On the Evolution of the tRNA-Dependent Amidotransferases, GatCAB and GatDE

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Abstract

Glutaminyl-tRNA synthetase and asparaginyl-tRNA synthetase evolved from glutamyl-tRNA synthetase and aspartyl-tRNA synthetase, respectively, after the split in the last universal communal ancestor (LUCA). Glutaminyl-tRNAGln and asparaginyl-tRNAAsn were likely formed in LUCA by amidation of the mischarged species, glutamyl-tRNAGln and aspartyl-tRNAAsn, by tRNA-dependent amidotransferases, as is still the case in most bacteria and all known archaea. The amidotransferase GatCAB is found in both domains of life, while the heterodimeric amidotransferase GatDE is found only in Archaea. The GatB and GatE subunits belong to a unique protein family that includes Pet112 that is encoded in the nuclear genomes of numerous eukaryotes. GatE was thought to have evolved from GatB after the emergence of the modern lines of decent. Our phylogenetic analysis though places the split between GatE and GatB, prior to the phylogenetic divide between Bacteria and Archaea, and Pet112 to be of mitochondrial origin. In addition, GatD appears to have emerged prior to the bacterial–archaeal phylogenetic divide. Thus, while GatDE is an archaeal signature protein, it likely was present in LUCA together with GatCAB. Archaea retained both amidotransferases, while Bacteria emerged with only GatCAB. The presence of GatDE has favored a unique archaeal tRNAGln that may be preventing the acquisition of glutaminyl-tRNA synthetase in Archaea. Archaeal GatCAB, on the other hand, has not favored a distinct tRNAAsn, suggesting that tRNAAsn recognition is not a major barrier to the retention of asparaginyl-tRNA synthetase in many Archaea.

Introduction

Aminoacyl-tRNA synthetases (aaRSs) are the enzymes that are primarily responsible for accurately pairing an amino acid with its cognate tRNA, a requirement for high-fidelity protein synthesis.1 In most bacteria2 and all known archaea,3 glutaminyl-tRNA synthetase (GlnRS) is absent, and Gln-tRNAGln is formed via an indirect route. These organisms take advantage of an aaRS with relaxed tRNA specificity, a nondiscriminating glutamyl-tRNA synthetase (ND-GluRS), to form Glu-tRNAGln.4 The Glu moiety on the tRNAGln is then converted into Gln via an amidation reaction catalyzed by a glutamyl-tRNAGln amidotransferase (Glu-AdT) in these species.5 Similarly, in the many bacteria and archaea that lack asparaginyl-tRNA synthetase (AsnRS),2, 3, 6 a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS)7 and an aspartyl-tRNAAsn amidotransferase (Asp-AdT) are used to synthesize Asn-tRNAAsn.8, 9

Two different tRNA-dependent amidotransferases (AdTs) are found in nature: GatCAB10 and GatDE.3 The latter is an archaeal signature protein11 and acts only as a Glu-AdT.3 GatCAB is present in most bacteria and archaea.2, 3, 6 In vitro, the bacterial GatCAB can transamidate both Glu-tRNAGln and Asp-tRNAAsn.2, 12, 13, 14, 15 Its role in vivo is determined by which mischarged tRNA species (Glu-tRNAGln and/or Asp-tRNAAsn) is present.2, 12, 13, 14, 16, 17 Archaeal GatCAB is unable to use archaeal Glu-tRNAGln as a substrate (see Sheppard, K., Sherrer, R.L., and Söll, D., Methanothermobacter thermautotrophicus tRNAGln confines the amidotransferase GatCAB to asparaginyl-tRNAAsn formation, this issue), is only present in Archaea lacking an AsnRS3, 6 and is thought to act as an Asp-AdT in vivo.

Both AdTs catalyze three distinct reactions in order to transamidate their mischarged tRNA substrates, Glu-tRNAGln and/or Asp-tRNAAsn.18 (1) They phosphorylate the γ- or β-carboxyl group of the Glu or Asp moiety attached to the tRNA in an ATP-dependent manner to form an activated intermediate.19 This kinase activity is the province of the B- and E-subunits of the respective holoenzymes.20, 21, 22 (2) The AdTs have a glutaminase subunit (GatA and GatD, respectively) that liberates ammonia from an amide donor such as Gln or Asn.2, 20, 23, 24 GatD belongs to the type I l-asparaginase (AnsA) family,3, 20, 23 while GatA is an amidase.2, 10, 21, 24 GatC, a small protein of approximately 100 amino acids, is required by GatA for proper folding10 and binding to GatB.21 (3) The AdTs amidate the activated intermediate using the ammonia liberated by the glutaminase subunit.

In the last universal communal ancestor (LUCA), the diverse community of ancient organisms that the three modern domains of life are hypothesized to have evolved from,25 it is thought that the indirect paths for Gln-tRNAGln and Asn-tRNAAsn syntheses were extant, since the specific aaRSs for these amino acids (GlnRS and AsnRS) evolved at a later stage.6, 26, 27, 28, 29 Given that GatCAB is present in both Archaea and Bacteria while GatDE is archaeal specific, it was speculated the AdT in LUCA was GatCAB, with GatDE evolving later in early archaea.30 To address this hypothesis and to gain a better understanding of the evolution of the two AdTs, we examined the phylogenetic relationship between the kinase subunits of the two AdTs (GatB and GatE). We constructed phylogenetic trees for four additional components of tRNA-dependent Gln and Asn syntheses: GatA; AnsA and the GatD family; tRNAGln; and tRNAAsn. This comparative phylogenetic approach reveals the coevolution of the subunits of GatDE and GatCAB, as well as the coevolution with their tRNA substrates, and resolves the evolutionary connection between the two distantly related AdTs. Our results suggest that, although GatDE is an archaeal signature protein, it evolved before the archaeal–bacterial phylogenetic divide and was present in LUCA.

Section snippets

Ancient divergence between GatB and GatE

GatB and GatE belong to a unique enzyme family that includes Pet112 (Fig. 1).3, 21, 22, 23, 31 The latter enzymes are encoded in the nuclear genomes of many eukaryotes.31 In Saccharomyces cerevisiae, Pet112 is essential for mitochondrial translation.31

Because GatDE is confined to the Archaea, it was assumed that GatE arose from a duplication of either an archaeal GatB or a bacterial GatB that was horizontally transferred to the Archaea.30 Pet112, on the other hand, is predicted to be of

Model of the evolution of the two AdTs

The hypothesis that GatDE is an archaeal invention30 is not supported by our phylogenetic results. In the unrooted phylogeny of the GatB and GatE core, GatE sequences did not cluster within one of the GatB branches, but rather were on a long branch away from the GatB sequences (Fig. 2). Without rooting the phylogeny, we cannot rule out a specific archaeal GatB/GatE relationship, with GatE evolving at an accelerated rate as compared to GatB. However, the phylogeny of the tail domain of the

Conclusion

While GlnRS and AsnRS were late inventions, evolving from GluRS and AspRS, respectively, after the split in LUCA, Gln and Asn codings are ancient.6, 26, 27, 28, 29 The primordial route to amide aa-tRNA formation was likely via the indirect pathway.66 The ancestor to GatE and GatB probably recognized both Asp-tRNAAsn and Glu-tRNAGln. While GatDE is an archaeal signature protein, it likely evolved before the phylogenetic split between Archaea and Bacteria and was present in LUCA together with

Phylogenetic analysis

The bacterial and the archaeal GatA, GatB, AnsA, and AnsB, along with the archaeal GatD and GatE, the eukaryotic homologs of GatB, and YqeY sequences, were taken from the National Center for Biotechnology Information database. The AspRS-like insertion domain in the GatE sequences was manually removed prior to alignment with GatB and Pet112 using Geneious v2.5.3†67 based on the structural alignment of the Pyrococcus abyssi GatE with the S. aureus GatB.21 GatA sequences

Acknowledgements

We thank Patrick O'Donoghue and Michael Hohn for advice and stimulating discussions. This work was supported by grants from the National Institute of General Medical Sciences (GM22854) and the Department of Energy (DE-FG02-98ER20311).

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