Amino acid activation analysis of primitive aminoacyl-tRNA synthetases encoded by both strands of a single gene using the malachite green assay

Onodera, Kazaha; Suganuma, Nana; Takano, Haruka; Sugita, Yu; Shoji, Tomoko; Minobe, Ayaka; Yamaki, Narumi; Otsuka, Riku; Mutsuro‐Aoki, Hiromi; Umehara, Takuya; Tamura, Koji

doi:10.1016/j.biosystems.2021.104481

Cited by 22 publications

(31 citation statements)

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“…As we had described [12], in their hands the Class I protozyme catalyzed amino acid activation by ATP significantly more than maltose-binding protein (MBP) alone when incubated with various amino acids, whereas the Class II protozyme's activity was closer to, but still greater than, that of MBP alone. That independent validation of our protozyme results [15] underscores the relevance of our work. Furthermore, both protozymes exhibited greater activity in the absence of any amino acid than did MBP alone.…”

Section: Introductionsupporting

confidence: 78%

“…Estimates of the active fraction of LeuAC molecules (0.4-0.6) in Figure 1c are more than 4000 fold greater than this value. Moreover, the contribution of maltose-binding protein to the amino acid activation reaction has been measured at less than 10 −5 times values reported here [12,15]. Thus, neither full-length LeuRS contamination nor MBP can account for any catalytic activities documented here.…”

Section: The Mbp-leuac Fusion Is Pure Enough To Exclude Contaminating...mentioning

confidence: 51%

“…Until recently, we alone have attempted to overcome them. However, Koji Tamura's group at Tokyo University of Science [15] have now re-examined the catalysis we reported from protozymes encoded by opposite strands of a designed gene [12]. As we had described [12], in their hands the Class I protozyme catalyzed amino acid activation by ATP significantly more than maltose-binding protein (MBP) alone when incubated with various amino acids, whereas the Class II protozyme's activity was closer to, but still greater than, that of MBP alone.…”

Section: Introductionmentioning

confidence: 99%

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A Leucyl-tRNA Synthetase Urzyme: Authenticity of tRNA Synthetase Catalytic Activities and Promiscuous Phosphorylation of Leucyl-5′AMP

Hobson

et al. 2022

IJMS

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Aminoacyl-tRNA synthetase (aaRS)/tRNA cognate pairs translate the genetic code by synthesizing specific aminoacyl-tRNAs that are assembled on messenger RNA by the ribosome. Deconstruction of the two distinct aaRS superfamilies (Classes) has provided conceptual and experimental models for their early evolution. Urzymes, containing ~120–130 amino acids excerpted from regions where genetic coding sequence complementarities have been identified, are key experimental models motivated by the proposal of a single bidirectional ancestral gene. Previous reports that Class I and Class II urzymes accelerate both amino acid activation and tRNA aminoacylation have not been extended to other synthetases. We describe a third urzyme (LeuAC) prepared from the Class IA Pyrococcus horikoshii leucyl-tRNA synthetase. We adduce multiple lines of evidence for the authenticity of its catalysis of both canonical reactions, amino acid activation and tRNALeu aminoacylation. Mutation of the three active-site lysine residues to alanine causes significant, but modest reduction in both amino acid activation and aminoacylation. LeuAC also catalyzes production of ADP, a non-canonical enzymatic function that has been overlooked since it first was described for several full-length aaRS in the 1970s. Structural data suggest that the LeuAC active site accommodates two ATP conformations that are prominent in water but rarely seen bound to proteins, accounting for successive, in situ phosphorylation of the bound leucyl-5′AMP phosphate, accounting for ADP production. This unusual ATP consumption regenerates the transition state for amino acid activation and suggests, in turn, that in the absence of the editing and anticodon-binding domains, LeuAC releases leu-5′AMP unusually slowly, relative to the two phosphorylation reactions.

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Section: Introductionsupporting

confidence: 78%

Section: The Mbp-leuac Fusion Is Pure Enough To Exclude Contaminating...mentioning

confidence: 51%

Section: Introductionmentioning

confidence: 99%

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A Leucyl-tRNA Synthetase Urzyme: Authenticity of tRNA Synthetase Catalytic Activities and Promiscuous Phosphorylation of Leucyl-5′AMP

Hobson

et al. 2022

IJMS

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“…Experimental Michaelis-Menten parameters of all four protozymes-Class I and II; native and bidirectional-are, remarkably, the same within experimental error [14]. Tamura's laboratory [38] have replicated those results. Experimental [14,17,39] and bioinformatic evidence [40] therefore support the hypothesis of Rodin and Ohno [22,41] that the two aaRS Classes descended from opposite strands of a single bidirectional gene.…”

Section: Introductionmentioning

confidence: 74%

Multidimensional Phylogenetic Metrics Identify Class I Aminoacyl-tRNA Synthetase Evolutionary Mosaicity and Inter-Modular Coupling

Carter

Popinga

Bouckaert

et al. 2022

IJMS

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The role of aminoacyl-tRNA synthetases (aaRSqianguize) in the emergence and evolution of genetic coding poses challenging questions concerning their provenance. We seek evidence about their ancestry from curated structure-based multiple sequence alignments of a structurally invariant “scaffold” shared by all 10 canonical Class I aaRS. Three uncorrelated phylogenetic metrics—mutation frequency, its uniformity, and row-by-row cladistic congruence—imply that the Class I scaffold is a mosaic assembled from successive genetic sources. Metrics for different modules vary in accordance with their presumed functionality. Sequences derived from the ATP– and amino acid– binding sites exhibit specific two-way coupling to those derived from Connecting Peptide 1, a third module whose metrics suggest later acquisition. The data help validate: (i) experimental fragmentations of the canonical Class I structure into three partitions that retain catalytic activities in proportion to their length; and (ii) evidence that the ancestral Class I aaRS gene also encoded a Class II ancestor in frame on the opposite strand. A 46-residue Class I “protozyme” roots the Class I tree prior to the adaptive radiation of the Rossmann dinucleotide binding fold that refined substrate discrimination. Such rooting implies near simultaneous emergence of genetic coding and the origin of the proteome, resolving a conundrum posed by previous inferences that Class I aaRS evolved after the genetic code had been implemented in an RNA world. Further, pinpointing discontinuous enhancements of aaRS fidelity establishes a timeline for the growth of coding from a binary amino acid alphabet.

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“…Experimental Michaelis-Menten parameters of all four protozymes-Class I and II; native and bidirectional-are, remarkably, the same within experimental error [14]. Tamura's laboratory [36] have replicated those results. Experimental [14,17,37] and bioinformatic evidence [38] therefore support the hypothesis of Rodin and Ohno [21,39] that the two aaRS Classes descended from opposite strands of a single bidirectional gene.…”

Section: Introductionmentioning

confidence: 74%

High-Resolution, Multidimensional Phylogenetic Metrics Identify Class I Aminoacyl-tRNA Synthetase Evolutionary Mosaicity and Inter-modular Coupling

Carter¹,

Popinga²,

Bouckaert³

et al. 2021

Preprint

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The provenance of the aminoacyl-tRNA synthetases (aaRS) poses challenging questions because of their role in the emergence and evolution of genetic coding. We investigate evidence about their ancestry from curated structure-based multiple sequence alignments of a structurally invariant “scaffold” shared by all 10 canonical Class I aaRS. Three uncorrelated phylogenetic metrics—residue-by-residue conservation, its variance, and row-by-row cladistic congruence—imply that the Class I scaffold is a mosaic assembled from distinct, successive genetic sources. These data are especially significant in light of: (i) experimental fragmentations of the Class I scaffold into three partitions that retain catalytic activities in proportion to their length; and (ii) evidence that two of these partitions arose from an ancestral Class I aaRS gene encoding a Class II ancestor in frame on the opposite strand. Phylogenetic metrics of different modules vary in accordance with their presumed functionality. A 46-residue Class I “protozyme” roots the Class I molecular tree prior to the adaptive radiation of the Rossmann dinucleotide binding fold that refined substrate discrimination. Such rooting is consistent with near simultaneous emergence of genetic coding and the origin of the proteome, resolving a conundrum posed by previous inferences that Class I aaRS evolved long after the genetic code had been implemented in an RNA world. Further, pinpointing discontinuous enhancements of aaRS fidelity establishes a timeline for the growth of coding from a binary amino acid alphabet.

show abstract

Amino acid activation analysis of primitive aminoacyl-tRNA synthetases encoded by both strands of a single gene using the malachite green assay

Cited by 22 publications

References 50 publications

A Leucyl-tRNA Synthetase Urzyme: Authenticity of tRNA Synthetase Catalytic Activities and Promiscuous Phosphorylation of Leucyl-5′AMP

A Leucyl-tRNA Synthetase Urzyme: Authenticity of tRNA Synthetase Catalytic Activities and Promiscuous Phosphorylation of Leucyl-5′AMP

Multidimensional Phylogenetic Metrics Identify Class I Aminoacyl-tRNA Synthetase Evolutionary Mosaicity and Inter-Modular Coupling

High-Resolution, Multidimensional Phylogenetic Metrics Identify Class I Aminoacyl-tRNA Synthetase Evolutionary Mosaicity and Inter-modular Coupling

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