FEBS Letters 476 (2000) 140^144 FEBS 23821 View metadata, citation and similar papers at core.ac.uk brought to you by CORE The heterotrimeric Thermus thermophilus Asp-tRNAAsn amidotransferaseprovided by Elsevier - Publisher Connector can also generate Gln-tRNAGln Hubert D. Beckera;1, Bokkee Mina;1, Carsten Jacobib, Gregory Raczniaka, Joanne Pelaschiera, Herve¨ Royc, Sylvain Kleinc, Daniel Kernc, Dieter So«lla;d;* aDepartment of Molecular Biophysics and Biochemistry, Yale University, P.O. Box 208114, 266 Whitney Avenue, New Haven, CT 06520-8114, USA bGo«ttingen Genomics Laboratory, Institute of Microbiology and Genetics, Grisebachstrasse 8, D-37077 Go«ttingen, Germany cInstitut de Biologie Mole¨culaire et Cellulaire du C.N.R.S., 15 Rue Rene¨ Descartes, F-67084 Strasbourg Cedex, France dDepartment of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06511, USA Received 8 May 2000 Edited by Horst Feldmann The conversions of Asp-tRNAAsn or Glu-tRNAGln into Abstract Thermus thermophilus strain HB8 is known to have a heterodimeric aspartyl-tRNAAsn amidotransferase (Asp-AdT) their cognate translational substrates requires a tRNA-depen- capable of forming Asn-tRNAAsn [Becker, H.D. and Kern, D. dent amidotransferase (AdT). The Bacillus subtilis enzyme has (1998) Proc. Natl. Acad. Sci. USA 95, 12832^12837]. Here we recently been shown to be heterotrimeric, with the subunits show that, like other bacteria, T. thermophilus possesses the encoded by the gatA, gatB and gatC genes [5]. These genes are canonical set of amidotransferase (AdT) genes (gatA, gatB and markers used to screen whole genome sequences, and their gatC). We cloned and sequenced these genes, and constructed an presence indicates the use of the indirect pathway in the de- artificial operon for overexpression in Escherichia coli of the ¢ned organism. The gatC, gatA and gatB genes have been thermophilic holoenzyme. The overproduced T. thermophilus found in many of the recently released genome sequences Gln Asn AdT can generate Gln-tRNA as well as Asn-tRNA . Thus, and in all living kingdoms [6], however, only B. subtilis Glu- the T. thermophilus tRNA-dependent AdT is a dual-specific Asp/ AdT, and Thermus thermophilus [7] and Deinococcus radiodur- Glu-AdT resembling other bacterial AdTs. In addition, we observed that removal of the 44 carboxy-terminal amino acids of ans [8] Asp-AdTs have been biochemically characterized. Fur- the GatA subunit only inhibits the Asp-AdT activity, leaving the thermore, it has recently been shown that the B. subtilis and Glu-AdT activity of the mutant AdT unaltered; this shows that D. radiodurans AdTs have in vitro both Asp-tRNAAsn and Asp-AdT and Glu-AdT activities can be mechanistically Glu-tRNAGln amidation activities, whereas in vivo they are separated. ß 2000 Federation of European Biochemical Soci- strictly restricted to Gln-tRNAGln and Asn-tRNAAsn forma- eties. Published by Elsevier Science B.V. All rights reserved. tion, respectively [8]. B. subtilis contains a non-discriminating GluRS capable of generating Glu-tRNAGln but possesses a Key words: Aminoacyl-tRNA; tRNA-dependent amidation; discriminating AspRS unable to generate the misacylated Thermus thermophilus; tRNA speci¢city Asp-tRNAAsn intermediate; therefore the gatCAB-encoded AdT only serves as a Glu-AdT in this bacterium. Conversely, Thermus and Deinococcus contain a discriminating GluRS but 1. Introduction two AspRS enzymes, one being of archaeal type ([9], J. Pe- laschier, personal communication) capable of making the The high speci¢city of aminoacyl-tRNA (AA-tRNA) syn- Asp-tRNAAsn intermediate used by the AdT which is there- thesis guarantees the faithful decoding of messenger RNA. fore an Asp-AdT. However, it is still unknown if the double Two di¡erent pathways lead to AA-tRNA; the direct charg- speci¢city applies to every AdT, or if it is restricted to only ing of the tRNA with the cognate amino acid by an AA- bacterial enzymes, or a subset of them. Our knowledge of the tRNA synthetase (AARS) or a two-step reaction involving role, contribution and requirement of the di¡erent subunits to mischarging of a tRNA followed by conversion of the amino the AdT activity also remains fragmentary. acid while bound to tRNA [1]. While such tRNA-dependent Since thermostable proteins have the remarkable ability to amino acid transformation pathways had long been known to crystallize, we decided to study T. thermophilus Asp-AdT to account for the formation of formylmethionyl-tRNA [2] and initiate structural investigations. However, unlike B. subtilis or selenocysteinyl-tRNA [3], it is now clear that this route is also D. radiodurans, no genome of any of the Thermus species has used extensively for Gln-tRNAGln and Asn-tRNAAsn [4] syn- been published thus far. Here, we report the cloning, sequenc- thesis. ing and tRNA speci¢city of T. thermophilus HB8 Asp-AdT. We show that although the enzyme is active as a GatAB heterodimer [7], T. thermophilus possesses the three canonical gatC, gatA and gatB set of genes. We establish that the over- expressed AdT exhibits both Asp-tRNAAsn and Glu-tRNAGln transamidation activities. Finally, we show that removal of the 44 carboxy-terminal amino acids of the GatA subunit *Corresponding author. Fax: (1)-203-432 6202. abolishes the enzyme's capacity to catalyze transamidation E-mail: [email protected] of Asp-tRNAAsn, without altering its Glu-AdT activity; thus 1 Both authors contributed equally to this work and are listed in the Asp-AdT and Glu-AdT activities can be physically sepa- alphabetical order. rated. 0014-5793 / 00 / $20.00 ß 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved. PII: S0014-5793(00)01697-5 FEBS 23821 30-6-00 H.D. Becker et al./FEBS Letters 476 (2000) 140^144 141 2. Materials and methods durans tRNAAsn with 0.3 nmol of pure AspRS2 from D. radiodurans and 50 WMof[14C]aspartate (216 mCi/mmol, 50 WCi/ml). Aminoacy- 2.1. Cloning and sequencing of T. thermophilus AdT genes lations were conducted in a 200 Wl standard reaction mixture [7] dur- ing 30 min at 37³C. The AA-tRNA was extracted with acid-bu¡ered The preliminary genomic sequence of T. thermophilus strain HB27 phenol, followed by a chloroform:isoamyl alcohol (24:1) extraction was searched for gat genes based on amino acid similarity with the B. subtilis Glu-AdT [5]. PCR primers were constructed based on the and an ethanol precipitation. sequences obtained. The genes were then ampli¢ed from genomic T. thermophilus strain HB8 DNA, and the clones were sequenced. 2.3. Preparation of extracts The GenBank accession numbers of T. thermophilus gat genes are Proteins were extracted by sonication of the cells at 4³C in 50 mM AF202446, AF202447 and AF202448 for gatC, gatA and gatB, re- Tris^HCl, pH 8.0, containing 5 mM 2-mercaptoethanol, 1 mM ben- spectively. zamidine, 0.1 mM Na2EDTA. S100 extracts were prepared by ultra- centrifugation at 100 000Ug for 2 h. Flocculated E. coli extracts were 2.2. Preparation of AA-tRNAs obtained by heat treatment of S100 samples at 70³C for 10 min fol- B. subtilis tRNAGln was overproduced in Escherichia coli as de- lowed by 10 min centrifugation at 15 000Ug at 4³C removing the scribed [5]. [14C]Glu-tRNAGln was prepared by aminoacylation of precipitated thermolabile proteins, which generally yields a 10-fold 0.6 nmol of overproduced B. subtilis tRNAGln in total tRNA from puri¢cation. E. coli with 63 WgofaB. subtilis tRNA-free extract (Q-Sepharose fraction) and 25 WM[14C]glutamate (251 mCi/mmol, 50 WCi/ml). 2.4. tRNA-dependent amidation assays D. radiodurans tRNAAsn was overexpressed in E. coli as described tRNA-dependent amidation reactions were performed at 37³C for [5]. [14C]Asp-tRNAAsn was prepared by aminoacylation of D. radio- 50 min in a 40 Wl standard reaction mixture [7], using 2 mM of Fig. 1. Sequence of T. thermophilus Asp-AdT subunits. The amino acid sequences of the GatA, GatB and GatC proteins are identi¢ed by ttga- tA, ttgatB and ttgatC. The amino acids in capital letters in the consensus sequence (shown below T. thermophilus genes) indicate full conserva- tion (among the samples compared). The numbers of bacterial and archeal and eukaryotic proteins used in these comparisons were, respec- tively, for GatA (14, 2, 0), for GatB (14, 2, 1), for GatC (9, 0, 0). FEBS 23821 30-6-00 142 H.D. Becker et al./FEBS Letters 476 (2000) 140^144 glutamine and 5 Wl of the enzyme preparation (see above). The AA- tRNAs were extracted, precipitated and deacylated as described pre- viously [7], and amino acids were analyzed by thin layer chromatog- raphy (TLC) in ammonia:water:chloroform:methanol (2:1:6:6). [14C]Amino acids on dried TLC plates were revealed by scanning with a Fuji Bioimager of the 12 h exposed image plate and veri¢ed by ninhydrin assay using non-radiolabeled standards (50 nmol). 3. Results 3.1. T. thermophilus strain HB8 AdT genes We searched a database (Go«ttingen Genomics Laboratory, Go«ttingen, Germany) containing preliminary genomic se- quence information of T. thermophilus strain HB27 for the gat genes encoding bacterial AdTs based on amino acid sim- ilarity with the three B. subtilis Glu-AdT subunits [5]. The search revealed the presence of not only gatA and gatB genes Fig. 3. Speci¢city of T. thermophilus AdT mutant GatDv44. Phos- in Thermus, but also of gatC. As was seen in D. radiodurans phoimages of TLC plates revealing the tRNA-dependent conversion 14 [8], an organism speci¢cally related to Thermus, the gat genes of C-radiolabeled aspartate (A and B) and glutamate (C) into as- paragine and glutamine, respectively.