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When Contemporary Aminoacyl-Trna Synthetases Invent Their Cognate Amino Acid Metabolism

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When Contemporary Aminoacyl-Trna Synthetases Invent Their Cognate Amino Acid Metabolism When contemporary aminoacyl-tRNA synthetases SEE COMMENTARY invent their cognate amino acid metabolism Herve´ Roy, Hubert Dominique Becker, Joseph Reinbolt, and Daniel Kern* De´partement Me´canismes et Macromole´cules de la Synthe`se Prote´ique et Cristallogene`se, Unite´Propre de Recherche 9002, Institut de Biologie Mole´culaire et Cellulaire du Centre National de la Recherche Scientifique, 15 Rue Rene´Descartes, F-67084 Strasbourg Ce´dex, France Edited by Paul R. Schimmel, The Scripps Research Institute, La Jolla, CA, and approved June 9, 2003 (received for review April 14, 2003) Faithful protein synthesis relies on a family of essential enzymes genomes drew our attention to the fact that all archaea that called aminoacyl-tRNA synthetases, assembled in a piecewise fash- possess AsnRS (7 of 13) display a second ORF highly homolo- ion. Analysis of the completed archaeal genomes reveals that all gous to the catalytic core of AsnRS (AsnRS2) but lacking the archaea that possess asparaginyl-tRNA synthetase (AsnRS) also anticodon-binding domain. We analyzed the functional proper- display a second ORF encoding an AsnRS truncated from its ties of AsnRS2 from Pyrococcus abyssi, a hyperthermophilic anticodon binding-domain (AsnRS2). We show herein that Pyro- archaeon. The results show that this truncated AsnRS does not coccus abyssi AsnRS2, in contrast to AsnRS, does not sustain form Asn-tRNAAsn but produces Asn by amidation of Asp. asparaginyl-tRNAAsn synthesis but is instead capable of converting Phylogeny suggests this asparagine synthetase (AS) appeared aspartic acid into asparagine. Functional analysis and complemen- recently in archaea and allowed emergence of a direct and tation of an Escherichia coli asparagine auxotrophic strain show autonomous pathway of tRNA asparaginylation. This study that AsnRS2 constitutes the archaeal homologue of the bacterial describes an example of emergence of a new tRNA aminoacy- ammonia-dependent asparagine synthetase A (AS-A), therefore lation specificity and of the enzyme forming the homologous aa named archaeal asparagine synthetase A (AS-AR). Primary se- from a contemporary aaRS. quence- and 3D-based phylogeny shows that an archaeal AspRS ancestor originated AS-AR, which was subsequently transferred Materials and Methods Ϫ Ϫ into bacteria by lateral gene transfer in which it underwent General. Escherichia coli ER strain (asnA , asnB ) was from structural changes producing AS-A. This study provides evidence Genetic Stock Center (Yale University, New Haven, CT), and P. that a contemporary aminoacyl-tRNA synthetase can be recruited abyssi DNA was a gift from J.-C. Thierry (Institut de Ge´ne´tique to sustain amino acid metabolism. et de Biologie Mole´culaireet Cellulaire, Illkirch, France). The pKKET vector is a modified version of pKK223-3 (Amersham ccurate protein translation requires a complete set of 20 Pharmacia) in which the EcoRI site was replaced by the NdeI Asp Asn Aspecies of perfectly paired aminoacyl-tRNAs (aa-tRNAs). site. Thermus thermophilus tRNA , tRNA , and AspRS2 It was therefore expected that each organism should possess 20 were purified from E. coli overproducing strains. aa-tRNA synthetases (aaRSs), each capable of matching a particular amino acid (aa) to the cognate tRNA (reviewed in ref. Cloning of the P. abyssi asnS2 Gene. The ORF was amplified by 1). However, the vast number of sequences that emerged from PCR of genomic DNA with 22- and 20-nt-long sense and genome sequencing and biochemical investigations revealed antisense primers extended by NdeI and PstIorKpnI restriction anomalies that forced a revision of this assumption (2). Archaea sites for cloning of the genes in pKKET and pET vectors, respectively. AsnRS2 was expressed in E. coli ER strain (ER͞ and various eubacteria are deprived of glutaminyl-tRNA syn- ͞ thetase and more exceptionally of asparaginyl-tRNA synthetase pKKET-asnS2) and in BL21-CodonPlus-RIL strain (BL21 (AsnRS). The homologous aa-tRNAs are formed indirectly by pET-asnS2). Gln CELL BIOLOGY amidation of Glu and Asp, respectively, mischarged on tRNA ͞ and tRNAAsn by a glutamyl-tRNA synthetase (GluRS) or an Overexpression and Purification of AsnRS2. The BL21 pET-asnS2 aspartyl-tRNA synthetase (AspRS) of relaxed specificity (3–6). strain was grown overnight at 37°C in LB medium containing ͞ ampicillin, and AsnRS2 was expressed by induction with isopro- In bacteria of the Thermus Deinococcus group, deprived of the ␤ enzymes forming free asparagine (Asn), the indirect route to pyl -D-thiogalactoside. The cells were disrupted by sonication; the thermolabile proteins were flocculated by heat treatment of tRNA asparaginylation also constitutes the sole pathway of Asn ϫ biosynthesis (6, 7). the 105,000 g supernatant during 30 min at 70°C and removed The functional interrelation between the direct and the indi- by centrifugation. AsnRS2 was further purified by chromatog- rect pathways of amide aa-tRNA formation is not well under- raphies on DEAE-cellulose and hydroxyapatite followed by stood, and the phylogenetic relationship of their partners has not FPLC chromatography on an UNO-Q6 (Bio-Rad) column. been explored. Emergence of the direct pathways of tRNA From 27 g of cells, 80 mg of pure protein was isolated. glutaminylation and asparaginylation had to be accompanied by AsnRS2 Activities. ATP–PP exchange. concomitant apparition of the enzymes forming the free aa and i The reaction mixture con- tained 100 mM NaHepes (pH 7.2), 10 mM MgCl2,2mM the homologous aa-tRNA. The paths leading to the emergence 32 [ P]PPi,2mMATP,5mML-Asp (0.05–1.35 mM for Km of glutaminyl-tRNA synthetase and AsnRS have been docu- ␮ mented (8, 9), but the origin of the Gln- and Asn-forming measurements) or L-Asn, and 1 M P. abyssi AsnRS2. For Ki enzymes remains unclear. In particular, it is not known whether measurements, the concentration of Asn varied from 12 to 95 the aaRS and the enzyme forming the cognate free aa originated from the same ancestor. This paper was submitted directly (Track II) to the PNAS office. The microbial genomes are sprinkled with polypeptide chains Abbreviations: aa, amino acid; aa-tRNA, aminoacyl-tRNA; aaRS, aa-tRNA synthetase; consisting of one of the functional domains of aaRSs (10). In AsnRS, asparaginyl-tRNA synthetase; AspRS, aspartyl-tRNA synthetase; AS, asparagine some cases, the participation of these domains in a variety of synthetase; AS-A, bacterial ammonia-dependent AS; AS-AR, archaeal AS-A; AdT, tRNA- cellular processes, such as translation regulation (11), stimula- dependent amidotransferase. tion of DNA polymerase processivity (12), and aa synthesis (13), See commentary on page 9650. could be assessed. Analysis of the aaRS ORFs in archaeal *To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.1632156100 PNAS ͉ August 19, 2003 ͉ vol. 100 ͉ no. 17 ͉ 9837–9842 Downloaded by guest on September 24, 2021 Table 1. Occurrence of AsnRS2 and enzymes involved in Asn and Asn-tRNAAsn synthesis in archaea Organism asnA* asnB* gatCAB*† asnS† asnS2 Crenarchaeota Aeropyrum pernix Ϫϩ ϩ ϪϪ Sulfolobus solfataricus ϪϪ ϩ ϪϪ Sulfolobus tokodaii Ϫϩ ϩ ϪϪ Pyrobaculum aerophilum Ϫϩ Ϫ ϩϩ Euryarchaeota Archaeoglobus fulgidus Ϫϩ ϩ ϪϪ Halobacterium sp. NRC1 Ϫϩ ϩ ϪϪ Methanobacterium thermoautotrophicum Ϫϩ ϩ ϪϪ Methanococcus jannaschii Ϫϩ ϩ ϪϪ Methanosarcina barkeri Ϫϩ ϩ ϪϪ P. abyssi Ϫϩ Ϫ ϩϩ Pyrococcus furiosus Ϫϩ Ϫ ϩϩ Pyrococcus horikoshii Ϫϩ Ϫ ϩϩ Ferroplasma acidarmanus Ϫϩ Ϫ ϩϩ Thermoplasma acidophilum Ϫϩ Ϫ ϩϩ Thermoplasma volcanium ϪϪ Ϫ ϩϩ Only archaeal genome sequences for which the presence (ϩ) or absence (Ϫ) of the genes could be ascertained are listed. Genes encoding AsnRS2 (asn2) and the enzymes involved in either Asn (*) or Asn-tRNAAsn (†) formation, asnA, asnB, gatCAB, and asnS encoding, respectively, asparagine synthetases A and B, amidotransferase (AdT), and AsnRS are presented. ␮M, and Asp was fixed at 88, 176, or 293 ␮M. The [32P]ATP that Version 4.0.2 (15). Trees were edited with the TREEVIEW pro- formed after 2–10 min at 70°C was determined in 20-␮l gram, Version 1.5. aliquots (6). Aminoacylation. The reaction mixture contained 100 mM NaHepes Results ␮ 14 (pH 7.2), 30 mM KCl, 10 mM MgCl2,10 M L-[ C]Asp or Existence of Two Genes Encoding AsnRS in P. abyssi. Analysis of the L-[3H]Asn, 320 ␮M unfractionated E. coli tRNA or 30 ␮M annotated genome from P. abyssi reveals two ORFs related to enriched Thermus thermophilus tRNAAsp or tRNAAsn, and 0.1 AsnRS, one encoding the full-length enzyme and the other one, ␮M P. abyssi AsnRS2 or Thermus thermophilus AspRS2. The a shorter isoform, identified as AsnRS2 sharing 39% identity aa-tRNA formed after 1–30 min at 50°C was determined in 40-␮l with the canonical AsnRS. The gene encoding this truncated aliquots (6). AsnRS (asnS2) was found in six other archaeal genomes (16) but Amidation. The reaction mixture contained 100 mM NaHepes (pH not in bacterial or eukaryal genomes. Surprisingly, all archaea 7.2), 10 mM MgCl2,10mMNH4Cl, L-Asn or L-Gln, 10 mM ATP, that encode AsnRS2 also possess the full-length AsnRS (Table ␮ 0.5 mM L-Asp (0.5–10 mM for Km measurements), and 2 M 1). Alignment of AsnRS2 with the archaeal AsnRS and AspRS AsnRS2. The reaction was conducted at 70°C. For character- shows this isoform lacks the 100 first aa, which in other AsnRS ization of Asn, [14C]Asp was used and 4.5-␮l aliquots were are organized in the anticodon-binding domain (Fig. 1); never- transferred in 1 ␮l of acetic acid after a 5-min to 3-h incubation. theless, the catalytic core displays the three consensus motifs For characterization of the ATP products, the reaction mixture characterizing class II aaRS in which the invariant and semi- contained 40 ␮M[␥-or␣-32P]ATP, 40 ␮M L-Asp, and 0.6 ␮M invariant residues are mostly conserved (17).
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