Transfer RNA-Dependent Amino Acid Biosynthesis: an Essential Route to Asparagine Formation
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Transfer RNA-dependent amino acid biosynthesis: An essential route to asparagine formation Bokkee Min†, Joanne T. Pelaschier†, David E. Graham‡, Debra Tumbula-Hansen†, and Dieter So¨ ll†§¶ †Departments of Molecular Biophysics and Biochemistry and §Chemistry, Yale University, New Haven, CT 06520-8114; and ‡Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0308 Contributed by Dieter So¨ll, January 11, 2001 Biochemical experiments and genomic sequence analysis showed thesis occurs in many bacteria as the sole synthetic route to this that Deinococcus radiodurans and Thermus thermophilus do not essential amino acid. possess asparagine synthetase (encoded by asnA or asnB), the enzyme forming asparagine from aspartate. Instead these organ- Materials and Methods isms derive asparagine from asparaginyl-tRNA, which is made from Chemicals and Enzymes. Oligonucleotides were synthesized, and aspartate in the tRNA-dependent transamidation pathway [Becker, PCR products were sequenced by the Keck Foundation Bio- H. D. & Kern, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12832–12837; technology Resource Laboratory at Yale University. Uniformly and Curnow, A. W., Tumbula, D. L., Pelaschier, J. T., Min, B. & So¨ll, labeled [14C]asparagine [228.4 mCi͞mmol (1 Ci ϭ 37 GBq)] was D. (1998) Proc. Natl. Acad. Sci. USA 95, 12838–12843]. A genetic from NEN Life Science Products. Uniformly labeled [14C]as- knockout disrupting this pathway deprives D. radiodurans of the partate (213 mCi͞mmol) and [3H]aspartate (28 mCi͞mmol) ability to synthesize asparagine and confers asparagine auxotro- were from Amersham Pharmacia. phy. The organism’s capacity to make asparagine could be restored by transformation with Escherichia coli asnB. This result demon- Strains, Plasmids, and Bacterial Growth. D. radiodurans strain R1 strates that in Deinococcus, the only route to asparagine is via was obtained from John Battista (Louisiana State University, asparaginyl-tRNA. Analysis of the completed genomes of many Baton Rouge, LA). Plasmids pMD66 (13) and pMD405 were bacteria reveal that, barring the existence of an unknown pathway kindly provided by Michael J. Daly (Uniformed Services Uni- of asparagine biosynthesis, a wide spectrum of bacteria rely on the versity of the Health Sciences, Bethesda, MD). pMD66 repli- tRNA-dependent transamidation pathway as the sole route to cates autonomously in both Escherichia coli and D. radiodurans. asparagine. pMD405 contains ampicillin-resistance and kanamycin- resistance markers and an origin of replication for E. coli but no sparagine, one of the 21 cotranslationally inserted amino replication capacity for D. radiodurans. D. radiodurans was grown acids that make up proteins, is known to be synthesized at 30°C with vigorous shaking or on medium containing 1.5% A agar. Complex medium for D. radiodurans was TGY (0.8% from aspartate in an ATP-dependent amidation reaction (1). ͞ ͞ Two mechanistically distinct asparagine synthetases are known tryptone 0.4% yeast extract 0.1% glucose). Minimal medium for D. radiodurans (14) was 20 mM potassium phosphate (pH (2–4). The one encoded by asnA utilizes ammonia as amide ͞ ͞ ͞ ͞ donor, whereas the asnB-derived protein works with glutamine. 8.0) 0.2 mM MgCl2 0.1 mM CaCl2 5 M manganese acetate 5 M (NH )Mo O ͞5 M FeSO ͞10 g/ml methionine͞25 g/ml These enzymes are present in organisms of all domains, the 4 7 24 4 histidine͞30 g/ml cysteine͞1 g/ml nicotinic acid͞2 mg/ml major one being asparagine synthetase B, which is encoded in fructose. Where necessary, the medium was supplemented with different organisms by a small number of related genes. Both 10 g/ml kanamycin͞2.5 g/ml tetracycline͞3 g/ml chloram- enzymes are well studied biochemically (5), and their crystal phenicol͞20 g/ml asparagine. E. coli strain DH5␣ was grown at structures are known (6, 7). Until recently they were assumed to 37°C on LB medium (1% tryptone͞0.5% yeast extract͞0.5% be the sole biosynthetic route to asparagine. However, Thermus NaCl͞1.5% agar) supplemented where necessary with 50 g͞ml thermophilus and Deinococcus radiodurans lack these enzymes; ampicillin and 30 g͞ml tetracycline. E. coli strain JF448 (15), instead they employ a tRNA-dependent transamidation mech- obtained from the E. coli Genetic Stock Center at Yale Uni- anism for conversion of aspartate to asparagine (8, 9). versity, has an asparagine auxotrophic phenotype (asnA and Two routes of Asn-tRNA synthesis exist in D. radiodurans asnB). It was grown for 3 days at 37°C on M9 minimal agar plates (Fig. 1). Similar to many bacteria, Deinococcus contains a containing 1 g͞ml thiamine and 30 g͞ml asparagine. tRNA-dependent two-step pathway of Asn-tRNA formation. In the first step a nondiscriminating aspartyl-tRNA synthetase Preparation of tRNA Transcripts. tRNAAsp and tRNAAsn genes were (AspRS)2 generates the misacylated Asp-tRNAAsn species, Asn identified in the preliminary D. radiodurans genomic DNA which then is amidated to the correctly charged Asn-tRNA by Asp Asn sequence by the program TRNASCAN (16). The tRNA gene was the heterotrimeric Asp-tRNA amidotransferase (Asp-AdT; produced by PCR using the Pfu Turbo polymerase (Stratagene) encoded by the gatCAB genes) with glutamine serving as the and subcloned into pUC119. The tRNAAsn gene was constructed amide donor (9). In addition, the organism also contains aspar- in pUC119 from six overlapping oligonucleotides. To enable in aginyl-tRNA synthetase (AsnRS; ref. 10), which is active and vitro transcription of tRNAAsn with T7 RNA polymerase, the first produces Asn-tRNA in the canonical aminoacylation reaction base pair was changed to G1-C72. The 5Ј end of each construct (9). The close Deinococcus relative T. thermophilus has similar contained the T7 promoter, and the 3Ј end contained a BstNI site enzymes and presumably uses the same asparagine biosynthetic routes (8, 11, 12). It was suggested earlier (8, 9) that the role of Asp-AdT in D. radiodurans and T. thermophilus is to synthesize Abbreviations: AspRS, aspartyl-tRNA synthetase; Asp-AdT, Asp-tRNAAsn amidotransferase; the cell’s entire supply of asparagine, because no asnA or asnB AsnRS, asparaginyl-tRNA synthetase. orthologs are present in the genome (10), and because biochem- ¶To whom reprint requests should be addressed at: Department of Molecular Biophysics and Biochemistry, Yale University, P.O. Box 208114, 266 Whitney Avenue, New Haven, CT ical analysis of crude cell extracts did not reveal the presence of 06520-8114. E-mail: [email protected]. any tRNA-independent asparagine synthetase activity (8, 9). The publication costs of this article were defrayed in part by page charge payment. This Here we present data from D. radiodurans that prove this role to article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. be correct and propose that tRNA-dependent asparagine syn- §1734 solely to indicate this fact. 2678–2683 ͉ PNAS ͉ March 5, 2002 ͉ vol. 99 ͉ no. 5 www.pnas.org͞cgi͞doi͞10.1073͞pnas.012027399 Downloaded by guest on September 30, 2021 Aminoacyl-tRNA Synthetase Assays. Reactions (100 l) were per- formed as described (9) at 30°C and contained 100 mM Hepes (pH 7.2), 50 mM KCl, 10 mM MgCl2,5mMDTT,6 M [3H]aspartate, 2.5 mM unlabeled aspartate, 2 mM ATP, and 100 nM AspRS1 or 200 nM AspRS2. Aliquots (10 l) of the reaction were removed at various time intervals and spotted onto a 3MM filter disk (Whatman) that was immersed in 10% trichloroacetic acid for 10 min and then washed twice with 5% trichloroacetic acid. The filters were rinsed in ethanol, dried-and counted in 3 ml of Ultima Gold scintillation mixture fluid (Packard). For kinetic analysis, a higher specific activity aspartate mix was composed of 16 M[3H]aspartate and 2.5 mM unlabeled Fig. 1. Redundant pathways of Asn-tRNAAsn synthesis in D. radiodurans. aspartate. Aliquots were taken every 15 sec, and initial rates Shown are the direct pathway (top half of diagram) and the transamidation were determined from a minimum of five time points in the pathway (bottom half of diagram). linear range of duplications of the assay. The enzyme concen- trations were held at 40 nM AspRS1 and 100 nM AspRS2. The that, after BstNI digestion, allowed generation of the correct 3Ј concentrations of ATP and aspartate were at least 5-fold above end of the DNA template for in vitro transcription. Reactions their determined apparent Km values (2 mM ATP and 0.5 mM aspartate in the AspRS1 reactions, and 2 mM ATP and 1.25 mM were performed as described (17); the resulting RNA was to 2.5 mM aspartate for AspRS2). When tRNAAsn was used, the ethanol-precipitated, resuspended in loading buffer [10 mM K of AspRS2 for aspartate was 2-fold higher than that when Hepes (pH 7.3)͞1mMNa-EDTA͞7 M urea], and heated to m 2 tRNAAsp was used. Therefore, for AspRS2, aspartate was kept 85°C for 10 min before loading onto a Q Sepharose column at 1.25 mM for tRNAAsp and 2.5 mM for tRNAAsn. Transcript (Amersham Pharmacia). Transcripts were eluted from the col- concentrations were at 3 M for AspRS1 and 20 M for umn at 1.3 M NaCl. Fractions were concentrated, desalted, AspRS2. For AspRS1, the concentration of aspartate was varied ethanol-precipitated, and resuspended in 10 mM Hepes from 2 to 250 M, ATP was varied from 0.05 to 5 mM, and (pH 7.2). tRNAAsp transcript was varied from 0.01 to 0.5 M. For AspRS2, aspartate was varied from 0.02 to 1.25 mM for tRNAAsp and 0.02 Preparation of Unfractionated and in Vivo Expressed tRNA. Unfrac- to 2.5 mM for tRNAAsn; ATP was varied from 13 M to 1.6 mM; tionated D. radiodurans tRNA was prepared as described (9).