The Helicobacter pylori amidotransferase GatCAB is equally efficient in glutamine-dependent transamidation of Asp-tRNAAsn and Glu-tRNAGln

The amide aminoacyl-tRNAs, Gln-tRNA(Gln) and Asn-tRNA(Asn), are formed in many bacteria by a pretranslational tRNA-dependent amidation of the mischarged tRNA species, Glu-tRNA(Gln) or Asp-tRNA(Asn). This conversion is catalyzed by a heterotrimeric amidotransferase GatCAB in the presence of ATP and an amide donor (Gln or Asn). Helicobacter pylori has a single GatCAB enzyme required in vivo for both Gln-tRNA(Gln) and Asn-tRNA(Asn) synthesis. In vitro characterization reveals that the enzyme transamidates Asp-tRNA(Asn) and Glu-tRNA(Gln) with similar efficiency (k(cat)/K(m) of 1368.4 s(-1)/mM and 3059.3 s(-1)/mM respectively). The essential glutaminase activity of the enzyme is a property of the A-subunit, which displays the characteristic amidase signature sequence. Mutations of the GatA catalytic triad residues (Lys(52), Ser(128), Ser(152)) abolished glutaminase activity and consequently the amidotransferase activity with glutamine as the amide donor. However, the latter activity was rescued when the mutant enzymes were presented with ammonium chloride. The presence of Asp-tRNA(Asn) and ATP enhances the glutaminase activity about 22-fold. H. pylori GatCAB uses the amide donor glutamine 129-fold more efficiently than asparagine, suggesting that GatCAB is a glutamine-dependent amidotransferase much like the unrelated asparagine synthetase B. Genomic analysis suggests that most bacteria synthesize asparagine in a glutamine-dependent manner, either by a tRNA-dependent or in a tRNA-independent route. However, all known bacteria that contain asparagine synthetase A form Asn-tRNA(Asn) by direct acylation catalyzed by asparaginyl-tRNA synthetase. Therefore, bacterial amide aminoacyl-tRNA formation is intimately tied to amide amino acid metabolism.     

A single amidotransferase forms asparaginyl-tRNA and glutaminyl-tRNA in Chlamydia trachomatis

Aminoacyl-tRNA is generally formed by aminoacyl-tRNA synthetases, a family of 20 enzymes essential for accurate protein synthesis. However, most bacteria generate one of the two amide aminoacyl-tRNAs, Asn-tRNA or Gln-tRNA, by transamidation of mischarged Asp-tRNA(Asn) or Glu-tRNA(Gln) catalyzed by a heterotrimeric amidotransferase (encoded by the gatA, gatB, and gatC genes). The Chlamydia trachomatis genome sequence reveals genes for 18 synthetases, whereas those for asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase are absent. Yet the genome harbors three gat genes in an operon-like arrangement (gatCAB). We reasoned that Chlamydia uses the gatCAB-encoded amidotransferase to generate both Asn-tRNA and Gln-tRNA. C. trachomatis aspartyl-tRNA synthetase and glutamyl-tRNA synthetase were shown to be non-discriminating synthetases that form the misacylated tRNA(Asn) and tRNA(Gln) species. A preparation of pure heterotrimeric recombinant C. trachomatis amidotransferase converted Asp-tRNA(Asn) and Glu-tRNA(Gln) into Asn-tRNA and Gln-tRNA, respectively. The enzyme used glutamine, asparagine, or ammonia as amide donors in the presence of either ATP or GTP. These results suggest that C. trachomatis employs the dual specificity gatCAB-encoded amidotransferase and 18 aminoacyl-tRNA synthetases to create the complete set of 20 aminoacyl-tRNAs.     

The nondiscriminating aspartyl-tRNA synthetase from Helicobacter pylori: anticodon-binding domain mutations that impact tRNA specificity and heterologous toxicity

Divergent tRNA substrate recognition patterns distinguish the two distinct forms of aspartyl-tRNA synthetase (AspRS) that exist in different bacteria. In some cases, a canonical, discriminating AspRS (D-AspRS) specifically generates Asp-tRNA(Asp) and usually coexists with asparaginyl-tRNA synthetase (AsnRS). In other bacteria, particularly those that lack AsnRS, AspRS is nondiscriminating (ND-AspRS) and generates both Asp-tRNA(Asp) and the noncanonical, misacylated Asp-tRNA(Asn); this misacylated tRNA is subsequently repaired by the glutamine-dependent Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase (Asp/Glu-Adt). The molecular features that distinguish the closely related bacterial D-AspRS and ND-AspRS are not well-understood. Here, we report the first characterization of the ND-AspRS from the human pathogen Helicobacter pylori (H. pylori or Hp). This enzyme is toxic when heterologously overexpressed in Escherichia coli. This toxicity is rescued upon coexpression of the Hp Asp/Glu-Adt, indicating that Hp Asp/Glu-Adt can utilize E. coli Asp-tRNA(Asn) as a substrate. Finally, mutations in the anticodon-binding domain of Hp ND-AspRS reduce this enzyme's ability to misacylate tRNA(Asn), in a manner that correlates with the toxicity of the enzyme in E. coli.     

Co-evolution of the archaeal tRNA-dependent amidotransferase GatCAB with tRNA(Asn)

The important identity elements in tRNA(Gln) and tRNA(Asn) for bacterial GatCAB and in tRNA(Gln) for archaeal GatDE are the D-loop and the first base pair of the acceptor stem. Here we show that Methanothermobacter thermautotrophicus GatCAB, the archaeal enzyme, is different as it discriminates Asp-tRNA(Asp) and Asp-tRNA(Asn) by use of U49, the D-loop and to a lesser extent the variable loop. Since archaea possess the tRNA(Gln)-specific amidotransferase GatDE, the archaeal GatCAB enzyme evolved to recognize different elements in tRNA(Asn) than those recognized by GatDE or by the bacterial GatCAB enzyme in their tRNA substrates.     

Assays for transfer RNA-dependent amino acid biosynthesis

Selenocysteinyl-tRNA(Sec), cysteinyl-tRNA(Cys), glutaminyl-tRNA(Gln), and asparaginyl-tRNA(Asn) in many organisms are formed in an indirect pathway in which a non-cognate amino acid is first attached to the tRNA. This non-cognate amino acid is then converted to the cognate amino acid by a tRNA-dependent modifying enzyme. The in vitro characterization of these modifying enzymes is challenging due to the fact the substrate, aminoacyl-tRNA, is labile and requires a prior enzymatic step to be synthesized. The need to separate product aa-tRNA from unreacted substrate is typically a labor- and time-intensive task; this adds another impediment in the investigation of these enzymes. Here, we review four different approaches for studying these tRNA-dependent amino acid modifications. In addition, we describe in detail a [32P]/nuclease P1 assay for glutaminyl-tRNA(Gln) and asparaginyl-tRNA(Asn) formation which is sensitive, enables monitoring of the aminoacyl state of the tRNA, and is less time consuming than some of the other techniques. This [32P]/nuclease P1 method should be adaptable to studying tRNA-dependent selenocysteine and cysteine synthesis.     

The asparagine-transamidosome from Helicobacter pylori: a dual-kinetic mode in non-discriminating aspartyl-tRNA synthetase safeguards the genetic code

Helicobacter pylori catalyzes Asn-tRNA(Asn) formation by use of the indirect pathway that involves charging of Asp onto tRNA(Asn) by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS), followed by conversion of the mischarged Asp into Asn by the GatCAB amidotransferase. We show that the partners of asparaginylation assemble into a dynamic Asn-transamidosome, which uses a different strategy than the Gln-transamidosome to prevent the release of the mischarged aminoacyl-tRNA intermediate. The complex is described by gel-filtration, dynamic light scattering and kinetic measurements. Two strategies for asparaginylation are shown: (i) tRNA(Asn) binds GatCAB first, allowing aminoacylation and immediate transamidation once ND-AspRS joins the complex; (ii) tRNA(Asn) is bound by ND-AspRS which releases the Asp-tRNA(Asn) product much slower than the cognate Asp-tRNA(Asp); this kinetic peculiarity allows GatCAB to bind and transamidate Asp-tRNA(Asn) before its release by the ND-AspRS. These results are discussed in the context of the interrelation between the Asn and Gln-transamidosomes which use the same GatCAB in H. pylori, and shed light on a kinetic mechanism that ensures faithful codon reassignment for Asn.     

Rational design and directed evolution of a bacterial-type glutaminyl-tRNA synthetase precursor

Protein biosynthesis requires aminoacyl-transfer RNA (tRNA) synthetases to provide aminoacyl-tRNA substrates for the ribosome. Most bacteria and all archaea lack a glutaminyl-tRNA synthetase (GlnRS); instead, Gln-tRNA(Gln) is produced via an indirect pathway: a glutamyl-tRNA synthetase (GluRS) first attaches glutamate (Glu) to tRNA(Gln), and an amidotransferase converts Glu-tRNA(Gln) to Gln-tRNA(Gln). The human pathogen Helicobacter pylori encodes two GluRS enzymes, with GluRS2 specifically aminoacylating Glu onto tRNA(Gln). It was proposed that GluRS2 is evolving into a bacterial-type GlnRS. Herein, we have combined rational design and directed evolution approaches to test this hypothesis. We show that, in contrast to wild-type (WT) GlnRS2, an engineered enzyme variant (M110) with seven amino acid changes is able to rescue growth of the temperature-sensitive Escherichia coli glnS strain UT172 at its non-permissive temperature. In vitro kinetic analyses reveal that WT GluRS2 selectively acylates Glu over Gln, whereas M110 acylates Gln 4-fold more efficiently than Glu. In addition, M110 hydrolyzes adenosine triphosphate 2.5-fold faster in the presence of Glu than Gln, suggesting that an editing activity has evolved in this variant to discriminate against Glu. These data imply that GluRS2 is a few steps away from evolving into a GlnRS and provides a paradigm for studying aminoacyl-tRNA synthetase evolution using directed engineering approaches.     

Characterization of the glutamyl-tRNA(Gln)-to-glutaminyl-tRNA(Gln) amidotransferase reaction of Bacillus subtilis

In Bacillus subtilis, the formation of glutaminyl-tRNA is accomplished by first charging tRNA(Gln) with glutamate, which is then amidated. Glutamine was preferred over asparagine and ammonia as the amide donor in vitro. There is a functional analogy of this reaction to that catalyzed by glutamine synthetase. Homogeneous glutamine synthetase, from either B. subtilis or Escherichia coli, catalyzed the amidotransferase reaction but only about 3 to 5% as well as a partially purified preparation from B. subtilis. Several classes of glutamine synthetase mutants of B. subtilis, however, were unaltered in the amidotransferase reaction. In addition, there was no inhibition by inhibitors of either glutamine synthetase or other amidotransferases. A unique, rather labile activity seems to be required for this reaction.     

A dual-specific Glu-tRNA(Gln) and Asp-tRNA(Asn) amidotransferase is involved in decoding glutamine and asparagine codons in Acidithiobacillus ferrooxidans.

The gatC, gatA and gatB genes encoding the three subunits of glutamyl-tRNA^Gln amidotransferase from Acidithiobacillus ferrooxidans, an acidophilic bacterium used in bioleaching of minerals, have been cloned and expressed in Escherichia coli. As in Bacillus subtilis the three gat genes are organized in an operon-like structure in A. ferrooxidans. The heterologously overexpressed enzyme converts Glu-tRNA^Gln to Gln-tRNA^Gln and Asp-tRNA^Asn to Asn-tRNA^Asn. Biochemical analysis revealed that neither glutaminyl-tRNA synthetase nor asparaginyl-tRNA synthetase is present in A. ferrooxidans, but that glutamyl-tRNA synthetase and aspartyl-tRNA synthetase enzymes are present in the organism. These data suggest that the transamidation pathway is responsible for the formation of Gln-tRNA and Asn-tRNA in A. ferrooxidans.     

Conserved discrimination against misacylated tRNAs by two mesophilic elongation factor Tu orthologs

Elongation factor Tu (EF-Tu) binds and loads elongating aminoacyl-tRNAs (aa-tRNAs) onto the ribosome for protein biosynthesis. Many bacteria biosynthesize Gln-tRNA (Gln) and Asn-tRNA (Asn) by an indirect, two-step pathway that relies on the misacylated tRNAs Glu-tRNA (Gln) and Asp-tRNA (Asn) as intermediates. Previous thermodynamic and experimental analyses have demonstrated that Thermus thermophilus EF-Tu does not bind Asp-tRNA (Asn) and predicted a similar discriminatory response against Glu-tRNA (Gln) [Asahara, H., and Uhlenbeck, O. (2005) Biochemistry 46, 6194-6200; Roy, H., et al. (2007) Nucleic Acids Res. 35, 3420-3430]. By discriminating against these misacylated tRNAS, EF-Tu plays a direct role in preventing misincorporation of aspartate and glutamate into proteins at asparagine and glutamine codons. Here we report the characterization of two different mesophilic EF-Tu orthologs, one from Escherichia coli, a bacterium that does not utilize either Glu-tRNA (Gln) or Asp-tRNA (Asn), and the second from Helicobacter pylori, an organism in which both misacylated tRNAs are essential. Both EF-Tu orthologs discriminate against these misacylated tRNAs, confirming the prediction that Glu-tRNA (Gln), like Asp-tRNA (Asn), will not form a complex with EF-Tu. These results also demonstrate that the capacity of EF-Tu to discriminate against both of these aminoacyl-tRNAs is conserved even in bacteria like E. coli that do not generate either misacylated tRNA.     

Mechanism of a GatCAB amidotransferase: aspartyl-tRNA synthetase increases its affinity for Asp-tRNA(Asn) and novel aminoacyl-tRNA analogues are competitive inhibitors

The trimeric GatCAB aminoacyl-tRNA amidotransferases catalyze the amidation of Asp-tRNAAsn and/or Glu-tRNAGln to Asn-tRNAAsn and/or Gln-tRNAGln, respectively, in bacteria and archaea lacking an asparaginyl-tRNA synthetase and/or a glutaminyl-tRNA synthetase. The two misacylated tRNA substrates of these amidotransferases are formed by the action of nondiscriminating aspartyl-tRNA synthetases and glutamyl-tRNA synthetases. We report here that the presence of a physiological concentration of a nondiscriminating aspartyl-tRNA synthetase in the transamidation assay decreases the Km of GatCAB for Asp-tRNAAsn. These conditions, which were practical for the testing of potential inhibitors of GatCAB, also allowed us to discover and characterize two novel inhibitors, aspartycin and glutamycin. These analogues of the 3'-ends of Asp-tRNA and Glu-tRNA, respectively, are competitive inhibitors of the transamidase activity of Helicobacter pylori GatCAB with respect to Asp-tRNAAsn, with Ki values of 134 microM and 105 microM, respectively. Although the 3' end of aspartycin is similar to the 3' end of Asp-tRNAAsn, this analogue was neither phosphorylated nor transamidated by GatCAB. These novel inhibitors could be used as lead compounds for designing new types of antibiotics targeting GatCABs, since the indirect pathway for Asn-tRNAAsn or Gln-tRNAGln synthesis catalyzed by these enzymes is not present in eukaryotes and is essential for the survival of the above-mentioned bacteria.     

The archaeal transamidosome for RNA-dependent glutamine biosynthesis

Archaea make glutaminyl-tRNA (Gln-tRNA^Gln) in a two-step process; a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) forms Glu-tRNA^Gln, while the heterodimeric amidotransferase GatDE converts this mischarged tRNA to Gln-tRNA^Gln. Many prokaryotes synthesize asparaginyl-tRNA (Asn-tRNA^Asn) in a similar manner using a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) and the heterotrimeric amidotransferase GatCAB. The transamidosome, a complex of tRNA synthetase, amidotransferase and tRNA, was first described for the latter system in Thermus thermophilus [Bailly, M., Blaise, M., Lorber, B., Becker, H.D. and Kern, D. (2007) The transamidosome: a dynamic ribonucleoprotein particle dedicated to prokaryotic tRNA-dependent asparagine biosynthesis. Mol. Cell, 28, 228–239.]. Here, we show a similar complex for Gln-tRNA^Gln formation in Methanothermobacter thermautotrophicus that allows the mischarged Glu-tRNA^Gln made by the tRNA synthetase to be channeled to the amidotransferase. The association of archaeal ND-GluRS with GatDE (K_D = 100 ± 22 nM) sequesters the tRNA synthetase for Gln-tRNA^Gln formation, with GatDE reducing the affinity of ND-GluRS for tRNA^Glu by at least 13-fold. Unlike the T. thermophilus transamidosome, the archaeal complex does not require tRNA for its formation, is not stable through product (Gln-tRNA^Gln) formation, and has no major effect on the kinetics of tRNA^Gln glutamylation nor transamidation. The differences between the two transamidosomes may be a consequence of the fact that ND-GluRS is a class I aminoacyl-tRNA synthetase, while ND-AspRS belongs to the class II family.     

Mutation analysis of carbamoyl phosphate synthetase: Does the structurally conserved glutamine amidotransferase triad act as a functional dyad?

Evolutionarily conserved triad glutamine amidotransferase (GAT) domains catalyze the cleavage of glutamine to yield ammonia and sequester the ammonia in a tunnel until delivery to a variety of acceptor substrates in synthetase domains of variable structure. Whereas a conserved hydrolytic triad (Cys/His/Glu) is observed in the solved GAT structures, the specificity pocket for glutamine is not apparent, presumably because its formation is dependent on the conformational change that couples acceptor availability to a greatly increased rate of glutamine cleavage. In Escherichia coli carbamoyl phosphate synthetase (eCPS), one of the best characterized triad GAT members, the Cys269 and His353 triad residues are essential for glutamine hydrolysis, whereas Glu355 is not critical for eCPS activity. To further define the glutamine-binding pocket and possibly identify an alternative member of the catalytic triad that is situated for this role in the coupled conformation, we have analyzed mutations at Gln310, Asn311, Asp334, and Gln351, four conserved, but not yet analyzed residues that might potentially function as the third triad member. Alanine substitution of Gln351, Asn311, and Gln310 yielded respective K(m) increases of 145, 27, and 15, suggesting that Gln351 plays a key role in glutamine binding in the coupled conformation, and that Asn311 and Gln310 make less significant contributions. None of the mutant k (cat) values varied significantly from those for wild-type eCPS. Combined with previously reported data on other conserved eCPS residues, these results strongly suggest that Cys269 and His353 function as a catalytic dyad in the GAT site of eCPS.     

Protein synthesis in Escherichia coli with mischarged tRNA

Two types of aspartyl-tRNA synthetase exist: the discriminating enzyme (D-AspRS) forms only Asp-tRNA(Asp), while the nondiscriminating one (ND-AspRS) also synthesizes Asp-tRNA(Asn), a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). On the basis of the E. coli trpA34 missense mutant transformed with heterologous ND-aspS genes, we developed a system with which to measure the in vivo formation of Asp-tRNA(Asn) and its acceptance by elongation factor EF-Tu. While large amounts of Asp-tRNA(Asn) are detrimental to E. coli, smaller amounts support protein synthesis and allow the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase.     

On the evolution of the tRNA-dependent amidotransferases, GatCAB and GatDE

Glutaminyl-tRNA synthetase and asparaginyl-tRNA synthetase evolved from glutamyl-tRNA synthetase and aspartyl-tRNA synthetase, respectively, after the split in the last universal communal ancestor (LUCA). Glutaminyl-tRNA^Gln and asparaginyl-tRNA^Asn were likely formed in LUCA by amidation of the mischarged species, glutamyl-tRNA^Gln and aspartyl-tRNA^Asn, by tRNA-dependent amidotransferases, as is still the case in most bacteria and all known archaea. The amidotransferase GatCAB is found in both domains of life, while the heterodimeric amidotransferase GatDE is found only in Archaea. The GatB and GatE subunits belong to a unique protein family that includes Pet112 that is encoded in the nuclear genomes of numerous eukaryotes. GatE was thought to have evolved from GatB after the emergence of the modern lines of decent. Our phylogenetic analysis though places the split between GatE and GatB, prior to the phylogenetic divide between Bacteria and Archaea, and Pet112 to be of mitochondrial origin. In addition, GatD appears to have emerged prior to the bacterial-archaeal phylogenetic divide. Thus, while GatDE is an archaeal signature protein, it likely was present in LUCA together with GatCAB. Archaea retained both amidotransferases, while Bacteria emerged with only GatCAB. The presence of GatDE has favored a unique archaeal tRNA^Gln that may be preventing the acquisition of glutaminyl-tRNA synthetase in Archaea. Archaeal GatCAB, on the other hand, has not favored a distinct tRNA^Asn, suggesting that tRNA^Asn recognition is not a major barrier to the retention of asparaginyl-tRNA synthetase in many Archaea.     

Aspartyl-tRNA synthetase requires a conserved proline in the anticodon-binding loop for tRNA(Asn) recognition in vivo

Most prokaryotes require Asp-tRNA(Asn) for the synthesis of Asn-tRNA(Asn). This misacylated tRNA species is synthesized by a non-discriminating aspartyl-tRNA synthetase (AspRS) that acylates both tRNA(Asp) and tRNA(Asn) with aspartate. In contrast, a discriminating AspRS forms only Asp-tRNA(Asp). Here we show that a conserved proline (position 77) in the L1 loop of the non-discriminating Deinococcus radiodurans AspRS2 is required for tRNA(Asn) recognition in vivo. Escherichia coli trpA34 was transformed with DNA from a library of D. radiodurans aspS2 genes with a randomized codon 77 and then subjected to in vivo selection for Asp-tRNA(Asn) formation by growth in minimal medium. Only proline codons were found at position 77 in the aspS2 genes isolated from 21 of the resulting viable colonies. However, when the aspS temperature-sensitive E. coli strain CS89 was transformed with the same DNA library and then screened for Asp-tRNA(Asp) formation in vivo by growth at the non-permissive temperature, codons for seven other amino acids besides proline were identified at position 77 in the isolates examined. Thus, replacement of proline 77 by cysteine, isoleucine, leucine, lysine, phenylalanine, serine, or valine resulted in mutant D. radiodurans AspRS2 enzymes still capable of forming Asp-tRNA(Asp) but unable to recognize tRNA(Asn). This strongly suggests that proline 77 is responsible for the non-discriminatory tRNA recognition properties of this enzyme.     

Purification and functional characterization of the Glu-tRNA(Gln) amidotransferase from Chlamydomonas reinhardtii

The formation of glutaminyl-tRNA (Gln-tRNA) in Bacilli, chloroplasts, and mitochondria occurs in a two-step reaction. This involves misacylation of tRNA(Gln) with glutamate by glutamyl-tRNA synthetase and subsequent amidation of Glu-tRNA(Gln) to the correctly acylated Gln-tRNA(Gln) by a specific amidotransferase (Sch?n, A., Kannangara, C. G., Gough, S., and S?ll, D. (1988) Nature 331, 187-190). Here we demonstrate the existence of this pathway in green algae and describe the purification of the Glu-tRNA(Gln) amidotransferase from Chlamydomonas reinhardtii. The purified enzyme showed an Mr of approximately 120,000 when analyzed by glycerol gradient sedimentation and gel filtration. An apparent Mr of 63,000 of the denatured protein was demonstrated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. This indicates that the enzyme possesses an alpha 2 structure. The substrate for the purified enzyme is Glu-tRNA(Gln) but not Glu-tRNA(Glu). The enzyme requires ATP, Mg2+, and an amide donor for the conversion. Acceptable amide donors are glutamine, asparagine, and ammonia. Blocking of the glutamine-dependent reaction by alkylation of the protein with 6-diazo-5-oxonorleucine did not inhibit the ammonia-dependent reaction, suggesting that the enzyme has separate glutamine and ammonia binding sites. As suggested by Wilcox (Wilcox, M. (1969) Eur. J. Biochem. 11, 405-412) the amidation reaction may involve glutamyl-phosphate formation, since ATP is cleaved to ADP when the enzyme is incubated with Glu-tRNA(Gln) and ATP. In common with other glutamine amidotransferases, the enzyme also possesses low glutaminase activity. The purified Glu-tRNA(Gln) amidotransferase forms a stable complex with Glu-tRNA(Gln) in the presence of ATP and Mg2+ but in the absence of the amide donor as determined by gradient centrifugation.