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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 AminoAcid 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.

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  • 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.

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  • 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.

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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 AminoAcid 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.

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  • 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.

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  • 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…

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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.

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  • 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.

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  • 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.

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