Crystal structure of a non-discriminating glutamyl-tRNA synthetase

Error-free protein biosynthesis is dependent on the reliable charging of each tRNA with its cognate amino acid. Many bacteria, however, lack a glutaminyl-tRNA synthetase. In these organisms, tRNA(Gln) is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase (ND-GluRS). This enzyme thus charges both tRNA(Glu) and tRNA(Gln) with glutamate. Discriminating GluRS (D-GluRS), found in some bacteria and all eukaryotes, exclusively generates Glu-tRNA(Glu). Here we present the first crystal structure of a non-discriminating GluRS from Thermosynechococcus elongatus (ND-GluRS(Tel)) in complex with glutamate at a resolution of 2.45 A. Structurally, the enzyme shares the overall architecture of the discriminating GluRS from Thermus thermophilus (D-GluRS(Tth)). We confirm experimentally that GluRS(Tel) is non-discriminating and present kinetic parameters for synthesis of Glu-tRNA(Glu) and of Glu-tRNA(Gln). Anticodons of tRNA(Glu) (34C/UUC36) and tRNA(Gln) (34C/UUG36) differ only in base 36. The pyrimidine base of C36 is specifically recognized in D-GluRS(Tth) by the residue Arg358. In ND-GluRS(Tel) this arginine residue is replaced by glycine (Gly366) presumably allowing both cytosine and the bulkier purine base G36 of tRNA(Gln) to be tolerated. Most other ND-GluRS share this structural feature, leading to relaxed substrate specificity.     

Synthesis of an inhibitor of glutamyl-tRNA synthetase

A specific inhibitor of glutamyl-tRNA synthetase, analog of glutamyl-adenylate, the N⁶-benzoyl-L-glutamol AMP, has been synthesized. This compound does not inhibit glutaminyl-tRNA synthetase.     

Bifunctional isocitrate-homoisocitrate dehydrogenase: a missing link in the evolution of beta-decarboxylating dehydrogenase

Beta-decarboxylating dehydrogenases comprise 3-isopropylmalate dehydrogenase, isocitrate dehydrogenase, and homoisocitrate dehydrogenase. They share a high degree of amino acid sequence identity and occupy equivalent positions in the amino acid biosynthetic pathways for leucine, glutamate, and lysine, respectively. Therefore, not only the enzymes but also the whole pathways should have evolved from a common ancestral pathway. In Pyrococcus horikoshii, only one pathway of the three has been identified in the genomic sequence, and PH1722 is the sole beta-decarboxylating dehydrogenase gene. The organism does not require leucine, glutamate, or lysine for growth; the single pathway might play multiple (i.e., ancestral) roles in amino acid biosynthesis. The PH1722 gene was cloned and expressed in Escherichia coli and the substrate specificity of the recombinant enzyme was investigated. It exhibited activities on isocitrate and homoisocitrate at near equal efficiency, but not on 3-isopropylmalate. PH1722 is thus a novel, bifunctional beta-decarboxylating dehydrogenase, which likely plays a dual role in glutamate and lysine biosynthesis in vivo.     

Transitional fossil earwigs - a missing link in Dermaptera evolution

Background  

The Dermaptera belongs to a group of winged insects of uncertain relationship within Polyneoptera, which has expanded anal region and adds numerous anal veins in the hind wing. Evolutional history and origin of Dermaptera have been in contention.     

Complex formation between glutamyl-tRNA synthetase and glutamyl-tRNA reductase during the tRNA-dependent synthesis of 5-aminolevulinic acid in Chlamydomonas reinhardtii.

The formation of a stable complex between glutamyl-tRNA synthetase and the first enzyme of chlorophyll biosynthesis glutamyl-tRNA reductase was investigated in the green alga Chlamydomonas reinhardtii. Apparently homogenous enzymes, purified after previously established purification protocols were incubated in various combinations with ATP, glutamate, tRNA(Glu) and NADPH and formed complexes were isolated via glycerol gradient centrifugation. Stable complexes were detected only after the preincubation of glutamyl-tRNA synthetase, glutamyl-tRNA reductase with either glutamyl-tRNA or free tRNA(Glu), ATP and glutamate, indicating the obligatory requirement of aminoacylated tRNA(Glu) for complex formation. The further addition of NADPH resulting in the reduction of the tRNA-bound glutamate to glutamate 1-semialdehyde led to the dissociation of the complex. Once complexed to the two enzymes tRNA(Glu) was found to be partially protected from ribonuclease digestion. Escherichia coli, Bacillus subtilis and Synechocystis 6803 tRNA(Glu) were efficiently incorporated into the protein-RNA complex. The detected complexes provide the chloroplast with a potential channeling mechanism for Glu-tRNA(Glu) into chlorophyll synthesis in order to compete with the chloroplastic protein synthesis machinery.     

Gln-tRNAGln formation from Glu-tRNAGln requires cooperation of an asparaginase and a Glu-tRNAGln kinase

Gln-tRNA(Gln) is synthesized from Glu-tRNA(Gln) in most microorganisms by a tRNA-dependent amidotransferase in a reaction requiring ATP and an amide donor such as glutamine. GatDE is a heterodimeric amidotransferase that is ubiquitous in Archaea. GatD resembles bacterial asparaginases and is expected to function in amide donor hydrolysis. We show here that Methanothermobacter thermautotrophicus GatD acts as a glutaminase but only in the presence of both Glu-tRNA(Gln) and the other subunit, GatE. The fact that only Glu-tRNA(Gln) but not tRNA(Gln) could activate the glutaminase activity of GatD suggests that glutamine hydrolysis is coupled tightly to transamidation. M. thermautotrophicus GatDE enzymes that were mutated in GatD at each of the four critical asparaginase-active site residues lost the ability to hydrolyze glutamine and were unable to convert Glu-tRNA(Gln) to Gln-tRNA(Gln) when glutamine was the amide donor. However, ammonium chloride rescued the activities of these mutants, suggesting that the integrity of the ATPase and the transferase activities in the mutant GatDE enzymes was maintained. In addition, pyroglutamyl-tRNA(Gln) accumulated during the reaction catalyzed by the glutaminase-deficient mutants or by GatE alone. The pyroglutamyl-tRNA is most likely a cyclized by-product derived from gamma-phosphoryl-Glu-tRNA(Gln), the proposed high energy intermediate in Glu-tRNA(Gln) transamidation. That GatE alone could form the intermediate indicates that GatE is a Glu-tRNA(Gln) kinase. The activation of Glu-tRNA(Gln) via gamma-phosphorylation bears a similarity to the mechanism used by glutamine synthetase, which may point to an ancient link between glutamine synthesized for metabolism and translation.     

Identification and functional verification of archaeal-type phosphoenolpyruvate carboxylase, a missing link in archaeal central carbohydrate metabolism

Despite the fact that phosphoenolpyruvate carboxylase (PEPC) activity has been measured and in some cases even purified from some Archaea, the gene responsible for this activity has not been elucidated. Using sensitive sequence comparison methods, we detected a highly conserved, uncharacterized archaeal gene family that is distantly related to the catalytic core of the canonical PEPC. To verify the predicted function of this archaeal gene family, we cloned a representative from the hyperthermophilic acidophile Sulfolobus solfataricus and functionally produced the corresponding enzyme as a fusion with the Escherichia coli maltose-binding protein. The purified fusion protein indeed displayed highly thermostable PEPC activity. The structural and biochemical properties of the characterized archaeal-type PEPC (atPEPC) from S. solfataricus are in good agreement with previously reported biochemical analyses of other archaeal PEPC enzymes. The newly identified atPEPC, with its distinct properties, constitutes yet another example of the versatility of the enzymes of the central carbon metabolic pathways in the archaeal domain.     

Community-level birth rate: a missing link between ecology, evolution and diversity

We propose a conceptual model to explain the variation in species richness in local communities and in build-up of regional species pools over time. The idea is that the opportunity for new species to enter a community (its invasibility) determines the present richness of that community as well as the long-term build-up of a species pool by speciation and migration. We propose that a community's invasibility is determined by the turnover rate of reproductive genets in the community, which we call the 'community-level birth rate'. The faster the turn-over, the more species will accumulate per unit time and per unit community size (number of genets) at a given per-birth rate of immigration and speciation. Spatially discrete communities inhabiting similar environments sum up to metacommunities, whose inhabitant species constitute the regional species pool. We propose that the size of a regional species pool is determined by the aggregate community-level birth rate, the size of the metacommunity through time and age of the metacommunity. Thus, the novel contribution is our proposal of a direct effect of local environment on the build-up rate of species pools. The relative importance of immigrating species and neospecies originating locally will change with the temporal and spatial scale under consideration. We propose that the diversification rate specific to evolutionary lineages and the build-up rate of species pools are two sides of the same coin, and that they are both depending on mean generation time. The proposed model offers a reconciliation of two contrasting paradigms in current community ecology, viz. one focussing on present-time ecological processes and one focussing on historical events governing the size of species pools which in turn determines local richness.     

X-ray structure of a two-domain type laccase: A missing link in the evolution of multi-copper proteins

A multi-copper protein with two cupredoxin-like domains was identified from our in-house metagenomic database. The recombinant protein, mgLAC, contained four copper ions/subunits, oxidized various phenolic and non-phenolic substrates, and had spectroscopic properties similar to common laccases. X-ray structure analysis revealed a homotrimeric architecture for this enzyme, which resembles nitrite reductase (NIR). However, a difference in copper coordination was found at the domain interface. mgLAC contains a T2/T3 tri-nuclear copper cluster at this site, whereas a mononuclear T2 copper occupies this position in NIR. The trimer is thus an essential part of the architecture of two-domain multi-copper proteins, and mgLAC may be an evolutionary precursor of NIR.     

Growth inhibition of Escherichia coli during heterologous expression of Bacillus subtilis glutamyl-tRNA synthetase that catalyzes the formation of mischarged glutamyl-tRNA1 Gln

It is known that Bacillus subtilis glutamyl-tRNA synthetase (GluRS) mischarges E. coli tRNA1 Gln with glutamate in vitro. It has also been established that the expression of B. subtilis GluRS in Escherichia coli results in the death of the host cell. To ascertain whether E. coli growth inhibition caused by B. subtilis GluRS synthesis is a consequence of Glu-tRNA1 Ghn formation, we constructed an in vivo test system, in which B. subtilis GluRS gene expression is controlled by IPTG. Such a system permits the investigation of factors affecting E. coli growth. Expression of E. coli glutaminyl-tRNA synthetase (GlnRS) also ameliorated growth inhibition, presumably by competitively preventing tRNA1 Gln misacylation. However, when amounts of up to 10 mM L-glutamine, the cognate amino acid for acylation of tRNA1 Gln, were added to the growth medium, cell growth was unaffected. Overexpression of the B. subtilis gatCAB gene encoding Glu-tRNAGln amidotransferase (Glu-AdT) rescued cells from toxic effects caused by the formation of the mischarging GluRS. This result indicates that B. subtilis Glu-AdT recognizes the mischarged E. coli GlutRNA1 Gln, and converts it to the cognate Gln-tRNA1 Gln species. B. subtilis GluRS-dependent Glu-tRNA1 Gln formation may cause growth inhibition in the transformed E. coli strain, possibly due to abnormal protein 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.     

Reconstructing a missing link in the evolution of a recently diverged phosphotriesterase by active-site loop remodeling

Only decades after the introduction of organophosphate pesticides, bacterial phosphotriesterases (PTEs) have evolved to catalyze their degradation with remarkable efficiency. Their closest known relatives, lactonases, with promiscuous phosphotriasterase activity, dubbed PTE-like lactonases (PLLs), share only 30% sequence identity and also differ in the configuration of their active-site loops. PTE was therefore presumed to have evolved from a yet unknown PLL whose primary activity was the hydrolysis of quorum sensing homoserine lactones (HSLs) (Afriat et al. (2006) Biochemistry45, 13677-13686). However, how PTEs diverged from this presumed PLL remains a mystery. In this study we investigated loop remodeling as a means of reconstructing a homoserine lactonase ancestor that relates to PTE by few mutational steps. Although, in nature, loop remodeling is a common mechanism of divergence of enzymatic functions, reproducing this process in the laboratory is a challenge. Structural and phylogenetic analyses enabled us to remodel one of PTE's active-site loops into a PLL-like configuration. A deletion in loop 7, combined with an adjacent, highly epistatic, point mutation led to the emergence of an HSLase activity that is undetectable in PTE (k(cat)/K(M) values of up to 2 × 10(4)). The appearance of the HSLase activity was accompanied by only a minor decrease in PTE's paraoxonase activity. This specificity change demonstrates the potential role of bifunctional intermediates in the divergence of new enzymatic functions and highlights the critical contribution of loop remodeling to the rapid divergence of new enzyme functions.     

Cloning of the gene for Escherichia coli glutamyl-tRNA synthetase

The structural gene for the glutamyl-tRNA synthetase of Escherichia coli has been cloned in E. coli strain JP1449, a thermosensitive mutant altered in this enzyme. Ampicillin-resistant and tetracycline-sensitive thermoresistant colonies were selected following the transformation of JP1449 by a bank of hybrid plasmids containing fragments from a partial Sau3A digest of chromosomal DNA inserted into the BamHI site of pBR322. One of the selected clones, HS7611, has a level of glutamyl-tRNA synthetase activity more than 20 times higher than that of a wild-type strain. The overproduced enzyme has the same molecular weight and is as thermostable as that of a wild-type strain, indicating that the complete structural gene is present in the insert. These characteristics were lost by curing this clone of its plasmid with acridine orange, and were transferred with high efficiency to the mutant strain JP1449 by transformation with the purified plasmid. A physical map of the plasmid, which contains an insert of about 2.7 kb in length, is presented.     

Monomeric structure of glutamyl-tRNA synthetase in Escherichia coli.

An investigation of the subunit structure of glutamyl-tRNA synthetase (EC 6.1.1.17) from Escherichia coli indicates that this enzyme is a monomer. The enzyme purified to apparent homogeneity is a single polypeptide chain with a molecular weight of 62,000 ± 3,000 and K^Glu_m ? 50 μM in the aminoacylation reaction. Analytical gel electrophoretic procedures were used to determine the molecular weight of species exhibiting glutamyl-tRNA synthetase activity in freshly prepared extracts of several strains of E. coli, which had been grown under various nutritional conditions and harvested at different stages of growth. In all cases, glutamyl-tRNA synthetase activity was associated with a protein having about the same molecular weight and K^Glu_m as the purified enzyme. Thus, no evidence of an oligomeric form of glutamyl-tRNA synthetase with a greater affinity for l-glutamate was obtained, in contrast to a previous report of J. Lapointe and D. Söll (J. Biol. Chem.247, 4966–4974, 1972).