Top PDF Discovery of Aminoacyl-tRNA Synthetase Mutants for the Incorporation of Non Canonical Amino Acids into Proteins

Discovery of Aminoacyl-tRNA Synthetase Mutants for the Incorporation of Non Canonical Amino Acids into Proteins

Discovery of Aminoacyl-tRNA Synthetase Mutants for the Incorporation of Non Canonical Amino Acids into Proteins

Although Aha is ideal for identification of newly synthesized proteins in cultures containing a single cell type, because it is such a good substrate of wild- type MetRS, it does not allow directing the labeling to only proteins in certain members of a complex cell mixture. Alternatively, using a reactive amino acid that requires an engineered MetRS for incorporation, labeling can be directed to individual cells that carry this engineered enzyme. In order to allow incorporation of reactive amino acids that cannot be activated by the wild-type cellular machinery, Link and co-workers devised a fluorescence-activated cell sorter (FACS) based high-throughput screen and demonstrated its utility by identifying E. coli MetRS mutants that can activate the methionine surrogate, azidonorleucine (Anl) from a four-position saturation-mutagenesis library [20]. (Figure 2.1) Cells carrying MetRS mutants that are active toward Anl can display this residue on their surface, on an E. coli outer-membrane protein C (OmpC) variant. (Figure 2.2) Using a strain-promoted version of the [3+2] azide-alkyne cycloaddition [21], which eliminates the copper cytotoxicity associated with the Cu(I)-catalyzed reaction, the azides displayed on cells were ligated to biotin and bound to fluorescent avidin. Clones carrying active MetRS mutants were then isolated with FACS, and screened further. Analysis of enriched clones revealed three MetRS mutants that allowed approximately 50% incorporation of Anl into Met sites when protein expression was carried out in the presence of 8 mM Anl. Recognizing that the L13G mutation was common to all MetRS mutants identified through the screen, the investigators tested this single mutant to discover that this enzyme allows near complete replacement of Met sites at 1 mM Anl.
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Synthetic Biology Tools for Targeted Incorporation of Non Canonical Amino Acids into Cellular Proteins

Synthetic Biology Tools for Targeted Incorporation of Non Canonical Amino Acids into Cellular Proteins

approaches provide unbiased and comprehensive datasets that are ideally suited to machine- learning and data-analytics techniques that are being increasingly used in the biological sciences. Proteomics platforms have rapidly advanced in the last decade. Advances in sequencing and proteomics have and will continue to beat Moore’s law in the years to come. 12-14 These improvements are complemented by the development of new chemical and synthetic biology tools that are specifically designed to leverage and utilize the capabilities of new instruments. Whereas instrumentation advances have enabled increased coverage, throughput, and precision, better chemical reporters and metabolic tags have enhanced the specificity and spatiotemporal resolution of labeling and detection. Here we provide an overview of new approaches to profile protein synthesis in cells. We also highlight a selective subset of chemical and synthetic techniques that enable the identification and quantitation of newly made proteins in different cell types. Notably, these techniques enable study of protein synthesis with varied spatial and temporal resolution in multicellular contexts as well as sub-cellular scales. We first review recent advances in translational profiling approaches that enable cell targeted and organelle-specific profiling. We then provide an overview of amino acid metabolic labeling methods to analyze newly synthesized proteins and highlight the use of bio-orthogonal chemistries to develop chemical reporters and improve quantitative proteomics capabilities. We close by providing an outlook on future directions to enhance the labeling, detection, and quantitation of proteome dynamics in cells that will ultimately facilitate biological discovery and inform subsequent engineering efforts.
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Structure-based design of mutant proteins: I. Molecular docking studies of amino acid binding to wild-type aminoacyl-tRNA synthetases. II. Structure-based design of mutant aminoacyl-tRNA synthetases for non-natural amino acid incorporation

Structure-based design of mutant proteins: I. Molecular docking studies of amino acid binding to wild-type aminoacyl-tRNA synthetases. II. Structure-based design of mutant aminoacyl-tRNA synthetases for non-natural amino acid incorporation

methyl L-tyrosine (OMe-Tyr) site-specifically in protein in response to an amber nonsense codon (14). A few other non-natural amino acids were incorporated by the same group using the same apparatus since then. Such procedures have tremendous potential to expand the genetic codes in living cells, but the current combinatorial experiments, which considered 5 20 mutation trials on five residues expected to be at the binding site of the tyrosine ligand, can become cumbersome. In this chapter we summarize the result of using the Clash-Opportunity Progressive Design algorithm (denoted as COP) to redesign the binding site of mj-TyrRS for the preferential binding of OMe-Tyr, Naphthyl-Ala and p-keto-Phe over natural amino acids. The design for OMe- Tyr leads to three mutants, of which the best mutant [Y32Q, D158A] is expected to bind OMe-Tyr strongly while discriminating against Tyr. This mutant is similar to the one [Y32Q, D158A, E107T, L162P] designed by Wang et al using combinatorial experiments. We predict that the new mutant will have much greater activity while retaining significant discrimination between OMe-Tyr and Tyr.
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The Use of Non-Canonical Amino Acids as a Novel Biocontainment Strategy

The Use of Non-Canonical Amino Acids as a Novel Biocontainment Strategy

Typically, directed evolution is used to create the novel tRNA and aminoacyl tRNA synthetase by encoding them on a plasmid. Then, they can be mutated either through error-prone PCR or the use of degenerate primers. Error-prone PCR uses a Taq polymerase that does not have a proof-reading capability, and the fidelity of the incorporation of nucleotides is controlled by altering the reaction buffer. Likewise, degenerate primers can be used to increase the mutation rate; they are a population of oligonucleotide sequences in which some positions can contain several possible bases. They cover all possible nucleotide sequence combinations for the targeted protein (Iserte et al. 2013). Following mutation, the library of tRNA/synthetase pairs can be examined via positive and negative selection to find those that display orthogonality. In the positive selection process, the plasmid is transferred to bacterial cells that contain genes that confer antibiotic resistance only in the presence of the ncAA. If a proper and functional orthogonal pair is expressed, the ncAA will be incorporated into an antibiotic resistance gene. The gene will be functional, and confer resistance to the antibiotic, allowing the bacteria to survive. In negative selection, the plasmid is transferred to cells that contain an essential gene that contains a premature amber stop codon. In the presence of the ncAA, the novel tRNA/synthetase pair successfully incorporates the ncAA, resulting in an essential, functional gene and the survival of the bacteria (Kato et al. 2015).
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Predicted class-I aminoacyl tRNA synthetase-like proteins in non-ribosomal peptide synthesis

Predicted class-I aminoacyl tRNA synthetase-like proteins in non-ribosomal peptide synthesis

Andrei Osterman, Burnham Institute, La Jolla, CA, United States A new study by L. Aravind and colleagues on evolution and functional diversification of the aminoacyl-tRNA synthetase (AAtRS) family is truly fascinating in a number of ways. Using a sophisticated comparative genomics approach, which combines the distant homology and genomic context analysis, they substantially expanded the boundaries of this family including a discovery of its previously unrecognized relationship with cyclodipeptide synthetases (CDPS). This discovery provided crucial evolutionary and mechanistic insights into this “ lost tribe ” of the AA-tRS clan and demonstrated an amazing functional versatility of the respective fold. The insightful analysis of conserved chromosomal clusters associated with some of the uncharacterized CDPSs opened a Pandora ’ s box of completely unknown and unforeseen biochemical transformation generating a plethora of novel secondary metabolites. Experimental elucidation of these new pathways and their products, which has been enabled by this study, would strongly impact our knowledge of various signaling systems (e.g. host-pathogen interactions) and lead to a discovery of new classes of bioactive compounds. However, as if it was not enough, this paper takes us further, to the analysis of a widespread and extremely interesting family of Met-tRS paralogs, which leads to further expansion of a biochemical landscape of nonribosomal peptidoids. As in the CDPS case, an ingenious combination of phylogenetic and genomic context analyses (already a standard in the field) with fully innovative chemical reasoning allows Aravind et al. to build a very solid case for a number of predicted biosynthetic pathways. The predicted pathways share common themes (eg MettRS-driven condensation of Met and Lys) accompanied by species- specific variations (novel oxidative and nonoxidative modifications of both amino acids). Going back to the overall impact of this paper, one can ’ t help notice that it is not only another triumphant example of creative comparative genomics contributing to better understanding of an important protein family. Its tremendous added value is in providing an incredible opportunity for a small army of experimental biochemists to uncover new processes and molecules with tremendous biomedical potential.
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Expanding the biosynthetic capacity of the aminoacyl-tRNA synthetases

Expanding the biosynthetic capacity of the aminoacyl-tRNA synthetases

By manipulating the editing pocket of E. coli ValRS, Schimmel and co-workers were able to expand the amino acid set of E. coli (91). A selection was designed to screen for chromosomal mutations to favor the charging of cysteine to tRNA Val . The authors found that all identified mutations resided in the editing domain of ValRS. One such editing disabled mutant E. coli strain was able to incorporate more than 20% non- canonical amino acid α -aminobutyrate into cellular proteins at valine codon sites, suggesting that editing may play an important role in restricting the genetic code to 20 amino acids and suppressing the genetic code ambiguity. Further experiments showed that cell viability is heavily reliant upon the accuracy of translation imposed by ValRS: the concentration of α -aminobutyrate to arrest the growth of E. coli cells inversely correlates with the impaired level of editing function (92). In Chapter 4 of this thesis, I will present an example where we found a non-canonical amino acid able to escape editing by ValRS and infiltrate into valine codon sites. In Chapter 7 of this thesis, I will describe an editing impaired mutant LeuRS to facilitate incorporation of a novel amino acid that otherwise could be edited by wild-type LeuRS.
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Non-canonical amino acid labeling in proteomics and biotechnology

Non-canonical amino acid labeling in proteomics and biotechnology

A major hurdle for biotechnology applications where stoichiometric labeling is desired is that ncAA incorpor- ation efficiency for site-specific protein modification often varies by the incorporation site. Elucidating factors determining site-dependence will enable the more effect- ive design of ncAA-modified proteins, for example, by targeting bases that flank suppressed codons [129]. Add- itionally, investigation of mechanisms involved in ribo- some stoppage, where polypeptide synthesis stalls or terminates prematurely, may also provide illumination towards efficient modification site selection. Develop- ment of novel cell strains lacking factors inhibitory to ncAA incorporation may also improve labeling effi- ciency. Such strains have already been developed in E. coli by knocking out release factor components respon- sible for competition with nonsense suppression at amber stop codons to reduce premature termination [125, 130, 131]. However, development of such strains for other organisms or ncAA incorporation methods may be challenging as the rarely used amber stop codon required significant mutation before a viable E. coli strain was produced [125, 130, 131].
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A human leucyl-tRNA synthetase as an anticancer target

A human leucyl-tRNA synthetase as an anticancer target

is often misregulated in human cancers, and the gene plays a role as a tumor suppressor or as an oncogene, depending on the cellular context and circumstances. On the other hand, as CHX inhibits protein synthesis by interfering with the translocation step in protein synthesis including p21 protein, p21 expression is predicted to be inhibited during treatment by LARS inhibitors. In this study, compound 2 was found to have an early induction of p21 expression, indicating the protective signaling of cell starvation at 12 hours treatment. The repression of p21 expression at 48 hours suggests general repression of protein synthesis at a later time. Moreover, compound 2 was shown to induce apoptosis in a dose- dependent manner within 24 hours. Meanwhile, the CHX, as a protein synthesis inhibitor, did not lead to activation of p21 signal transduction pathway, but inhibited protein synthesis in a dose-dependent manner. Therefore, we suggest that the performance of the LARS inhibitor is quite different from that of the general inhibitors of protein synthesis such as CHX. Moreover, the inhibition of LARS caused an increase in uncharged leucine-specific tRNA, which triggered a chain of malnutrition signaling despite a normal supply of leucine
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Site specific incorporation of synthetic amino acids into functioning ion channels

Site specific incorporation of synthetic amino acids into functioning ion channels

uncharged tRNA. The degree to which read-through is observed is rather variable, as may be seen by comparing the relatively light band intensity in Figure 3.13 with the currents observed in oocytes injected with uncharged tRNA in Figure 3.14. Indeed, during the course of these studies, an extremely surprising observation was made. As indicated earlier, the co-injection of mRNA with non-aminoacylated tRNA is a routine null control. These tRNA’s are ligated to dCA, so that they are treated in exactly the same way as charged tRNA’s. Typically, this 76 nt tRNA-dCA gives much larger currents than mRNA injected alone, but much smaller currents than in cells injected with aminoacylated tRNA carrying a natural or unnatural amino acid. In attempting to identify the source of full-length read-through protein, tRNA which had not been ligated to dCA (i.e. 74 nt tRNA missing the critical CCA motif found at the 3’ end of all tRNA) was co-injected with mRNA containing a stop codon. To our great surprise, this 74mer produced larger currents than tRNA-dCA, although still much smaller than those from oocytes injected with charged tRNA. A comparison of a number of tRNA constructs was made. (Figure 3.14)
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A MUTANT OF YEAST WITH A DEFECTIVE METHIONYL-tRNA SYNTHETASE

A MUTANT OF YEAST WITH A DEFECTIVE METHIONYL-tRNA SYNTHETASE

In forty-five of the forty-seven tetrads from hybrid HI9 le- : le+ segregated 2 : 2 and the ts- 296 mutation (methionyl-tRNA synthetase) displayed a second division segr[r]

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Protein engineering via site specific incorporation of nonnatural amino acids

Protein engineering via site specific incorporation of nonnatural amino acids

amino acid, Trp, due to the enlarged binding pocket generated by the T415G mutation. 13 The relaxed substrate specificity of yPheRS (T415G) showed several drawbacks. Cell growth rate decreased, likely due to misincorporation of Trp at Phe sites in essential proteins. Leaky expression of target protein even under uninduced conditions was prominent, perhaps due to the impaired repressor proteins. Furthermore, misincorporation of Trp as well as 2Nal was observed at Phe residues encoded as UUU codons, which prevented high fidelity incorporation of 2Nal at programmed sites. These drawbacks prompted us to pursue more selective yPheRS variants. Previously we reported that an other rationally designed yPheRS (T415A) showed 10-fold higher activity toward pBrF than Trp. However, yPheRS (T415A) did not exhibit enhanced selectivity toward pBrF against Phe. 13 Therefore, we need to explore different approaches to obtain highly selective yPheRS variants. Aminoacyl-tRNA synthetases are known to be readily evolvable. Schultz and co-workers have developed powerful screening methods to change the substrate specificity of tyrosyl-tRNA synthetase (mjTyrRS) derived from Methanococcus jannaschii toward nonnatural amino acids. 14,18,22,29 Recently our lab reported a novel screening method to adapt E. coli methionyl-tRNA synthetase to a reactive methionine analog, azidonorleucine. 7 However, until now there have been no reports about evolving eukaryotic aminoacyl-tRNA synthetases to change their substrate specificity toward a nonnatural amino acid. In this report, we describe the high- throughput screening of a yPheRS library to obtain yPheRS variants of which substrate specificity is changed to a nonnatural amino acid, 2Nal.
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Viral Hijacking of Mitochondrial Lysyl-tRNA Synthetase

Viral Hijacking of Mitochondrial Lysyl-tRNA Synthetase

Mitochondrial LysRS accounts for less than 10% of total LysRS. In order to be able to distinguish between cyto-LysRS and mito-LysRS by Western blotting on HIV extracts, we raised antibodies in rabbit against peptides specific for the cytoplasmic or mitochondrial form of LysRS. Peptides of 19 amino acid residues, located within the cytoplasm-specific se- quence encoded by exon 1, and of 18 residues, located within the mitochondrion-specific sequence encoded by exon 2 and positioned 8 residues after the putative cleavage site of the import presequence (Fig. 1B), were synthesized, conjugated to ovalbumin, and used for immunization. As shown Fig. 3a, the rabbit antisera proved to be specific for the human cytoplasmic (anti-cKRS antibodies) or mitochondrial (anti-mKRS antibod- ies) LysRS expressed in yeast. As expected, the LysRS com- ponent of the multi-aminoacyl-tRNA synthetase complex iso- lated from rabbit liver could be revealed only with anti-cKRS antibodies (Fig. 3a). With anti-cKRS antibodies, a single polypeptide was revealed in total extracts of human HeLa and U937 cells in culture (Fig. 3b). With anti-mKRS antibodies, a FIG. 2. Expression of human cyto-LysRS and mito-LysRS in yeast. Total extracts from the yeast diploid strain ccdYK or from the yeast haploid strains cdYK ⫻ pyHKm and cdYK ⫻ pyHKc, lacking the yeast KRS1 gene but complemented with human mito-KRS or cyto-KRS, respectively, were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue (left panel) or subjected to Western blotting using antibodies directed to yeast LysRS (IgG ⫻ yKRS) or raised against the full-length cytoplasmic LysRS of mammalian origin (IgG ⫻ KRS). The polypeptides corresponding to mito-LysRS and cyto-LysRS, visualized by Coomassie blue staining, are indicated by dots. MARS, the multi-aminoacyl-tRNA synthetase complex purified from rabbit containing cyto-LysRS (indicated by an arrow). ⴱ , a polypeptide cross-reacting with anti-yKRS antibodies.
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Protein modification through in vivo incorporation of noncanonical amino acids

Protein modification through in vivo incorporation of noncanonical amino acids

Genetic engineering provides a tool with which one can prepare complex macromolecules possessing both precisely controlled architectures and specific catalytic or biological activity. Recent work has shown the advantages of using the biosynthetic machinery to produce new materials (for a review see reference [1]). The use of monomers other than the twenty canonical amino acids enables the introduction of new functionality into proteins, creating the potential for novel physical and chemical properties. Analogues of many of the canonical amino acids have been incorporated into proteins in E. coli using the wild-type biosynthetic machinery, e.g. [2, 3], while modifications of that machinery have permitted the incorporation of a still broader set of non-canonical amino acids [4-14]. Increasing the number of amino acid monomers that can be incorporated into proteins, and thereby the range of physical properties and chemistries available, requires detailed understanding of the biosynthetic apparatus.
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Computational Insights into the Accuracy and Editing of Aminoacyl tRNA Synthetases

Computational Insights into the Accuracy and Editing of Aminoacyl tRNA Synthetases

Proteins# are# ubiquitous# in# nature,# carrying# out# many# of# life’s# functions.# However,# for# these# biomolecules# to# function# correctly# they# must# be# accurately# synthesized.#The#task#of#ensuring#the#desired#accuracy#is#achieved#falls#to#Aminoacyl# tRNA#Synthetases#(aaRS).#These#enzymes#catalyze#the#activation#and#transfer#of#amino# acids# to# tRNA.# In# addition# to# carrying# out# these# reactions,# they# also# edit# against# the# incorporation# of# incorrect# amino# acids.# Within# this# thesis# the# many# facets# of# aaRS# editing#and#accuracy#are#examined#in#detail.#In#particular,#the#editing#mechanism#of# Hcy# and# Hse# in# MetRS# is# elucidated# in# detail.# These# results# allow# us# to# infer# generalizations#of#the#editing#of#Hcy#within#many#aaRS's.#Zn(II)'s#role#in#catalysis#and# accuracy# for# SerRS,# CysRS# and# ThrRS# is# also# examined# in# some# detail# and# gives# key# insights#into#a#new#potential#role#it#may#play.##
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Isolation of aminoacyl-trna and its labeling with stable-isotope

Isolation of aminoacyl-trna and its labeling with stable-isotope

Values of plasma a-KIC 13C labeling in arterial or venous blood were greater than the mixed tissue free leucine (P < 0.05) but not signif-. icantly different from the labeling of bloo[r]

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IN THE NUCLEIC ACIDS. PROTEINS AND AMINO ACIDS IN THE DEVELOPING' MANGO BUDS

IN THE NUCLEIC ACIDS. PROTEINS AND AMINO ACIDS IN THE DEVELOPING' MANGO BUDS

The results obtained in the present study clearly indicate that in the mango buds of Dashehari variety taken from March/April flush, the levels of the metabolites, v[r]

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Computational Insights into the High-Fidelily Catalysis of Aminoacyl-tRNA Synthetases

Computational Insights into the High-Fidelily Catalysis of Aminoacyl-tRNA Synthetases

Despite the unparalleled success in the application of DFT functionals, it is unfortunate to know that they suffer from four major challenges. 65 The largest error is the self-interaction error which arises from the electron interacting with itself in the columbic term described by the DFT Hamiltonian. 66 This error directly influences the underestimation of barrier heights, but can be somewhat addressed by including additional contribution from the exact HF exchange. 67 Also, another major limitation is the inability of these functionals to describe non-covalent long-range (van der Waals) interactions. 68 Presently, there is ongoing progress towards eliminating this shortcoming through introducing empirical dispersion corrections such as Grimme’s empirical formula. 69 Interestingly, either geometry optimization of the chemical models or single point energy calculations using B3LYP-D3 were found to be equally successful in minimizing this error in different systems. 70 One more critical drawback of DFT functionals is that they all are ground-state methods and are unable to provide reasonable results for excited state applications. Finally, error arises from the inaccurate description of chemical systems containing transition metals, increasing with increasing the %XC included contrary to a solution to self-interaction error. 71 To illustrate, M06L (with no HF exchange) led to accurate predictions in the excitation energies in some systems. 72
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Biosynthesis of food constituents: Amino acids: 4  Non protein amino acids – a review

Biosynthesis of food constituents: Amino acids: 4 Non protein amino acids – a review

results in the release of alliinase and subsequent α,β-elimination of S-alk(en)ylcysteine sulfoxides, affording the corresponding alk(en)ylsulfenic acids and α-aminoacrylic acid. The latter compound spontaneously decomposes to yield ammonia and pyruvic acid (via α-iminoacrylic acid). Conden- sation of the arising alk(en)ylsulfenic acids leads to the formation of thiosulfinates, the flavour principles of freshly disrupted Allium vegetables (Figure 11). The most typical amino acid of onion, S-(prop-1-en-1-yl)cysteine sulfoxide (isoalliin), enzymatically decomposes yielding prop-1-en- 1-ylsulfenic acid (Figure 12). This sulfenic acid can be either spontaneously transformed into prop-1-en-1-yl-containing thiosulfinates or, by the action of the lachrymatory factor synthase, it yields the irritating lachrymatory factor of onion, (Z)-propanethial S-oxide (IMAI et al. 2002). The arising S-alk(en)yl alkanethiosulfinates can par- ticipate in an astonishing variety of subsequent reactions which strongly depend on the conditions (particularly on the polarity of medium and tem- perature), and which afford miscellaneous types of organosulfur compounds, such as sulfides, vi- nyldithiins, ajoenes, etc. (BLOCK 1992) (Figure 13). These compounds exhibit a broad spectrum of health-promoting activities, e.g. hypolipidemic, antithrombotic, antioxidant, hypocholesterolemic, cancer-preventive and anticancer effects.
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Stable isotope studies reveal pathways for the incorporation of non essential amino acids in Acyrthosiphon pisum (pea aphids)

Stable isotope studies reveal pathways for the incorporation of non essential amino acids in Acyrthosiphon pisum (pea aphids)

Phloem-feeding insects benefit from having evolved rapid and efficient pathways for the uptake and metabolic conversion of glutamine, glutamic acid, asparagine and aspartic acid into essential amino acids. Aphids accomplish this in part through close interactions with endosymbiont bacteria, Buchnera aphidicola , that are contained in specialized cells called bacteriocytes (Buchner, 1965). Although all Buchnera synthesize essential nutrients for their aphid hosts, there is variation among different aphid species and even lineages within a species in the metabolic functions of their bacterial endosymbionts and their utilization of host plant nutrients (MacDonald et al., 2011; Vogel and Moran, 2011). In addition to Buchnera , several aphid species have been shown to contain facultative endosymbionts that can contribute to survival and fitness (Leonardo and Muiru, 2003; Oliver et al., 2003), including through amino acid biosynthesis (Richards et al., 2010). Sequencing of the pea aphid ( Acyrthosiphon pisum ) and Buchnera genomes, gene expression profiling, and proteomic studies have provided new insight into the predicted pathways of amino acid biosynthesis in the A. pisum–Buchnera symbiosis (Brinza et al., 2010; Hansen and Moran, 2011; Poliakov et al., 2011; Richards et al., 2010; Shigenobu et al., 2000). Notably, there are several examples of shared biosynthesis pathways for essential amino acids that are partly encoded by the endosymbiont bacteria and partly by the aphid host (Russell et al., 2013; Wilson et al., 2010). Genomic analyses indicate that aspartic acid and glutamic acid contribute to the biosynthesis of all amino acids in the aphid – Buchnera system by providing nitrogen and/or the carbon backbone (Hansen and Moran, 2011).
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Aminoacyl tRNA synthetases as malarial drug targets: a comparative bioinformatics study

Aminoacyl tRNA synthetases as malarial drug targets: a comparative bioinformatics study

The GluRS family also showed low conservation at the N-terminal with Motif 16 present in mammalian sequences at this terminal (Additional file 4D). The HIGH signature was found in Motif 3 as HIGH in all sequences analysed except for PfGluRS where it occurs as HVGH (Additional file  4). P. falciparum GluRS sequence has a glutamine rich N- terminal from residue 68 as opposed to other Plasmodium species. In mammals, including human, this enzyme is a bifunctional protein acting both as GluRS and ProRS. Thus it catalyses aminoacylation of both proline and glutamate [135]. On alignment with Plasmodium GluRS, the mammalian sequences showed a C-terminal extension indicating that it is the N- terminal end that catalyses glutamate aminoacylation. The human enzyme contains three motifs that link the two catalytic domains that function in formation of the multicomplex synthetase and play a role in protein-nucleic acid inter- actions [135, 136]. Similar motifs have been reported in other aaRS like GlyRS, HisRS and TrpRS though they occur at the N-or C-termini of the core domains as a sin- gle copy as opposed to the Glu/ProRS where they occur as tandem repeats linking the two catalytic domains [135–137]. Human IleRS has an extension at the C-termi- nal which was absent in Plasmodium sequences, but the core domain of this family was highly conserved (Addi- tional file 4E). Motif 19, 20 and 26 were conserved in the C-terminal of mammalian IleRS sequences but absent in Plasmodium sequences. The three tandem motifs in the human bifunctional Glu/ProRS have been shown to interact with two repeated motifs in IleRS at the C-ter- minal extension [138]. In IleRS, the HIGH signature was found in Motif 1 while the KMSKS signature was in Motif 3 occurring as HYGH and KMSKR, respectively (Addi- tional file  4E). Alignment and motif discovery of LeuRS family showed that this family of protein has low conser- vation even at the core domain (Additional file 4F). Motif 21, 25 and 27 were conserved in Plasmodium sequences. Only Motifs 3, 5, 6, 26 and 36 were conserved through all mammalian and Plasmodium sequences (Additional file 4F).
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