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

Abstract

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.

A Zymography technique to study amino acid activation by aminoacyl tRNA synthetases (aaRS): A broad spectrum, high-throughput tool to screen activities of aaRS and their “Urzyme” variants

“…Purification of the proteins was done as per Hobson et. al [20], Li et al [21], Williams et al [22].…”

“…Bacterial Cells were lysed, by sonication with 8 pulses of 10 second 70% amplitude sonic vibrations keeping 20 seconds of pause time, ensuring the tube remained in ice during sonication. Lysed cells were then centrifuged at 12000 rpm for 10 min at 4 °C and supernatants were collected, aliquoted in tubes, and stored at -80°C [20,[23][24][25].…”

Mesenchymal Stem Cell-Derived Apoptotic Bodies: Biological Functions and Therapeutic Potential

“…In general, amino acids binding to amino-tRNA could be transported to the ribosome for protein synthesis [ 60 ]. Activation of mitochondrial translation elongation factor Tu (TUFM), one of the mitochondrial protein translation elongation factors, promotes amino acid delivery from the aminoacyl-tRNA to the mitochondrial ribosomal A site, thereby increasing the rate of amino acid elongation [ 61 ]. In vitro experiments substantiated that the transportation of TUFM from dental pulp stem cell-derived ABs (DPSC-ABs) into ECs activated the TFEB-mediated ECs autophagy via TFEB nuclear translocation in lysosomes.…”

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

“…Genetic deconstruction and experimental biochemistry have suggested significant mosaicity within the Class I aaRS catalytic domains (Figure 1). Two segments nested within these domains-protozymes (from προτο = first [14]) and urzymes (from Ur = original, authentic [15][16][17])-represent successive intermediate evolutionary states of increasing length and dating from well before the Last Universal Common Ancestor [18,19]. [20] from coordinates (PDB ID 1I6L) for the tryptophanyl-tRNA synthetase, the smallest Class I aaRS.…”

“…"Connecting peptide 1" or CP1 [40,41] intersects the Class I aaRS Rossmann fold immediately after the protozyme between structurally homologous residues that are ~4.5 Å apart. CP1 can thus be replaced by a peptide bond without structural disruption to produce the Class I urzyme [15,17] as detailed in Figure 1. CP1 insertions include the editing domains of the aaRS for aliphatic amino acids, and thus largely account for the variable size of Class I catalytic domains.…”