When contemporary aminoacyl-tRNA synthetases invent their cognate amino acid metabolism

Abstract

Faithful protein synthesis relies on a family of essential enzymes called aminoacyl-tRNA synthetases, assembled in a piecewise fashion. Analysis of the completed archaeal genomes reveals that all archaea that possess asparaginyl-tRNA synthetase (AsnRS) also display a second ORF encoding an AsnRS truncated from its anticodon binding-domain (AsnRS2). We show herein that Pyrococcus abyssi AsnRS2, in contrast to AsnRS, does not sustain asparaginyl-tRNAAsn synthesis but is instead capable of converting aspartic acid into asparagine. Functional analysis and complementation of an Escherichia coli asparagine auxotrophic strain show that AsnRS2 constitutes the archaeal homologue of the bacterial ammonia-dependent asparagine synthetase A (AS-A), therefore named archaeal asparagine synthetase A (AS-AR). Primary sequence- and 3D-based phylogeny shows that an archaeal AspRS ancestor originated AS-AR, which was subsequently transferred into bacteria by lateral gene transfer in which it underwent structural changes producing AS-A. This study provides evidence that a contemporary aminoacyl-tRNA synthetase can be recruited to sustain amino acid metabolism.

Fig. 1.

Alignments of AsnRS2 with archaeal AsnRS and AspRS. The alignment of 7 archaeal AsnRS (N),19 archaeal AspRS (D), and 7 AsnRS2 (2) is summarized. The sequences of the enzymes of P. abyssi (P_abyssi) and the consensus sequences encompassing the consensus motifs 1, 2, and 3 of class II aaRSs are shown. The motifs are schematized by black boxes located at the top or bottom of the sequences, and the invariant residues are indicated in white letters. The percentage homology is symbolized by shading: black and dark-gray shadings denote 100% and 80% of homology, and light-gray shading denotes conservation of the chemical nature in at least 80% of residues; dots symbolize lack of a residue in the detailed sequences and homology lower than 80% in the consensus sequences. Nomenclature of the consensus sequences is the following: a, aromatic; h, hydrophobic; – and +, negatively and positively charged; p, polar or small residues; ^ refers to a gap. Residues interacting with the aa (♦) or the adenylate (•) moiety of Asp∼AMP or with both (▪) in the 3D structure of the complex of Pyrococcus AspRS (22) are indicated.

Fig. 2.

Identification of the end products formed by P. abyssi AsnRS2 by TLC. Phosphor images of TLC plates of the amidation mixture containing [¹⁴C]Asp without amide group donor (lane 2) or with NH₄Cl (lane 3) or containing unlabeled Asp and either [³H]Asn (lane 4) or [³H]Gln (lane 5); lanes 1 and 6, controls with labeled aa.

Fig. 3.

Kinetics of substrate consumption and of end-product formation by AsnRS2. (A) Consumption of [¹⁴C]Asp (▴) and formation of [¹⁴C]Asn (•). (B) Consumption of [α-³²P]ATP (♦) and formation of [³²P]AMP (▪) and [³²P]ADP (×). The labeled reactants were fractionated by TLC (Inset) and quantified by using the IMAGE GAUGE software (Fuji), and their amounts are expressed as the percentage of the total radioactivity in the assay.

Fig. 4.

Complementation of the Asn auxotrophy of E. coli ER strain by the P. abyssi asnS2 gene. The E. coli ER strain was transformed with either the pKKET vector or the recombined pKKET-asnS2 vector and grown on minimal M9 medium agar plates supplemented with ampicillin, and 0.5 mM isopropyl β-d-thiogalactoside in the presence (+) or absence (–) of 40 μg/liter l-Asn.

Fig. 5.

Attempt to pinpoint the origin of the archaeal ammonia-dependent Asn metabolism. The tree is based on an ungapped alignment done by CLUSTALX of 7 AsnRS2, 60 AspRS, 49 AsnRS, and 61 lysyl-tRNA synthetase (LysRS) sequences. LysRS sequences were used to root the tree. Nodes statistically relevant [bootstrap value of 100% (⋄) or >80% (○)] are indicated. The evolutionary path of AsnRS2 is indicated by thick bars. The arrows indicate the gene duplications. The scale bar represents 10 substitutions per 100 aa.

Fig. 6.

Alignment-based identification of P. abyssi AsnRS2 active site residues. (A) Comparative alignment of AS-A (A), AsnRS2 (2), and AspRS (D). Only blocks of sequences encompassing the active-site residues involved in binding of Asp (arrowheads) are shown. The equivalent of AspRS flipping- and motif 2-loops are indicated by black boxes located at the top of the alignment. Nomenclature for the alignment is indicated in the Fig. 1 legend. (B) Comparison of the 3D structures of Pyrococcus AspRS and E. coli AS-A active sites. Coordinates of AspRS complexed to Asp∼AMP (22) and of AS-A complexed to Asn and ATP (21) were used. Hydrogen bonds are symbolized by dashed lines and water molecules by small spheres, and ‡ refers to residues of the same chemical nature spatially conserved in AspRS and AS-A active sites. Residues in white letters over black boxes are those of P. abyssi AsnRS2 identified in the alignment with AS-A shown in A as potential candidates for recognition of Asp. (C) Schematized 2D representation comparing recognition of Asp by Pyrococcus AspRS and AsnRS2. The residues interacting with Asp in AspRS and suggested to interact with Asp in AsnRS2 are displayed. Residues in ellipses are spatially conserved in AspRS and AsnRS2; residues in white letters in black ellipses determine different orientations of Asp in the two active sites; hydrogen bonds are symbolized by dashed lines.