Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1998 Oct 27;95(22):12838–12843. doi: 10.1073/pnas.95.22.12838

Glutamyl-tRNAGln amidotransferase in Deinococcus radiodurans may be confined to asparagine biosynthesis

Alan W Curnow *,†, Debra L Tumbula *,†, Joanne T Pelaschier ‡, Bokkee Min *, Dieter Söll *,§,
PMCID: PMC23620  PMID: 9789001

Abstract

Asparaginyl-tRNA (Asn-tRNA) and glutaminyl-tRNA (Gln-tRNA) are essential components of protein synthesis. They can be formed by direct acylation by asparaginyl-tRNA synthetase (AsnRS) or glutaminyl-tRNA synthetase (GlnRS). The alternative route involves transamidation of incorrectly charged tRNA. Examination of the preliminary genomic sequence of the radiation-resistant bacterium Deinococcus radiodurans suggests the presence of both direct and indirect routes of Asn-tRNA and Gln-tRNA formation. Biochemical experiments demonstrate the presence of AsnRS and GlnRS, as well as glutamyl-tRNA synthetase (GluRS), a discriminating and a nondiscriminating aspartyl-tRNA synthetase (AspRS). Moreover, both Gln-tRNA and Asn-tRNA transamidation activities are present. Surprisingly, they are catalyzed by a single enzyme encoded by three ORFs orthologous to Bacillus subtilis gatCAB. However, the transamidation route to Gln-tRNA formation is idled by the inability of the discriminating D. radiodurans GluRS to produce the required mischarged Glu-tRNAGln substrate. The presence of apparently redundant complete routes to Asn-tRNA formation, combined with the absence from the D. radiodurans genome of genes encoding tRNA-independent asparagine synthetase and the lack of this enzyme in D. radiodurans extracts, suggests that the gatCAB genes may be responsible for biosynthesis of asparagine in this asparagine prototroph.

Aminoacyl-tRNA is the key intermediate in translation, providing the link between the genetic code and protein synthesis. Two routes to formation of the cognate aminoacyl-tRNA amino acid corresponding to the anticodon of the tRNA are known to exist (Fig. 1). The direct pathway, which is the more general route, involves aminoacyl-tRNA synthetases that esterify tRNA species with the correct amino acid. However, in organisms that lack specific synthetase activities for a subset of the 20 amino acids, tRNA-dependent transformation of mischarged aminoacyl-tRNA ensures the formation of a full complement of correctly charged tRNAs. As an example, in systems lacking discernible GlnRS activity, Gln-tRNAGln formation is catalyzed by two enzymes. The first is a nondiscriminating GluRS, i.e., one that mischarges tRNAGln with glutamate, forming Glu-tRNAGln. In the second step, the tRNA-bound glutamate is transamidated to glutamine by glutamyl-tRNAGln amidotransferase (Glu-AdT), a tRNA-dependent glutamine amidotransferase. This enzyme is best described in Bacillus subtilis, from which the enzyme was cloned and purified (1). Moreover, a genetic knockout of the 5′ portion of the gatCAB operon in B. subtilis is lethal (1). This pathway is widespread, having been identified in most Gram-positive and some Gram-negative bacteria, Rhizobium meliloti, cyanobacteria, archaea, and eukaryal organelles (2).

Figure 1.

Figure 1

Open in a new tab

Putative pathways to (Upper) Gln-tRNA and (Lower) Asn-tRNA formation in D. radiodurans in accordance with current genomic sequence data. Shown are the direct (top half of each diagram) and indirect (transamidation) pathways (bottom half of each diagram). Designations for both enzymes and genes are presented. The genes for Asp-AdT have yet to be identified in any organism. GlnRS, glutaminyl-tRNA synthetase.

A similar transamidation pathway to Asn-tRNA formation was described in Haloferax volcanii (3). In this archaeon, a misacylating AspRS results in the formation of Asp-tRNAAsn, then tRNA-dependent transamidation of aspartate by aspartyl-tRNAAsn amidotransferase (Asp-AdT) produces Asn-tRNAAsn. The crystal structure of Pyrococcus kodakaraensis AspRS has recently been solved, allowing for rigorous investigation of this archaeal synthetase (4). A relative of the archaeal AspRS has been studied in the hyperthermophilic bacterium Thermus thermophilus (5). The enzyme responsible for Asp-AdT activity in T. thermophilus has recently been identified (see the accompanying paper, ref. 6). The absence of homologs of the asnS gene in the genomes of several archaea (7–9) and in the bacterium Helicobacter pylori (10) suggests that this pathway supplies Asn-tRNA in these organisms. To date, direct and indirect pathways to the formation of a single aminoacyl-tRNA have not been observed in the same cellular compartment.

Examination of the preliminary Deinococcus radiodurans database suggested that both direct acylation of tRNAGln and tRNAAsn as well as indirect tRNA-dependent transamidation pathways may exist in this mesophilic bacterium. This organism is specifically but not closely related to T. thermophilus (11), from which many aminoacyl-tRNA synthetases have been purified and studied in biochemical and structural detail (12–14). The knowledge of its genomic sequence, together with its ease of growth and its suitability for molecular genetic experiments (15, 16), makes D. radiodurans an ideal system for an integrated study of the direct and indirect pathways of aminoacyl-tRNA formation. Furthermore, D. radiodurans, an asparagine prototroph, which grows in a defined medium lacking asparagine (17), may facilitate studies of the role tRNA plays in amino acid metabolism. We therefore characterized the enzymatic activities involved in the formation of both Asn-tRNA and Gln-tRNA in D. radiodurans.

MATERIALS AND METHODS

General.

Media for bacterial growth and molecular biology protocols were standard (18) unless otherwise noted. D. radiodurans R1, obtained from John R. Battista (Louisiana State University, Baton Rouge) and Kenneth W. Minton (University of the Armed Services, Bethesda, MD), was grown at 30°C in tryptone/yeast extract/glucose (TGY) medium (19). Chromosomal D. radiodurans DNA was purified as described (19). Uniformly labeled [3H]asparagine (1 Ci/mmol; 1 Ci = 37 GBq) was custom-synthesized by American Radiolabeled Chemicals (St. Louis). Amino acids had the following specific activities: [14C]aspartate, 203 mCi/mmol; [3H]glutamine, 48 Ci/mmol; [14C]glutamate, 258 mCi/mmol; and [3H]glutamate, 50 Ci/mmol. Oligonucleotides were synthesized in the Keck Foundation Research Biotechnology Resource Laboratory at Yale University.

DNA Sequence Analyses.

The preliminary sequence of the D.radiodurans genome (ftp://ftp.tigr.org/pub/data/ dradiodurans/) was searched by using the gapped blast algorithm (20) and the Wisconsin Package, Version 9 (Genetics Computer Group, Madison, WI).

Cloning and Expression of Selected Open Reading Frames from D. radiodurans.

Based on the D. radiodurans sequence data, the genes encoding AspRS1, AspRS2, and Glu-AdT subunits C, A, and B were PCR-amplified from genomic DNA. AspRS1 and AspRS2 were cloned into the commercially available vector, pCR2.1-TOPO (Invitrogen). The AspRS1 and AspRS2 genes were then subcloned into pET15b (Novagen) or pCYB2 (New England Biolabs), respectively, for heterologous expression in Escherichia coli. Recombinant AspRS1 was purified on nickel–nitrilotriacetate resin (Qiagen, Chatsworth, CA), and AspRS2 was purified by chitin-binding affinity chromatography. The gatC, gatA, and gatB orthologs were ligated to form an artificial operon [like the one found in B. subtilis (1)], PCR amplified, inserted into pCR2.1-TOPO, and then subcloned into pET20b (Novagen). The Glu-AdT activity was then partially purified by anion-exchange chromatography on Q-Sepharose (Pharmacia).

tRNA Preparation.

Unfractionated D. radiodurans tRNA was prepared from frozen cells harvested during logarithmic-phase growth. Cells (15 g) were resuspended in lysis buffer (20 mM Tris⋅HCl, pH 7.4/20 mM magnesium acetate). The suspension was extracted twice with phenol (equilibrated in 25 mM sodium acetate, pH 4.6/50 mM NaCl) and once with chloroform/isoamyl alcohol (24:1, vol/vol). Nucleic acids in the aqueous phase were precipitated in 0.3 M sodium acetate (pH 5.2) and 1 vol of 2-propanol, collected by centrifugation at 8,700 × g for 30 min at 4°C, and washed in 70% ethanol. The tRNA was deacylated by incubation at 37°C for 30 min in the presence of 0.2 M Tris⋅acetate (pH 9.0) and precipitated in 0.3 M sodium acetate (pH 5.2) and 2 vol of ethanol. High-molecular-weight nucleic acids were removed by precipitation in 0.1 M NaCl overnight at 4°C and centrifugation at 8,700 × g for 30 min at 4°C. Additional phenol and chloroform extractions were followed by ethanol precipitation. The tRNA was further purified by binding to DEAE-cellulose resin (DE52, Whatman) (1 g/A260 unit) in buffer 1 (20 mM Tris⋅HCl, pH 7.0) and washing with buffer 2 (20 mM Tris⋅HCl, pH 7.0/0.2 M NaCl). The tRNA was eluted with buffer 3 (20 mM Tris⋅HCl, pH 7.0/1 M NaCl) and recovered by ethanol precipitation. Aliquots resuspended in double-distilled water were stored at −70°C.

D. radiodurans tRNA-Free Cell Extract Preparation.

D. radiodurans cells (10 g) harvested from logarithmic-phase growth were suspended in 20 ml of buffer A (50 mM Hepes⋅KOH, pH 7.2/10 mM MgCl2/10% glycerol/5 mM DTT) with 0.5 mM phenylmethylsulfonyl fluoride (PMSF). The suspension was passed twice through a cooled (4°C) French pressure cell at 24,000 psi (1 psi = 6.89 kPa). After a low-speed centrifugation (8,700 × g) to remove cell debris, the supernatant was centrifuged for 90 min at 100,000 × g. The supernatant was applied to Q-Sepharose (Pharmacia) equilibrated with buffer A. The tRNA-free protein fraction was eluted with buffer A containing 300 mM NaCl.

Aminoacylation Assays.

The AsnRS, GlnRS, and GluRS activities were assayed in freshly prepared D. radiodurans cell-free extracts depleted of tRNA. Aminoacylation of unfractionated D. radiodurans tRNA was performed at 30°C in triplicate. Reactions (80 μl) contained 50 mM Hepes⋅KOH (pH 7.2), 25 mM KCl, 10 mM MgCl2, 5 mM DTT, 4 mM ATP, 45–65 μM tRNA, and 30–130 μg of extract protein. Aliquots (10 μl) taken at various time intervals were spotted onto a 3MM filter disk (Whatman) and immersed in 10% trichloroacetic acid with 5 mM of the corresponding amino acid for 10 min, then washed twice in 5% trichloroacetic acid. The filters were rinsed in ethanol, dried, and counted in 3 ml of Ultima Gold scintillation fluid (Packard). For GlnRS activity, the tRNA was heated to 90°C for 5 min and then annealed by allowing to cool to room temperature to reach a plateau of charging activity within 30 min. Conversion values for pmol and cpm of labeled amino acids were determined by counting prewashed filters that had been spotted with 10 μl of dilutions of the labeled amino acids. A value of 1600 pmol of tRNA/A260 unit was assumed for calculating the acceptor activity.

Amidotransferase Assays.

The assays of Asp-AdT and Glu-AdT were based on earlier protocols (1, 2). D. radiodurans [14C]Asp-tRNA was prepared by using purified D. radiodurans AspRS1 or AspRS2 as described above for the aminoacylation assays. The heterologous substrate B. subtilis [14C]Glu-tRNAGln was prepared by using an in vivo-expressed cloned tRNAGln gene and partially purified B. subtilis GluRS, as described earlier (1). Aminoacylated tRNA was isolated by phenol extraction and ethanol precipitation. The amidotransferase assay, performed at 30°C, contained 50 mM Hepes⋅KOH (pH 7.5), 25 mM KCl, 15 mM MgCl2, 5 mM DTT, 1 mM ATP, 2.5 mM glutamine, and a source of amidotransferase. The source of D. radiodurans AdT was partially purified recombinant enzyme (see above); B. subtilis Glu-AdT was the pure recombinant enzyme (1). In each reaction, 10 pmol of unfractionated D. radiodurans tRNA or 25 pmol of expressed B. subtilis tRNAGln was used. Following a 30-min incubation, sodium acetate (pH 4.5) was added to 0.3 M, and the aminoacyl-tRNA was isolated by phenol extraction and ethanol precipitation. The tRNA was deacylated in 25 mM KOH at 65°C for 10 min. After being dried and resuspended in water, the samples were separated by thin-layer chromatography on cellulose (Sigma) in chloroform/methanol/water/ammonium hydroxide (6:6:1:2). After drying, the plate was exposed to a phosphorimaging plate (Fuji) to detect the labeled amino acids. The locations of the amino acids were verified by running 20 nmol of the corresponding unlabeled amino acid in each lane followed by visualization with ninhydrin solution (0.2% in ethanol).

Asparagine Synthetase Assay.

tRNA-free S100 extracts of D. radiodurans and E. coli DH5α were prepared as described above for D. radiodurans. The assays contained 50 mM Hepes⋅KOH (pH 7.2), 25 mM KCl, 10 mM MgCl2, 4 mM DTT, 4 mM ATP, 2.5 mM glutamine, 2.5 μM [14C]aspartate, 1 mM [12C]aspartate, and cell extract protein from D. radiodurans (0.56 μg/ml) or E. coli (0.17 μg/ml). The assays were performed at 30°C for D. radiodurans or at 37°C for E. coli. Following a 45-min incubation, 2-μl aliquots were separated by thin-layer chromatography and analyzed by using phosphoimaging as described above for the amidotransferase assays.

RESULTS

The Genomic D. radiodurans Sequence Data Suggest the Presence of Redundant Pathways of Aminoacyl-tRNA Formation.

The recent interest in novel aminoacyl-tRNA synthetase genes and pathways in various organisms (2) prompted us to analyze the preliminary sequence data available for D. radiodurans. Gapped tblastn searches (20) revealed the presence of genes similar to those encoding proteins implicated in direct aminoacylation and tRNA-dependent amino acid transformation routes. With respect to Gln-tRNAGln formation, the search established that a glnS gene is present, suggesting the capacity of direct acylation of tRNAGln, as well as orthologs for all three Glu-AdT encoding genes (gatA, gatB, and gatC). Unlike in the Gram-positive bacteria, the genes were not present as an operon. This is not unusual, as the absence of an operon structure of these genes occurs in the archaea Methanococcus jannaschii (7), Methanobacterium thermoautotrophicum (9), and Archaeoglobus fulgidus (8) and in the bacteria Helicobacter pylori (10) and Aquifex aeolicus (21). As these organisms lack an identifiable glnS gene, these genes may provide the requisite Glu-AdT activity.

For formation of Asn-tRNAAsn, the D. radiodurans genome contains one asnS gene encoding a conventional AsnRS. Furthermore, two aspS genes are present. The aspS1 gene encodes a canonical, bacteria-like AspRS (576 amino acids). In comparison, aspS2 appears to encode a much smaller protein (435 amino acids) whose closest relatives are the archaeal AspRS enzymes (ref. 4; GenBank accession no. AB010464). The AspRS2 likely misacylates tRNAAsn, because the archaea-like AspRS2 of T. thermophilus has been shown to misacylate tRNAAsn (6).

Canonical tRNA Aminoacylation.

In Fig. 2 direct aminoacylation activities are shown for AsnRS (A), GlnRS (B), and GluRS (C) in D. radiodurans. The percentages of acceptor tRNAs present were 2.1% (tRNAAsn), 7.0% (tRNAGln), and 7.7% (tRNAGlu). That equal amounts of aminoacylated tRNA are formed by the direct incorporation of both glutamate and glutamine suggests that the GluRS is discriminating. The amount of Glu-tRNA formed would be doubled if the GluRS were able to recognize both tRNAGlu and tRNAGln.

Figure 2.

Figure 2

Open in a new tab

Direct aminoacylation activities of AsnRS (A), GlnRS (B), and GluRS (C) in D. radiodurans tRNA-free extracts by using 3H-labeled amino acids. Shown are complete reactions (•) and reactions omitting tRNA (○) or extract (×).

D. radiodurans Possesses Both Glu-AdT and Asp-AdT Activity.

Purified components from the B. subtilis transamidation pathway (1) were used in combination with D. radiodurans extract to determine whether D. radiodurans possesses Glu-AdT activity. When B. subtilis tRNAGln was mischarged with glutamate by using B. subtilis GluRS, conversion of Glu-tRNAGln to Gln-tRNAGln was detected with both B. subtilis Glu-AdT and D. radiodurans extract (Fig. 3A, lanes 2 and 3, respectively). However, when D. radiodurans extract was the source of GluRS in the assay, no conversion by B. subtilis Glu-AdT or D. radiodurans extract was detected (Fig. 3A, lanes 5 and 6). When B. subtilis GluRS was used to misacylate D. radiodurans total tRNA, conversion was detectable with B. subtilis Glu-AdT (Fig. 3B, lane 2) and to a lower degree with D. radiodurans extract (data not shown).

Figure 3.

Figure 3

Open in a new tab

Glu-AdT activity. Phosphorimages of thin-layer chromatographic separation of [14C]glutamine and [14C]glutamate recovered from transamidation assays in which (A) B. subtilis tRNAGln was aminoacylated by B. subtilis GluRS (lanes 1–3) or D. radiodurans tRNA-free S100 extract protein (lanes 4–6). No enzyme (lanes 1 and 4), recombinant B. subtilis Glu-AdT (lanes 2 and 5), or D. radiodurans tRNA-free S100 extract protein (lanes 3 and 6) was added. (B) Unfractionated D. radiodurans tRNA aminoacylated by B. subtilis GluRS; no enzyme (lane 1) or recombinant B. subtilis Glu-AdT (lane 2) was added.

To explore this result further, three D. radiodurans genes orthologous to the B. subtilis Glu-AdT subunit genes gatC, gatA, and gatB were cloned, expressed, and the resulting D. radiodurans Glu-AdT activity purified from E. coli. Extracts of cells transformed with this vector possessed Glu-AdT activity when B. subtilis [14C]Glu-tRNAGln was used as substrate (Fig. 4A, lane 3), whereas extracts from cells transformed with the empty vector showed no activity (data not shown). The partially purified recombinant D. radiodurans Glu-AdT also possessed Asp-AdT activity, as conversion of D. radiodurans Asp-tRNA to Asn-tRNA was detectable (Fig. 4C, lane 3). An unexpected result from this assay was that the pure B. subtilis Glu-AdT enzyme also possessed Asp-AdT activity. Moreover, this transamidation activity occurred with tRNA charged with AspRS1 and AspRS2 (Fig. 4 B and C, lanes 2). This somewhat relaxed tRNA specificity may be the result of the B. subtilis Glu-AdT having evolved in the context of Glu-tRNAGln to Gln-tRNAGln conversion, and under the conditions of Asp to Asn conversion this enzyme has a limited ability to discriminate between Asp-tRNAAsn and Asp-tRNAAsp. The D. radiodurans Asp-AdT activity depends on aminoacylation by AspRS2, since Asp-AdT activity was not observed when AspRS1 was used to charge the tRNA (Fig. 4B, lane 3). Furthermore, because D. radiodurans lacks discernible asparagine synthetase (asnA and asnB) genes, formation of asparagine depends on the presence of tRNA in this organism. As illustrated in Fig. 5 (lane 1), no asparagine is synthesized in the absence of tRNA in D. radiodurans extracts, whereas asparagine is formed in tRNA-free extracts of E. coli (Fig. 5, lane 2).

Figure 4.

Figure 4

Open in a new tab

Glu-AdT and Asp-AdT activity from the recombinant D. radiodurans gatCAB operon. Phosphorimages of thin-layer chromatographic separation of 14C-labeled amino acids recovered from transamidation assays where: B. subtilis tRNAGln was aminoacylated by B. subtilis GluRS (A) or unfractionated D. radiodurans tRNA was aminoacylated by recombinant D. radiodurans AspRS1 (B) or AspRS2 (C). In each panel no enzyme (lane 1), recombinant B. subtilis Glu-AdT (lane 2), or recombinant D. radiodurans Glu-AdT (lane 3) was added.

Figure 5.

Figure 5

Open in a new tab

Glutamine-dependent asparagine synthetase activity. Phosphorimages of thin-layer chromatography plates showing conversion of [14C]aspartate to [14C]asparagine recovered from incubations with D. radiodurans (lane 1) and E. coli (lane 2) tRNA-free S100 extract protein.

DISCUSSION

The Transamidation Pathway Versus Canonical Aminoacylation.

The necessity of transamidation pathways remains uncertain, considering that direct pathways of aminoacylation exist for each of the 20 standard amino acids. Currently available genomic data as well as biochemical analyses have shown the wide distribution in nature of the transamidation pathway to Gln-tRNA formation. GlnRS in bacteria appears to be derived by a horizontal transfer from eukarya and presumably accompanied by elimination of the indirect pathway (22). GlnRS catalyzes Gln-tRNA formation in eukaryal cytoplasm, in some proteobacteria of the γ-subdivision, and in some mitochondria; archaea and many bacteria employ the transamidation route (2). It has been suggested (1) that the indirect pathway may be advantageous in certain organisms for control of glutamine and glutamate levels (23). Did such metabolic advantage prevent the transfer of glnS to other families of bacteria or did the transfer happen only once? A comparison of the G+C content of the relevant genes in D. radiodurans does not suggest a recent transfer from another organism, as the G+C content (65–70%) of these genes is the same as that of unrelated Deinococcus genes.

The role, if any, of the Glu-AdT activity in D. radiodurans is unclear because the transamidation pathway depends on a nondiscriminating GluRS. Interestingly, examination of the D. radiodurans genomic sequence reveals a second GluRS-encoding gene. However, it is truncated and lacks the information for the C-terminal 190 amino acids. Thus, the encoded protein is likely inactive, as the missing sequence includes the vital anticodon-binding domain. The fragmentation may indicate that this gene is in the process of being lost from the genome. This is similar to the canonical Treponema pallidum lysyl-tRNA synthetase gene, which has lost its critical 5′ portion (24). In this organism, the lysyl-tRNA synthetase activity is probably supplied by a full-length lysS gene of the unusual class I type, as was demonstrated in the related organism Borrelia burgdorferi (25). Thus, is Glu-AdT a remnant of an indirect pathway that existed in an ancestor of D. radiodurans?

Although it is currently not understood structurally what determines the inclusive recognition of tRNAGlu and tRNAGln, it is clear from genomic or biochemical analysis that only a few bacteria (e.g., E. coli and D. radiodurans) have a discriminating GluRS enzyme, which is usually found in coexistence with a functional GlnRS. Because sequence analysis at present cannot distinguish between a discriminating and nondiscriminating GluRS, this case provides another example of the shortcomings of assigning pathways based on genomic analysis rather than on biochemical study.

A Single Enzyme Possesses Both Asp-AdT and Glu-AdT Activity in D. radiodurans.

The cloning and expression of the three D. radiodurans gat orthologs and the test of their transamidation activity demonstrates that Glu-AdT and Asp-AdT activity can reside in the same enzyme, making this enzyme the first of a family of glutamyl/aspartyl-tRNA amidotransferases. The A subunit is a tRNA-independent glutaminase (A.C., unpublished results); the B subunit is the putative tRNA-binding protein. In line with the dual-specificity transamidation activity data, the D. radiodurans genome contains only one gatB ortholog. The somewhat relaxed tRNA specificity of the D. radiodurans amidotransferase enzyme (recognizing tRNAAsn and tRNAGln) would not be manifested in vivo, because the substrate availability (Asp-tRNAAsn) allows only one final product. The fact that the B. subtilis Glu-AdT, in a heterologous reaction, also forms Asn-tRNAAsn shows relaxed tRNA specificity of this enzyme; this, however, is of no consequence in Bacillus, as the absence of the mischarged Asp-tRNAAsn confines Glu-AdT to Gln-tRNAGln formation.

However, there may well be organisms that require Asp-AdT and Glu-AdT activities by distinct enzymes. Some archaea do not possess asnS and glnS genes, but have gatB orthologs and paralogs. We suggested earlier (1) that Glu-AdT and Asp-AdT share the A and C subunits, but differ in the B subunit, which presumably recognizes the tRNA. Such organisms may carry two genes, gatB and gatB′, encoding variants of the B subunit. This type of multimeric system may provide the facility for more stringent tRNA recognition if one of the B subunits has specific discrimination capacity for tRNAAsn and the other is specific for tRNAGln. With the exception of H. pylori, this is consistent for all organisms in which Glu-AdT and Asp-AdT activity were shown (D.T., unpublished results) as these genomes carry orthologous and paralogous gatB genes.

Asparagine Biosynthesis in D. radiodurans May Require Asp-AdT-Catalyzed Asn-tRNA Formation.

A possible explanation for the unusual presence of redundant pathways of Asn-tRNAAsn formation in D. radiodurans involves a role for tRNA in amino acid biosynthesis. The only known biosynthetic route to asparagine is via asparagine synthetase, encoded by asnA or asnB (26). The glutamine-dependent asparagine synthetase, the product of the asnB gene, is present in bacteria and eukarya and uses ATP and glutamine in asparagine synthesis from aspartate with the formation of a β-aspartyl-AMP intermediate (27). A structurally different, ammonia-dependent enzyme encoded by asnA is present only in a few bacteria (28). A search for these enzyme activities in D. radiodurans extracts was unsuccessful (Fig. 5). tblastn searches of the preliminary sequence of D. radiodurans did not reveal orthologs of asnA or asnB in the genome. Further database searches did not uncover any organisms lacking these genes but containing the asnS and the gat genes. Because AsnRS in D. radiodurans is active in Asn-tRNA synthesis (Fig. 2), it is appealing to speculate that the Asp-AdT-catalyzed transamidation of Asp-tRNAAsn is present for the purpose of asparagine biosynthesis in this organism. The amide source in the transamidation reaction would be glutamine or ammonia (Fig. 1). Indeed, the genome of D. radiodurans has a well conserved glnA gene whose product, glutamine synthetase, could provide glutamine for the organism. In addition, glutamine synthetase activity has been demonstrated in T. thermophilus (6), a specific relative of D. radiodurans (11).

To have an effective transamidation pathway to asparagine formation, Asn-tRNA needs to be hydrolyzed. Although the chemical stability of the Asn-tRNA linkage is in the lower range of all aminoacyl-tRNA bonds (29), the cell may need a hydrolase to release asparagine from tRNA to make this pathway to asparagine formation more efficient. Such a hydrolase may exist; e.g., a protein that releases d-tyrosine from charged tRNA has been characterized (30). Alternatively, AsnRS may fill this role, as synthetase-catalyzed hydrolysis of aminoacyl-tRNA in the absence of the other substrates (AMP and PPi) is known (31).

Are the Enzymes of Asn and Asn-tRNA Formation Related?

Asp-AdT activity potentially serving as the sole source of asparagine formation in this organism focuses the question on the relationship between AspRS2, the nondiscriminating aspartyl-tRNA synthetase essential for asparagine formation, and asparagine synthetase. Biochemical (32) and structural analyses have shown a strong structural and evolutionary relatedness between the glutamine-dependent asparagine synthetase (33) and class II aminoacyl-tRNA synthetases, especially AspRS and AsnRS (14). Although asparagine synthetase does not share overall sequence similarity with the aminoacyl-tRNA synthetases, its structure contains a class II fold similar to that found in AspRS (14). Their structural similarity, probably dictated by common recognition requirements for aspartyl-AMP, suggests that these proteins were involved in a common ancestral process. Possibly an AspRS2-like aminoacyl-tRNA synthetase and the tRNA-dependent aspartate transamidation pathway was once a route to asparagine. Further evolution of the tRNA-independent asparagine synthetase may have made this pathway superfluous and established a direct route for asparagine formation. This is consistent with the idea that GlnRS (23) and possibly AsnRS (C. Woese and G. Olsen, personal communication) are recent transfers from eukarya to bacteria.

Evolution of tRNA Function.

Transfer RNA is a very old molecule (34). It was part of the early translation system, deciphering the genetic code and carrying activated amino acids for protein synthesis. Although these functions are still the main roles of tRNA, there might have been other tasks [e.g., genomic tagging (35) or initiation of reverse transcription (36)] that did not rely on charged tRNA. As a carrier of activated amino acids, tRNA might also have served roles in primitive metabolism (37). The formation by glutamyl-tRNA reductase of glutamate semialdehyde, the first precursor of porphyrin biosynthesis, is one example (38). Possibly, glutamine and asparagine synthesis by the tRNA-dependent transamidation route might once have been a prevailing route in intermediary metabolism. With the evolution of sophisticated metabolic pathways, such a role of tRNA may have been lost. However, in a few organisms some alternative tRNA roles may still be in use.

Acknowledgments

We thank Carl Woese and Howard Zalkin for review of the manuscript. We are indebted to John R. Battista, Nicole Shank, and Kenneth W. Minton for providing D. radiodurans R1 and for helpful advice on handling the organism. We recognize Michael J. Daly, Claire Fraser, Michael Ibba, and Owen White for helpful discussions. D.L.T. is a National Institutes of Health Postdoctoral Fellow (GM19278-01). This work was supported by grants from the National Institute of General Medical Sciences (GM22854 and GM55674) and the Department of Energy (DE-FGO2-98ER20311). We acknowledge the Institute of Genomic Research for making genomic sequence data available prior to publication.

ABBREVIATIONS

RS

tRNA synthetase

AdT

amidotransferase

References

  • 1. Curnow A W, Hong K-W, Yuan R, Kim S-I, Martins O, Winkler W, Henkin T M, Söll D. Proc Natl Acad Sci USA. 1997;94:11819–11826. doi: 10.1073/pnas.94.22.11819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Ibba M, Curnow A W, Söll D. Trends Biochem Sci. 1997;22:39–42. doi: 10.1016/s0968-0004(96)20033-7. [DOI] [PubMed] [Google Scholar]
  • 3.Curnow A W, Ibba M, Söll D. Nature (London) 1996;382:589–590. doi: 10.1038/382589b0. [DOI] [PubMed] [Google Scholar]
  • 4.Schmitt E, Moulinier L, Fujiwara S, Imanaka T, Thierry J-C, Moras D. EMBO J. 1998;17:5227–5237. doi: 10.1093/emboj/17.17.5227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Becker H D, Reinbolt J, Kreutzer R, Giegé R, Kern D. Biochemistry. 1997;36:8785–8797. doi: 10.1021/bi970392v. [DOI] [PubMed] [Google Scholar]
  • 6.Becker H D, Kern D. Proc Natl Acad Sci USA. 1998;95:12832–12837. doi: 10.1073/pnas.95.22.12832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Bult C J, White O, Olsen G J, Zhou L, Fleischmann R D, Sutton G G, Blake J A, FitzGerald L M, Clayton RA, et al. Science. 1997;273:1058–1073. doi: 10.1126/science.273.5278.1058. [DOI] [PubMed] [Google Scholar]
  • 8.Klenk H-P, Clayton R A, Tomb J-F, White O, Nelson K E, Ketchum KA, Dodson R J, Gwinn M, Hickey E K, Peterson J D, et al. Nature (London) 1997;390:364–370. doi: 10.1038/37052. [DOI] [PubMed] [Google Scholar]
  • 9.Smith D R, Doucette-Stamm L A, Deloughery C, Lee H, Dubois J, Aldredge T, Bashirzadeh R, Blakely D, Cook R, Gilbert K, et al. J Bacteriol. 1997;179:7135–7155. doi: 10.1128/jb.179.22.7135-7155.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Tomb J F, White O, Kerlavage A R, Clayton R A, Sutton G G, Fleischmann R D, Ketchum K A, Klenk H P, Gill S, Dougherty B A, et al. Nature (London) 1997;388:539–547. doi: 10.1038/41483. [DOI] [PubMed] [Google Scholar]
  • 11.Olsen G J, Woese C R, Overbeek R. J Bacteriol. 1994;176:1–6. doi: 10.1128/jb.176.1.1-6.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Delarue M, Poterszman A, Nikonov S, Garber M, Moras D, Thierry J C. EMBO J. 1994;13:3219–3229. doi: 10.1002/j.1460-2075.1994.tb06623.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Nureki O, Vassylyev D G, Katayanagi K, Shimizu T, Sekine S, Kigawa T, Miyazawa T, Yokoyama S, Morikawa K. Science. 1995;267:1958–1965. doi: 10.1126/science.7701318. [DOI] [PubMed] [Google Scholar]
  • 14.Berthet-Colominas C, Seignovert L, Hartlein M, Grotli M, Cusack S, Leberman R. EMBO J. 1998;17:2947–2960. doi: 10.1093/emboj/17.10.2947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Carroll J D, Daly M J, Minton K W. J Bacteriol. 1996;178:130–135. doi: 10.1128/jb.178.1.130-135.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Daly M J, Minton K W. J Bacteriol. 1996;178:4461–4471. doi: 10.1128/jb.178.15.4461-4471.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Raj H D, Duryee F L, Deeney A M, Wang C H, Anderson A W, Elliker P R. Can J Microbiol. 1960;6:289–298. doi: 10.1139/m60-033. [DOI] [PubMed] [Google Scholar]
  • 18.Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Plainview, NY: Cold Spring Harbor Lab. Press; 1989. [Google Scholar]
  • 19.Mattimore V, Udupa K S, Berne G A, Battista J R. J Bacteriol. 1995;177:5232–5237. doi: 10.1128/jb.177.18.5232-5237.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Altschul S F, Madden T L, Schaffer A A, Zhang J, Zhang Z, Miller W, Lipman D J. Nucleic Acids Res. 1997;25:3389–3402. doi: 10.1093/nar/25.17.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Deckert G, Warren P V, Gaasterland T, Young W G, Lenox A L, Graham D E, Overbeek R, Snead M A, Keller M, Aujay M, et al. Nature (London) 1998;392:353–358. doi: 10.1038/32831. [DOI] [PubMed] [Google Scholar]
  • 22.Siatecka M, Rozek M, Barciszewski J, Mirande M. Eur J Biochem. 1998;256:80–87. doi: 10.1046/j.1432-1327.1998.2560080.x. [DOI] [PubMed] [Google Scholar]
  • 23.Tempest D W, Meers J L, Brown C M. J Gen Microbiol. 1970;64:171–185. doi: 10.1099/00221287-64-2-171. [DOI] [PubMed] [Google Scholar]
  • 24.Fraser C M, Norris S J, Weinstock G M, White O, Sutton G G, Dodson R, Gwinn M, Hickey E K, Clayton R, Ketchum K A, et al. Science. 1998;281:375–388. doi: 10.1126/science.281.5375.375. [DOI] [PubMed] [Google Scholar]
  • 25.Ibba M, Bono J L, Rosa P A, Söll D. Proc Natl Acad Sci USA. 1997;94:14383–14388. doi: 10.1073/pnas.94.26.14383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Reitzer L J. In: Escherichia coli and Salmonella: Cellular and Molecular Biology. Neidhardt F C, Curtiss R, Ingraham J L, Lin E C C, Low K B, et al., editors. Washington, DC: Am. Soc. Microbiol.; 1996. pp. 391–407. [Google Scholar]
  • 27.Humbert R, Simoni R D. J Bacteriol. 1980;142:212–220. doi: 10.1128/jb.142.1.212-220.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Cedar H, Schwartz J H. J Bacteriol. 1968;96:2043–2048. doi: 10.1128/jb.96.6.2043-2048.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Hentzen D, Mandel P, Garel J P. Biochim Biophys Acta. 1972;281:228–232. doi: 10.1016/0005-2787(72)90174-8. [DOI] [PubMed] [Google Scholar]
  • 30.Calendar R, Berg P. J Mol Biol. 1967;26:39–54. doi: 10.1016/0022-2836(67)90259-8. [DOI] [PubMed] [Google Scholar]
  • 31.Ebel J P, Giege R, Bonnet J, Kern D, Befort N, Bollack C, Fasiolo F, Gangloff J, Dirheimer G. Biochimie. 1974;56:383–390. doi: 10.1016/s0300-9084(73)80415-8. [DOI] [PubMed] [Google Scholar]
  • 32.Hinchman S K, Henikoff S, Schuster S M. J Biol Chem. 1992;267:144–149. [PubMed] [Google Scholar]
  • 33.Nakatsu T, Kato H, Oda J. Nat Struct Biol. 1998;5:15–19. doi: 10.1038/nsb0198-15. [DOI] [PubMed] [Google Scholar]
  • 34.Eigen M, Winkler-Oswatitsch R. Naturwissenschaften. 1981;68:217–228. doi: 10.1007/BF01047323. [DOI] [PubMed] [Google Scholar]
  • 35.Weiner A M, Maizels N. Proc Natl Acad Sci USA. 1987;84:7383–7387. doi: 10.1073/pnas.84.21.7383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Cordell B, Swanstrom R, Goodman H M, Bishop J M. J Biol Chem. 1979;254:1866–1874. [PubMed] [Google Scholar]
  • 37.Di Giulio M. J Mol Evol. 1993;37:5–10. doi: 10.1007/BF00170456. [DOI] [PubMed] [Google Scholar]
  • 38.Jahn D, Verkamp E, Söll D. Trends Biochem Sci. 1992;17:215–218. doi: 10.1016/0968-0004(92)90380-r. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES