfer editing. Together, the data provide us with a further understanding of the organization and structure of LeuRS domains.

  • tRNA-independent pretransfer editing by class I leucyl-tRNA synthetase.
    Journal of Biological Chemistry, 2009
    Co-Authors: Bin Zhu, Peng Yao, Min Tan, Gilbert Eriani, En-duo Wang
    Abstract:

    Aminoacyl-tRNA synthetases catalyze the formation of Aminoacyl-tRNA in a two-step reaction starting with Amino Acid Activation followed by Aminoacyl group transfer to tRNA. To clear mistakes that occasionally occur, some of these enzymes carry out editing activities, acting on the misactivated Amino Acid (pretransfer editing) or after the transfer on the tRNA (post-transfer editing). The post-transfer editing pathway of leucyl-tRNA synthetase has been extensively studied by structural and biochemical approaches. Here, we report the finding of a tRNA-independent pretransfer editing pathway in leucyl-tRNA synthetases from Aquifex aeolicus. Using a CP1-mutant defective in its post-transfer editing function, we showed that this new editing pathway is distinct from the post-transfer editing site and may occur at the synthetic catalytic site, as recently proposed for other Aminoacyl-tRNA synthetases.

  • The CP2 domain of leucyl-tRNA synthetase is crucial for Amino Acid Activation and post-transfer editing.
    The Journal of biological chemistry, 2008
    Co-Authors: Xiao-long Zhou, Bin Zhu, En-duo Wang
    Abstract:

    Leucyl-tRNA synthetase (LeuRS) has an insertion domain, called connective peptide 2 (CP2), either directly preceding or following the editing domain (CP1 domain), depending on the species. The global structures of the CP2 domains from all LeuRSs are similar. Although the CP1 domain has been extensively explored to be responsible for hydrolysis of mischarged tRNALeu, the role of the CP2 domain remains undefined. In the present work, deletion of the CP2 domain of Giardia lamblia LeuRS (GlLeuRS) showed that the CP2 domain is indispensable for Amino Acid Activation and post-transfer editing and that it contributes to LeuRS-tRNALeu binding affinity. In addition, its functions are conserved in both eukaryotic/archaeal and prokaryotic LeuRSs from G. lamblia, Pyrococcus horikoshii (PhLeuRS), and Escherichia coli (EcLeuRS). Alanine scanning and site-directed mutagenesis assays of the CP2 domain identified several residues that are crucial for its various functions. Data from the chimeric mutants, which replaced the CP2 domain of GlLeuRS with either PhLeuRS or EcLeuRS, showed that the CP2 domain of PhLeuRS but not that of EcLeuRS can partially restore Amino Acid Activation and post-transfer editing functions, suggesting that the functions of the CP2 domain are dependent on its location in the primary sequence of LeuRS.

  • Arginyl-tRNA synthetase with signature sequence KMSK from Bacillus stearothermophilus.
    Biochemical Journal, 2003
    Co-Authors: Juan Li, En-duo Wang
    Abstract:

    ArgRS (arginyl-tRNA synthetase) belongs to the class I aaRSs (Aminoacyl-tRNA synthetases), though the majority of ArgRS species lack the canonical KMSK sequence characteristic of class I aaRSs. A DNA fragment of the ArgRS gene from Bacillus stearothermophilus was amplified using primers designed according to the conserved regions of known ArgRSs. Through analysis of the amplified DNA sequence and known tRNA(Arg)s with a published genomic sequence of B. stearothermophilus, the gene encoding ArgRS ( argS ') was amplified by PCR and the gene encoding tRNA(Arg) (ACG) was synthesized. ArgRS contained 557 Amino Acid residues including the canonical KMKS sequence. Recombinant ArgRS and tRNA(Arg) (ACG) were expressed in Escherichia coli. ArgRS purified by nickel-affinity chromatography had no ATPase activity. The kinetics of ArgRS and cross-recognition between ArgRSs and tRNA(Arg)s from B. stearothermophilus and E. coli were studied. The activities of B. stearothermophilus ArgRS mutated at Lys(382) and Lys(385) of the KMSK sequence and at Gly(136) upstream of the HIGH loop were determined. From the mutation results, we concluded that there was mutual compensation of Lys(385) and Gly(136) for the Amino Acid-Activation activity of B. stearothermophilus ArgRS.

Li Li - One of the best experts on this subject based on the ideXlab platform.

  • The Rodin-Ohno hypothesis that two enzyme superfamilies descended from one ancestral gene: an unlikely scenario for the origins of translation that will not be dismissed
    Biology Direct, 2014
    Co-Authors: Charles W. Carter, Violetta Weinreb, Li Li, Martha Collier, Katiria Gonzalez-rivera, Mariel Jimenez-rodriguez, Ozgün Erdogan, Brian Kuhlman, Xavier Ambroggio, Tishan Williams
    Abstract:

    Background Because Amino Acid Activation is rate-limiting for uncatalyzed protein synthesis, it is a key puzzle in understanding the origin of the genetic code. Two unrelated classes (I and II) of contemporary Aminoacyl-tRNA synthetases (aaRS) now translate the code. Observing that codons for the most highly conserved, Class I catalytic peptides, when read in the reverse direction, are very nearly anticodons for Class II defining catalytic peptides, Rodin and Ohno proposed that the two superfamilies descended from opposite strands of the same ancestral gene. This unusual hypothesis languished for a decade, perhaps because it appeared to be unfalsifiable. Results The proposed sense/antisense alignment makes important predictions. Fragments that align in antiparallel orientations, and contain the respective active sites, should catalyze the same two reactions catalyzed by contemporary synthetases. Recent experiments confirmed that prediction. Invariant cores from both classes, called Urzymes after Ur = primitive, authentic, plus enzyme and representing ~20% of the contemporary structures, can be expressed and exhibit high, proportionate rate accelerations for both Amino-Acid Activation and tRNA acylation. A major fraction (60%) of the catalytic rate acceleration by contemporary synthetases resides in segments that align sense/antisense. Bioinformatic evidence for sense/antisense ancestry extends to codons specifying the invariant secondary and tertiary structures outside the active sites of the two synthetase classes. Peptides from a designed, 46-residue gene constrained by Rosetta to encode Class I and II ATP binding sites with fully complementary sequences both accelerate Amino Acid Activation by ATP ~400 fold. Conclusions Biochemical and bioinformatic results substantially enhance the posterior probability that ancestors of the two synthetase classes arose from opposite strands of the same ancestral gene. The remarkable acceleration by short peptides of the rate-limiting step in uncatalyzed protein synthesis, together with the synergy of synthetase Urzymes and their cognate tRNAs, introduce a new paradigm for the origin of protein catalysts, emphasize the potential relevance of an operational RNA code embedded in the tRNA acceptor stems, and challenge the RNA-World hypothesis. Reviewers This article was reviewed by Dr. Paul Schimmel (nominated by Laura Landweber), Dr. Eugene Koonin and Professor David Ardell.

  • Aminoacylating urzymes challenge the rna world hypothesis
    Journal of Biological Chemistry, 2013
    Co-Authors: Li Li, Christopher S Francklyn, Charles W. Carter
    Abstract:

    Abstract We describe experimental evidence that ancestral peptide catalysts substantially accelerated development of genetic coding. Structurally invariant 120–130-residue Urzymes (Ur = primitive plus enzyme) derived from Class I and Class II Aminoacyl-tRNA synthetases (aaRSs) acylate tRNA far faster than the uncatalyzed rate of nonribosomal peptide bond formation from activated Amino Acids. These new data allow us to demonstrate statistically indistinguishable catalytic profiles for Class I and II aaRSs in both Amino Acid Activation and tRNA acylation, over a time period extending to well before the assembly of full-length enzymes and even further before the Last Universal Common Ancestor. Both Urzymes also exhibit ∼60% of the contemporary catalytic proficiencies. Moreover, they are linked by ancestral sense/antisense genetic coding, and their evident modularities suggest descent from even simpler ancestral pairs also coded by opposite strands of the same gene. Thus, aaRS Urzymes substantially pre-date modern aaRS but are, nevertheless, highly evolved. Their unexpectedly advanced catalytic repertoires, sense/antisense coding, and ancestral modularities imply considerable prior protein-tRNA co-evolution. Further, unlike ribozymes that motivated the RNA World hypothesis, Class I and II Urzyme·tRNA pairs represent consensus ancestral forms sufficient for codon-directed synthesis of nonrandom peptides. By tracing aaRS catalytic activities back to simpler ancestral peptides, we demonstrate key steps for a simpler and hence more probable peptide·RNA development of rapid coding systems matching Amino Acids with anticodon trinucleotides.

  • histidyl trna synthetase urzymes class i and ii Aminoacyl trna synthetase urzymes have comparable catalytic activities for cognate Amino Acid Activation
    Journal of Biological Chemistry, 2011
    Co-Authors: Li Li, Christopher S Francklyn, Violetta Weinreb, Charles W. Carter
    Abstract:

    Abstract Four minimal (119 - 145 residue) active site fragments of Escherichia coli Class II histidyl-tRNA synthetase were constructed, expressed as maltose-binding protein fusions, and assayed for histidine Activation as fusion proteins and after TEV cleavage, using the 32PPi exchange assay. All contain conserved Motifs 1 and 2. Two contain an N-terminal extension of Motif 1 and two contain Motif 3. Five experimental results argue strongly for the authenticity of the observed catalytic activities: (i) active site titration experiments showing high (∼0.1–0.55) fractions of active molecules, (ii) release of cryptic activity by TEV cleavage of the fusion proteins, (iii) reduced activity associated with an active site mutation, (iv) quantitative attribution of increased catalytic activity to the intrinsic effects of Motif 3, the N-terminal extension and their synergistic effect, and (v) significantly altered Km values for both ATP and histidine substrates. It is therefore plausible that neither the insertion domain nor Motif 3 were essential for catalytic activity in the earliest Class II Aminoacyl-tRNA synthetases. The mean rate enhancement of all four cleaved constructs is ∼109 times that of the estimated uncatalyzed rate. As observed for the tryptophanyl-tRNA synthetase (TrpRS) Urzyme, these fragments bind ATP tightly but have reduced affinity for cognate Amino Acids. These fragments thus likely represent Urzymes (Ur = primitive, original, earliest + enzyme) comparable in size and catalytic activity and coded by sequences proposed to be antisense to that coding the previously described Class I TrpRS Urzyme. Their catalytic activities provide metrics for experimental recapitulation of very early evolutionary events.

Shigeyuki Yokoyama - One of the best experts on this subject based on the ideXlab platform.

  • structural bases of transfer rna dependent Amino Acid recognition and Activation by glutamyl trna synthetase
    Structure, 2006
    Co-Authors: Shunichi Sekine, Mika Shichiri, Stephane Bernier, Robert Chenevert, Jacques Lapointe, Shigeyuki Yokoyama
    Abstract:

    Glutamyl-tRNA synthetase (GluRS) is one of the Aminoacyl-tRNA synthetases that require the cognate tRNA for specific Amino Acid recognition and Activation. We analyzed the role of tRNA in Amino Acid recognition by crystallography. In the GluRS*tRNA(Glu)*Glu structure, GluRS and tRNA(Glu) collaborate to form a highly complementary L-glutamate-binding site. This collaborative site is functional, as it is formed in the same manner in pretransition-state mimic, GluRS*tRNA(Glu)*ATP*Eol (a glutamate analog), and posttransition-state mimic, GluRS*tRNA(Glu)*ESA (a glutamyl-adenylate analog) structures. In contrast, in the GluRS*Glu structure, only GluRS forms the Amino Acid-binding site, which is defective and accounts for the binding of incorrect Amino Acids, such as D-glutamate and L-glutamine. Therefore, tRNA(Glu) is essential for formation of the completely functional binding site for L-glutamate. These structures, together with our previously described structures, reveal that tRNA plays a crucial role in accurate positioning of both L-glutamate and ATP, thus driving the Amino Acid Activation.

  • structural basis of nonnatural Amino Acid recognition by an engineered Aminoacyl trna synthetase for genetic code expansion
    Proceedings of the National Academy of Sciences of the United States of America, 2005
    Co-Authors: Takatsugu Kobayashi, Tetsuo Takimura, Ryo Sekine, Kenji Kamata, Kensaku Sakamoto, Susumu Nishimura, Kelly Vincent, Shigeyuki Yokoyama
    Abstract:

    The genetic code in a eukaryotic system has been expanded by the engineering of Escherichia coli tyrosyl-tRNA synthetase (TyrRS) with the Y37V and Q195C mutations (37V195C), which specifically recognize 3-iodo-l-tyrosine rather than l-tyrosine. In the present study, we determined the 3-iodo-l-tyrosine- and l-tyrosine-bound structures of the 37V195C mutant of the E. coli TyrRS catalytic domain at 2.0-A resolution. The γ-methyl group of Val-37 and the sulfur atom of Cys-195 make van der Waals contacts with the iodine atom of 3-iodo-l-tyrosine. The Val-37 and Cys-195 side chains are rigidly fixed by the neighboring residues forming the hydrophobic core of the TyrRS. The major roles of the two mutations are different for the 3-iodo-l-tyrosine-selective recognition in the first step of the Aminoacylation reaction (the Amino Acid Activation step): the Y37V mutation eliminates the fatal steric repulsion with the iodine atom, and the Q195C mutation reduces the l-tyrosine misrecognition. The structure of the 37V195C mutant TyrRS complexed with an l-tyrosyladenylate analogue was also solved, indicating that the 3-iodo-l-tyrosine and l-tyrosine side chains are similarly discriminated in the second step (the Aminoacyl transfer step). These results demonstrate that the Amino Acid-binding pocket on the 37V195C mutant is optimized for specific 3-iodo-l-tyrosine recognition.

  • Structural snapshots of the KMSKS loop rearrangement for Amino Acid Activation by bacterial tyrosyl-tRNA synthetase
    Journal of molecular biology, 2004
    Co-Authors: Takatsugu Kobayashi, Tetsuo Takimura, Ryo Sekine, Vincent P. Kelly, Kenji Kamata, Kensaku Sakamoto, Susumu Nishimura, Shigeyuki Yokoyama
    Abstract:

    Tyrosyl-tRNA synthetase (TyrRS) has been studied extensively by mutational and structural analyses to elucidate its catalytic mechanism. TyrRS has the HIGH and KMSKS motifs that catalyze the Amino Acid Activation with ATP. In the present study, the crystal structures of the Escherichia coli TyrRS catalytic domain, in complexes with l-tyrosine and a l-tyrosyladenylate analogue, Tyr-AMS, were solved at 2.0A and 2.7A resolution, respectively. In the Tyr-AMS-bound structure, the 2'-OH group and adenine ring of the Tyr-AMS are strictly recognized by hydrogen bonds. This manner of hydrogen-bond recognition is conserved among the class I synthetases. Moreover, a comparison between the two structures revealed that the KMSKS loop is rearranged in response to adenine moiety binding and hydrogen-bond formation, and the KMSKS loop adopts the more compact ("semi-open") form, rather than the flexible, open form. The HIGH motif initially recognizes the gamma-phosphate, and then the alpha and gamma-phosphates of ATP, with a slight rearrangement of the residues. The other residues around the substrate also accommodate the Tyr-AMS. This induced-fit form presents a novel "snapshot" of the Amino Acid Activation step in the Aminoacylation reaction by TyrRS. The present structures and the T.thermophilus TyrRS ATP-free and bound structures revealed that the extensive induced-fit conformational changes of the KMSKS loop and the local conformational changes within the substrate binding site form the basis for driving the Amino Acid Activation step: the KMSKS loop adopts the open form, transiently shifts to the semi-open conformation according to the adenosyl moiety binding, and finally assumes the rigid ATP-bound, closed form. After the Amino Acid Activation, the KMSKS loop adopts the semi-open form again to accept the CCA end of tRNA for the Aminoacyl transfer reaction.