Kelly Sheppard

From one amino acid to another: tRNA-dependent amino acid biosynthesis

Aminoacyl-tRNAs (aa-tRNAs) are the essential substrates for translation. Most aa-tRNAs are formed by direct aminoacylation of tRNA catalyzed by aminoacyl-tRNA synthetases. However, a smaller number of aa-tRNAs (Asn-tRNA, Gln-tRNA, Cys-tRNA and Sec-tRNA) are made by synthesizing the amino acid on the tRNA by first attaching a non-cognate amino acid to the tRNA, which is then converted to the cognate one catalyzed by tRNA-dependent modifying enzymes. Asn-tRNA or Gln-tRNA formation in most prokaryotes requires amidation of Asp-tRNA or Glu-tRNA by amidotransferases that couple an amidase or an asparaginase to liberate ammonia with a tRNA-dependent kinase. Both archaeal and eukaryotic Sec-tRNA biosynthesis and Cys-tRNA synthesis in methanogens require O-phosophoseryl-tRNA formation. For tRNA-dependent Cys biosynthesis, O-phosphoseryl-tRNA synthetase directly attaches the amino acid to the tRNA which is then converted to Cys by Sep-tRNA: Cys-tRNA synthase. In Sec-tRNA synthesis, O-phosphoseryl-tRNA kinase phosphorylates Ser-tRNA to form the intermediate which is then modified to Sec-tRNA by Sep-tRNA:Sec-tRNA synthase. Complex formation between enzymes in the same pathway may protect the fidelity of protein synthesis. How these tRNA-dependent amino acid biosynthetic routes are integrated into overall metabolism may explain why they are still retained in so many organisms.

Structural Basis of RNA-Dependent Recruitment of Glutamine to the Genetic Code

Glutaminyl–transfer RNA (Gln-tRNAGln) in archaea is synthesized in a pretranslational amidation of misacylated Glu-tRNAGln by the heterodimeric Glu-tRNAGln amidotransferase GatDE. Here we report the crystal structure of the Methanothermobacter thermautotrophicus GatDE complexed to tRNAGln at 3.15 angstroms resolution. Biochemical analysis of GatDE and of tRNAGln mutants characterized the catalytic centers for the enzymes three reactions (glutaminase, kinase, and amidotransferase activity). A 40 angstrom–long channel for ammonia transport connects the active sites in GatD and GatE. tRNAGln recognition by indirect readout based on shape complementarity of the D loop suggests an early anticodon-independent RNA-based mechanism for adding glutamine to the genetic code.

Archaeal 3′-phosphate RNA splicing ligase characterization identifies the missing component in tRNA maturation

Intron removal from tRNA precursors involves cleavage by a tRNA splicing endonuclease to yield tRNA 3′-halves beginning with a 5′-hydroxyl, and 5′-halves ending in a 2′,3′-cyclic phosphate. A tRNA ligase then incorporates this phosphate into the internucleotide bond that joins the two halves. Although this 3′-P RNA splicing ligase activity was detected almost three decades ago in extracts from animal and later archaeal cells, the protein responsible was not yet identified. Here we report the purification of this ligase from Methanopyrus kandleri cells, and its assignment to the still uncharacterized RtcB protein family. Studies with recombinant Pyrobaculum aerophilum RtcB showed that the enzyme is able to join spliced tRNA halves to mature-sized tRNAs where the joining phosphodiester linkage contains the phosphate originally present in the 2′,3′-cyclic phosphate. The data confirm RtcB as the archaeal RNA 3′-P ligase. Structural genomics efforts previously yielded a crystal structure of the Pyrococcus horikoshii RtcB protein containing a new protein fold and a conserved putative Zn2+ binding cleft. This structure guided our mutational analysis of the P. aerophilum enzyme. Mutations of highly conserved residues in the cleft (C100A, H205A, H236A) rendered the enzyme inactive suggesting these residues to be part of the active site of the P. aerophilum ligase. There is no significant sequence similarity between the active sites of P. aerophilum ligase and that of T4 RNA ligase, nor ligases from plants and fungi. RtcB sequence conservation in archaea and in eukaryotes implicates eukaryotic RtcB as the long-sought animal 3′-P RNA ligase.

The Helicobacter pylori Amidotransferase GatCAB Is Equally Efficient in Glutamine-dependent Transamidation of Asp-tRNAAsn and Glu-tRNAGln

The amide aminoacyl-tRNAs, Gln-tRNAGln and Asn-tRNAAsn, are formed in many bacteria by a pretranslational tRNA-dependent amidation of the mischarged tRNA species, Glu-tRNAGln or Asp-tRNAAsn. 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-tRNAGln and Asn-tRNAAsn synthesis. In vitro characterization reveals that the enzyme transamidates Asp-tRNAAsn and Glu-tRNAGln with similar efficiency (kcat/Km 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 (Lys52, Ser128, Ser152) 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-tRNAAsn 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-tRNAAsn by direct acylation catalyzed by asparaginyl-tRNA synthetase. Therefore, bacterial amide aminoacyl-tRNA formation is intimately tied to amide amino acid metabolism.

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.

Insights into tRNA-Dependent Amidotransferase Evolution and Catalysis from the Structure of the Aquifex aeolicus Enzyme

Many bacteria form Gln-tRNA(Gln) and Asn-tRNA(Asn) by conversion of the misacylated Glu-tRNA(Gln) and Asp-tRNA(Asn) species catalyzed by the GatCAB amidotransferase in the presence of ATP and an amide donor (glutamine or asparagine). Here, we report the crystal structures of GatCAB from the hyperthermophilic bacterium Aquifex aeolicus, complexed with glutamine, asparagine, aspartate, ADP, or ATP. In contrast to the Staphylococcus aureus GatCAB, the A. aeolicus enzyme formed acyl-enzyme intermediates with either glutamine or asparagine, in line with the equally facile use by the amidotransferase of these amino acids as amide donors in the transamidation reaction. A water-filled ammonia channel is open throughout the length of the A. aeolicus GatCAB from the GatA active site to the synthetase catalytic pocket in the B-subunit. A non-catalytic Zn(2+) site in the A. aeolicus GatB stabilizes subunit contacts and the ammonia channel. Judged from sequence conservation in the known GatCAB sequences, the Zn(2+) binding motif was likely present in the primordial GatB/E, but became lost in certain lineages (e.g., S. aureus GatB). Two divalent metal binding sites, one permanent and the other transient, are present in the catalytic pocket of the A. aeolicus GatB. The two sites enable GatCAB to first phosphorylate the misacylated tRNA substrate and then amidate the activated intermediate to form the cognate products, Gln-tRNA(Gln) or Asn-tRNA(Asn).

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.

Branchiostoma floridae has separate healing and sealing enzymes for 5′-phosphate RNA ligation

Animal cells have two tRNA splicing pathways: (i) a 5′-P ligation mechanism, where the 5′-phosphate of the 3′ tRNA half becomes the junction phosphate of the new phosphodiester linkage, and (ii) a 3′-P ligation process, in which the 3′-phosphate of the 5′ tRNA half turns into the junction phosphate. Although both activities are known to exist in animals, in almost three decades of investigation, neither of the two RNA ligases has been identified. Here we describe a gene from the chordate Branchiostoma floridae that encodes an RNA ligase (Bf RNL) with a strict requirement for RNA substrates with a 2′-phosphate terminus for the ligation of RNAs with 5′-phosphate and 3′-hydroxyl ends. Unlike the yeast and plant tRNA ligases involved in tRNA splicing, Bf RNL lacks healing activities and requires the action of a polynucleotide kinase (PNK) and a cyclic phosphodiesterase (CDPase) in trans. The activities of these two enzymes were identified in a single B. floridae protein (Bf PNK/CPDase). The combined activities of Bf RNL and Bf PNK/CPDase are sufficient for the joining of tRNA splicing intermediates in vitro, and for the functional complementation of a tRNA ligase-deficient Saccharomyces cerevisiae strain in vivo. Hence, these two proteins constitute the 5′-P RNA ligation pathway in an animal organism.

Rational design of an evolutionary precursor of glutaminyl-tRNA synthetase

The specificity of most aminoacyl-tRNA synthetases for an amino acid and cognate tRNA pair evolved before the divergence of the three domains of life. Glutaminyl-tRNA synthetase (GlnRS) evolved later and is derived from the archaeal-type nondiscriminating glutamyl-tRNA synthetase (GluRS), an enzyme with relaxed tRNA specificity capable of forming both Glu-tRNAGlu and Glu-tRNAGln. The archaea lack GlnRS and use a specialized amidotransferase to convert Glu-tRNAGln to Gln-tRNAGln needed for protein synthesis. We show that the Methanothermobacter thermautotrophicus GluRS is active toward tRNAGlu and the two tRNAGln isoacceptors the organism encodes, but with a significant catalytic preference for . The less active responds to the less common CAA codon for Gln. From a biochemical characterization of M. thermautotrophicus GluRS variants, we found that the evolution of tRNA specificity in GlnRS could be recapitulated by converting the M. thermautotrophicus GluRS to a tRNAGln specific enzyme, solely through the addition of an acceptor stem loop present in bacterial GlnRS. One designed GluRS variant is also highly specific for the isoacceptor, which responds to the CAG codon, and shows no activity toward . Because it is now possible to eliminate particular codons from the genome of Escherichia coli, additional codons will become available for genetic code engineering. Isoacceptor-specific aminoacyl-tRNA synthetases will enable the reassignment of more open codons while preserving accurate encoding of the 20 canonical amino acids.

Aminoacyl-tRNA synthesis by pre-translational amino acid modification.

Aminoacyl-tRNAs (aa-tRNAs) are essential substrates for ribosomal translation, and aregenerally synthesized by aminoacyl-tRNA synthetases (aaRSs). It was expected earlier thatevery organism would contain a complete set of twenty aaRSs, one for each canonical aminoacid. However, analysis of the many known genome sequences and biochemical studiesrevealed that most organisms lack asparaginyl- and glutaminyl-tRNA synthetases, and thus areunable to attach asparagine and glutamine directly onto their corresponding tRNA. Instead, apre-translational amino acid modification is required to convert Asp-tRNAAsn and Glu-tRNAGlnto the correctly charged Asn-tRNAAsn and Gln-tRNAGln, respectively. This transamidationpathway of amide aa-tRNA synthesis is common in most bacteria and archaea. Unexpectedresults from biochemical, genetic and genomic studies showed that a large variety of differentbacteria rely on tRNA-dependent transamidation for the formation of the amino acidasparagine. Pre-translational modifications are not restricted to asparagine and glutamine butare also found in the biosynthesis of some other aa-tRNAs, such as the initiator tRNA fmettRNAMetiand Sec-tRNASec specifying selenocysteine, the 21st co-translationally inserted aminoacid. tRNA-dependent amino acid modification is also involved in the generation ofaminolevulinic acid, the first precursor for porphyrin biosynthesis in many organisms.