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1.
Science ; 374(6573): eabm4805, 2021 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-34762488

RESUMO

Protein-protein interactions play critical roles in biology, but the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions not yet identified. We take advantage of advances in proteome-wide amino acid coevolution analysis and deep-learning­based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes within the Saccharomyces cerevisiae proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of yeast proteins, identify 1505 likely to interact, and build structure models for 106 previously unidentified assemblies and 806 that have not been structurally characterized. These complexes, which have as many as five subunits, play roles in almost all key processes in eukaryotic cells and provide broad insights into biological function.


Assuntos
Aprendizado Profundo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mapeamento de Interação de Proteínas , Proteoma/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Aciltransferases/química , Aciltransferases/metabolismo , Segregação de Cromossomos , Biologia Computacional , Simulação por Computador , Reparo do DNA , Evolução Molecular , Recombinação Homóloga , Ligases/química , Ligases/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Biossíntese de Proteínas , Conformação Proteica , Mapas de Interação de Proteínas , Proteoma/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/química , Ubiquitina/química , Ubiquitina/metabolismo
2.
mBio ; 12(4): e0110021, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34225495

RESUMO

Most bacteria employ a two-step indirect tRNA aminoacylation pathway for the synthesis of aminoacylated tRNAGln and tRNAAsn. The heterotrimeric enzyme GatCAB performs a critical amidotransferase reaction in the second step of this pathway. We have previously demonstrated in mycobacteria that this two-step pathway is error prone and translational errors contribute to adaptive phenotypes such as antibiotic tolerance. Furthermore, we identified clinical isolates of the globally important pathogen Mycobacterium tuberculosis with partial loss-of-function mutations in gatA, and demonstrated that these mutations result in high, specific rates of translational error and increased rifampin tolerance. However, the mechanisms by which these clinically derived mutations in gatA impact GatCAB function were unknown. Here, we describe biochemical and biophysical characterization of M. tuberculosis GatCAB, containing either wild-type gatA or one of two gatA mutants from clinical strains. We show that these mutations have minimal impact on enzymatic activity of GatCAB; however, they result in destabilization of the GatCAB complex as well as that of the ternary asparaginyl-transamidosome. Stabilizing complex formation with the solute trehalose increases specific translational fidelity of not only the mutant strains but also of wild-type mycobacteria. Therefore, our data suggest that alteration of GatCAB stability may be a mechanism for modulation of translational fidelity. IMPORTANCE Most bacteria use a two-step indirect pathway to aminoacylate tRNAGln and tRNAAsn, despite the fact that the indirect pathway consumes more energy and is error prone. We have previously shown that the higher protein synthesis errors from this indirect pathway in mycobacteria allow adaptation to hostile environments such as antibiotic treatment through generation of novel alternate proteins not coded by the genome. However, the precise mechanisms of how translational fidelity is tuned were not known. Here, we biochemically and biophysically characterize the critical enzyme of the Mycobacterium tuberculosis indirect pathway, GatCAB, as well as two mutant enzymes previously identified from clinical isolates that were associated with increased mistranslation. We show that the mutants dysregulate the pathway via destabilizing the enzyme complex. Importantly, increasing stability improves translational fidelity in both wild-type and mutant bacteria, demonstrating a mechanism by which mycobacteria may tune mistranslation rates.


Assuntos
Regulação Bacteriana da Expressão Gênica , Mutação , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/genética , Transferases de Grupos Nitrogenados/genética , Biossíntese de Proteínas/genética , Humanos , RNA de Transferência de Glutamina/metabolismo , Aminoacilação de RNA de Transferência , Tuberculose/microbiologia
3.
Genes (Basel) ; 12(3)2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33809136

RESUMO

The twenty amino acids in the standard genetic code were fixed prior to the last universal common ancestor (LUCA). Factors that guided this selection included establishment of pathways for their metabolic synthesis and the concomitant fixation of substrate specificities in the emerging aminoacyl-tRNA synthetases (aaRSs). In this conceptual paper, we propose that the chemical reactivity of some amino acid side chains (e.g., lysine, cysteine, homocysteine, ornithine, homoserine, and selenocysteine) delayed or prohibited the emergence of the corresponding aaRSs and helped define the amino acids in the standard genetic code. We also consider the possibility that amino acid chemistry delayed the emergence of the glutaminyl- and asparaginyl-tRNA synthetases, neither of which are ubiquitous in extant organisms. We argue that fundamental chemical principles played critical roles in fixation of some aspects of the genetic code pre- and post-LUCA.


Assuntos
Aminoácidos/genética , Aminoacil-tRNA Sintetases/genética , Animais , Aspartato-tRNA Ligase/genética , Código Genético/genética , Humanos , Aminoacil-RNA de Transferência/genética
4.
Enzymes ; 48: 39-68, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33837710

RESUMO

In this chapter we consider the catalytic approaches used by aminoacyl-tRNA synthetase (AARS) enzymes to synthesize aminoacyl-tRNA from cognate amino acid and tRNA. This ligase reaction proceeds through an activated aminoacyl-adenylate (aa-AMP). Common themes among AARSs include use of induced fit to drive catalysis and transition state stabilization by class-conserved sequence and structure motifs. Active site metal ions contribute to the amino acid activation step, while amino acid transfer to tRNA is generally a substrate-assisted concerted mechanism. A distinction between classes is the rate-limiting step for aminoacylation. We present some examples for each aspect of aminoacylation catalysis, including the experimental approaches developed to address questions of AARS chemistry.


Assuntos
Aminoácidos , Aminoacil-tRNA Sintetases , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Aminoacilação , Catálise , RNA de Transferência/genética
5.
Genes (Basel) ; 10(4)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30939863

RESUMO

The aminoacyl-tRNA synthetases (aaRSs) are well established as the translators of the genetic code, because their products, the aminoacyl-tRNAs, read codons to translate messenger RNAs into proteins. Consequently, deleterious errors by the aaRSs can be transferred into the proteome via misacylated tRNAs. Nevertheless, many microorganisms use an indirect pathway to produce Asn-tRNAAsn via Asp-tRNAAsn. This intermediate is produced by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) that has retained its ability to also generate Asp-tRNAAsp. Here we report the discovery that ND-AspRS and its discriminating counterpart, AspRS, are also capable of specifically producing Glu-tRNAGlu, without producing misacylated tRNAs like Glu-tRNAAsn, Glu-tRNAAsp, or Asp-tRNAGlu, thus maintaining the fidelity of the genetic code. Consequently, bacterial AspRSs have glutamyl-tRNA synthetase-like activity that does not contaminate the proteome via amino acid misincorporation.


Assuntos
Aspartato-tRNA Ligase/genética , Glutamato-tRNA Ligase/genética , RNA de Transferência de Asparagina/genética , RNA de Transferência de Ácido Aspártico/genética , Sequência de Aminoácidos/genética , Asparagina/química , Asparagina/genética , Aspartato-tRNA Ligase/química , Código Genético/genética , Glutamato-tRNA Ligase/química , Mycobacterium smegmatis/química , Mycobacterium smegmatis/genética , Conformação Proteica , Proteoma/química , Proteoma/genética , Aminoacil-RNA de Transferência/genética , RNA de Transferência de Asparagina/química , RNA de Transferência de Ácido Aspártico/química , Homologia de Sequência de Aminoácidos
6.
J Biol Chem ; 293(20): 7892-7893, 2018 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-29777017

RESUMO

The introduction of manmade chemicals, including the herbicide atrazine, into the environment has led to the emergence of microorganisms with new biodegradation pathways. Esquirol et al. demonstrate that the AtzE enzyme catalyzes a central step in atrazine degradation and that expression of AtzE requires coexpression of the small protein AtzG. Remarkably, AtzG and AtzE appear to have evolved from GatC and GatA, components of an ancient enzyme involved in indirect tRNA aminoacylation, providing an elegant demonstration of metabolic repurposing.


Assuntos
Atrazina , Herbicidas , Biodegradação Ambiental , Triazinas
7.
Curr Opin Chem Biol ; 41: 114-122, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29156229

RESUMO

The fact that most bacteria do not contain a full set of aminoacyl-tRNA synthetases (aaRS) is often underappreciated. In the absence of asparaginyl-tRNA and/or glutaminyl-tRNA synthetase (AsnRS and GlnRS), Asn-tRNAAsn and/or Gln-tRNAGln are produced by an indirect tRNA aminoacylation pathway that relies on misacylation of these two tRNAs by two different misacylating aaRSs, followed by transamidation by an amidotransferase (GatCAB in bacteria). This review highlights the central importance of indirect tRNA aminoacylation to accurate protein translation, mechanistic peculiarities that appear to be unique to this system, and the newly recognized connection between indirect tRNA aminoacylation and mistranslation as a strategy used by bacteria to respond to environmental stressors like antibiotics.


Assuntos
Fenótipo , Aminoacilação de RNA de Transferência , Amônia/metabolismo , Evolução Molecular , Humanos , Transferases de Grupos Nitrogenados/metabolismo
8.
Arch Biochem Biophys ; 633: 58-67, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28893510

RESUMO

Glycosylphosphatidylinositol transamidase (GPI-T) catalyzes the post-translational addition of the GPI anchor to the C-terminus of some proteins. In most eukaryotes, Gpi8, the active site subunit of GPI-T, is part of a hetero-pentameric complex containing Gpi16, Gaa1, Gpi17, and Gab1. Gpi8, Gaa1, and Gpi16 co-purify as a heterotrimer from Saccharomyces cerevisiae, suggesting that they form the core of the GPI-T. Details about the assembly and organization of these subunits have been slow to emerge. We have previously shown that the soluble domain of S. cerevisiae Gpi8 (Gpi823-306) assembles as a homodimer, similar to the caspases with which it shares weak sequence homology (Meitzler, J. L. et al., 2007). Here we present the characterization of a complex between the soluble domains of Gpi8 and Gaa1. The complex between GST-Gpi823-306 (α) and His6-Gaa150-343 (ß) was characterized by native gel analysis and size exclusion chromatography (SEC) and results are most consistent with an α2ß2 stoichiometry. These results demonstrate that Gpi8 and Gaa1 interact specifically without a requirement for other subunits, bring us closer to determining the stoichiometry of the core subunits of GPI-T, and lend further credence to the hypothesis that these three subunits assemble into a dimer of a trimer.


Assuntos
Aminoaciltransferases/química , Glicoproteínas de Membrana/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Motivos de Aminoácidos , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Solubilidade , Homologia Estrutural de Proteína , Especificidade por Substrato , Vibrionaceae/química , Vibrionaceae/enzimologia
9.
FEBS Lett ; 590(18): 3122-32, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27500385

RESUMO

The Helicobacter pylori Asp-tRNA(A) (sn) /Glu-tRNA(G) (ln) amidotransferase (GatCAB) utilizes an uncommonly hydrophilic, ~ 40 Å ammonia tunnel for ammonia/ammonium transport between isolated active sites. Hydrophilicity of this tunnel requires a distinct ammonia transport mechanism, which hypothetically occurs through a series of deprotonation and protonation steps. To explore the initiation of this relay mechanism, the highly conserved tunnel residue D185 (in the GatA subunit) was enzymatically and computationally investigated by comparing D185A, D185N, and D185E mutant enzymes to wild-type GatCAB. Our results indicate that D185 acts as an acid/base residue, participating directly in catalysis. To our knowledge, this is the first example of acid/base chemistry in a glutamine-dependent amidotransferase ammonia tunnel.


Assuntos
Amônia/metabolismo , Proteínas de Bactérias/metabolismo , Helicobacter pylori/enzimologia , Mutação de Sentido Incorreto , Transferases de Grupos Nitrogenados/metabolismo , Proteínas de Bactérias/genética , Domínio Catalítico , Simulação de Dinâmica Molecular , Transferases de Grupos Nitrogenados/química , Transferases de Grupos Nitrogenados/genética
10.
Biochem Mol Biol Educ ; 43(2): 68-75, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25727192

RESUMO

Recently, a requirement for directed responsible conduct in research (RCR) education has become a priority in the United States and elsewhere. In the US, both the National Institutes of Health and the National Science Foundation require RCR education for all students who are financially supported by federal awards. The guidelines produced by these agencies offer useful templates for the introduction of RCR materials into courses worldwide. Many academic programs already offer courses or workshops in RCR for their graduate students and for undergraduate science majors and/or researchers. Introducing RCR into undergraduate biochemistry and molecular biology laboratory curricula is another, highly practical way that students can be exposed to these important topics. In fact, a strong argument can be made for integrating RCR into laboratory courses because these classes often introduce students to a scientific environment like that they might encounter in their careers after graduation. This article focuses on general strategies for incorporating explicit RCR education into biochemistry and molecular biology laboratory coursework using the topics suggested by NIH as a starting point.


Assuntos
Pesquisa Biomédica/educação , Currículo , Educação Profissionalizante , Biologia Molecular/educação , Humanos
11.
Crit Rev Biochem Mol Biol ; 48(5): 446-64, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23978072

RESUMO

Cancer is second only to heart disease as a cause of death in the US, with a further negative economic impact on society. Over the past decade, details have emerged which suggest that different glycosylphosphatidylinositol (GPI)-anchored proteins are fundamentally involved in a range of cancers. This post-translational glycolipid modification is introduced into proteins via the action of the enzyme GPI transamidase (GPI-T). In 2004, PIG-U, one of the subunits of GPI-T, was identified as an oncogene in bladder cancer, offering a direct connection between GPI-T and cancer. GPI-T is a membrane-bound, multi-subunit enzyme that is poorly understood, due to its structural complexity and membrane solubility. This review is divided into three sections. First, we describe our current understanding of GPI-T, including what is known about each subunit and their roles in the GPI-T reaction. Next, we review the literature connecting GPI-T to different cancers with an emphasis on the variations in GPI-T subunit over-expression. Finally, we discuss some of the GPI-anchored proteins known to be involved in cancer onset and progression and that serve as potential biomarkers for disease-selective therapies. Given that functions for only one of GPI-T's subunits have been robustly assigned, the separation between healthy and malignant GPI-T activity is poorly defined.


Assuntos
Aminoaciltransferases/metabolismo , Biomarcadores Tumorais/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Glicoproteínas de Membrana/metabolismo , Neoplasias/metabolismo , Oncogenes/genética , Sequência de Aminoácidos , Aminoaciltransferases/química , Humanos , Dados de Sequência Molecular
12.
J Biol Chem ; 288(6): 3816-22, 2013 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-23258533

RESUMO

Many bacteria lack genes encoding asparaginyl- and/or glutaminyl-tRNA synthetase and consequently rely on an indirect path for the synthesis of both Asn-tRNA(Asn) and Gln-tRNA(Gln). In some bacteria such as Thermus thermophilus, efficient delivery of misacylated tRNA to the downstream amidotransferase (AdT) is ensured by formation of a stable, tRNA-dependent macromolecular complex called the Asn-transamidosome. This complex enables direct delivery of Asp-tRNA(Asn) from the non-discriminating aspartyl-tRNA synthetase to AdT, where it is converted into Asn-tRNA(Asn). Previous characterization of the analogous Helicobacter pylori Asn-transamidosome revealed that it is dynamic and cannot be stably isolated, suggesting the possibility of an alternative mechanism to facilitate assembly of a stable complex. We have identified a novel protein partner called Hp0100 as a component of a stable, tRNA-independent H. pylori Asn-transamidosome; this complex contains a non-discriminating aspartyl-tRNA synthetase, AdT, and Hp0100 but does not require tRNA(Asn) for assembly. Hp0100 also enhances the capacity of AdT to convert Asp-tRNA(Asn) into Asn-tRNA(Asn) by ∼35-fold. Our results demonstrate that bacteria have adopted multiple divergent methods for transamidosome assembly and function.


Assuntos
Amidinotransferases/metabolismo , Proteínas de Bactérias/metabolismo , Helicobacter pylori/enzimologia , Complexos Multienzimáticos/metabolismo , RNA Bacteriano/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Amidinotransferases/genética , Proteínas de Bactérias/genética , Helicobacter pylori/genética , Complexos Multienzimáticos/genética , RNA Bacteriano/genética , Aminoacil-RNA de Transferência/genética
13.
Biosci Rep ; 32(6): 577-86, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22938202

RESUMO

In eukaryotes, GPI (glycosylphosphatidylinositol) lipid anchoring of proteins is an abundant post-translational modification. The attachment of the GPI anchor is mediated by GPI-T (GPI transamidase), a multimeric, membrane-bound enzyme located in the ER (endoplasmic reticulum). Upon modification, GPI-anchored proteins enter the secretory pathway and ultimately become tethered to the cell surface by association with the plasma membrane and, in yeast, by covalent attachment to the outer glucan layer. This work demonstrates a novel in vivo assay for GPI-T. Saccharomyces cerevisiae INV (invertase), a soluble secreted protein, was converted into a substrate for GPI-T by appending the C-terminal 21 amino acid GPI-T signal sequence from the S. cerevisiae Yapsin 2 [Mkc7p (Y21)] on to the C-terminus of INV. Using a colorimetric assay and biochemical partitioning, extracellular presentation of GPI-anchored INV was shown. Two human GPI-T signal sequences were also tested and each showed diminished extracellular INV activity, consistent with lower levels of GPI anchoring and species specificity. Human/fungal chimaeric signal sequences identified a small region of five amino acids that was predominantly responsible for this species specificity.


Assuntos
Aminoaciltransferases/metabolismo , Ensaios Enzimáticos , Glicosilfosfatidilinositóis/metabolismo , Saccharomyces cerevisiae/enzimologia , beta-Frutofuranosidase/metabolismo , Sequência de Aminoácidos , Aminoaciltransferases/análise , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Ensaios Enzimáticos/métodos , Glicosilfosfatidilinositóis/análise , Humanos , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , beta-Frutofuranosidase/química
14.
Nucleic Acids Res ; 40(11): 4965-76, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22362756

RESUMO

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.


Assuntos
Aspartato-tRNA Ligase/metabolismo , Helicobacter pylori/enzimologia , Transferases de Grupos Nitrogenados/metabolismo , RNA de Transferência de Asparagina/metabolismo , Aminoacilação de RNA de Transferência , Asparagina/metabolismo , Ácido Aspártico/metabolismo , Código Genético , Cinética , RNA de Transferência de Ácido Aspártico/metabolismo
15.
Biochemistry ; 51(1): 273-85, 2012 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-22229412

RESUMO

The Helicobacter pylori (Hp) Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase (AdT) plays important roles in indirect aminoacylation and translational fidelity. AdT has two active sites, in two separate subunits. Kinetic studies have suggested that interdomain communication occurs between these subunits; however, this mechanism is not well understood. To explore domain-domain communication in AdT, we adapted an assay and optimized it to kinetically characterize the kinase activity of Hp AdT. This assay was applied to the analysis of a series of point mutations at conserved positions throughout the putative AdT ammonia tunnel that connects the two active sites. Several mutations that caused significant decreases in AdT's kinase activity (reduced by 55-75%) were identified. Mutations at Thr149 (37 Å distal to the GatB kinase active site) and Lys89 (located at the interface of GatA and GatB) were detrimental to AdT's kinase activity, suggesting that these mutations have disrupted interdomain communication between the two active sites. Models of wild-type AdT, a valine mutation at Thr149, and an arginine mutation at Lys89 were subjected to molecular dynamics simulations. A comparison of wild-type, T149V, and K89R AdT simulation results unmasks 59 common residues that are likely involved in connecting the two active sites.


Assuntos
Amônia/química , Aspartato-tRNA Ligase/química , Glutamina/deficiência , Helicobacter pylori/enzimologia , Mutagênese Sítio-Dirigida , Transferases de Grupos Nitrogenados/química , Aminoacil-RNA de Transferência/química , Asparagina/genética , Aspartato-tRNA Ligase/biossíntese , Aspartato-tRNA Ligase/genética , Ativação Enzimática/genética , Glutamina/biossíntese , Helicobacter pylori/genética , Lisina/genética , Simulação de Dinâmica Molecular , Transferases de Grupos Nitrogenados/biossíntese , Transferases de Grupos Nitrogenados/genética , Fosforilação/genética , Aminoacil-RNA de Transferência/biossíntese , Aminoacil-RNA de Transferência/genética , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Tirosina/genética
16.
Nucleic Acids Res ; 39(21): 9306-15, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21813455

RESUMO

In many bacteria and archaea, an ancestral pathway is used where asparagine and glutamine are formed from their acidic precursors while covalently linked to tRNA(Asn) and tRNA(Gln), respectively. Stable complexes formed by the enzymes of these indirect tRNA aminoacylation pathways are found in several thermophilic organisms, and are called transamidosomes. We describe here a transamidosome forming Gln-tRNA(Gln) in Helicobacter pylori, an ε-proteobacterium pathogenic for humans; this transamidosome displays novel properties that may be characteristic of mesophilic organisms. This ternary complex containing the non-canonical GluRS2 specific for Glu-tRNA(Gln) formation, the tRNA-dependent amidotransferase GatCAB and tRNA(Gln) was characterized by dynamic light scattering. Moreover, we observed by interferometry a weak interaction between GluRS2 and GatCAB (K(D) = 40 ± 5 µM). The kinetics of Glu-tRNA(Gln) and Gln-tRNA(Gln) formation indicate that conformational shifts inside the transamidosome allow the tRNA(Gln) acceptor stem to interact alternately with GluRS2 and GatCAB despite their common identity elements. The integrity of this dynamic transamidosome depends on a critical concentration of tRNA(Gln), above which it dissociates into separate GatCAB/tRNA(Gln) and GluRS2/tRNA(Gln) complexes. Ester bond protection assays show that both enzymes display a good affinity for tRNA(Gln) regardless of its aminoacylation state, and support a mechanism where GluRS2 can hydrolyze excess Glu-tRNA(Gln), ensuring faithful decoding of Gln codons.


Assuntos
Glutamato-tRNA Ligase/metabolismo , Helicobacter pylori/enzimologia , Transferases de Grupos Nitrogenados/metabolismo , Aminoacil-RNA de Transferência/metabolismo , RNA de Transferência de Glutamina/metabolismo , Helicobacter pylori/genética , Hidrólise , Interferometria , Cinética , Modelos Biológicos , Estabilidade de RNA
17.
Org Lett ; 12(9): 2080-3, 2010 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-20380381

RESUMO

Many eukaryotic proteins are modified with a glycosylphosphatidylinositol (GPI) anchor at their C-termini. This post-translational modification causes these proteins to be noncovalently tethered to the plasma membrane. The synthesis of truncated GPI anchor analogues is reported; these compounds were designed for use as soluble substrates for GPI transamidase (GPI-T), the enzyme that appends the GPI anchor onto proteins.


Assuntos
Glicosilfosfatidilinositóis/química , Fosfatidilinositóis/química , Configuração de Carboidratos , Sequência de Carboidratos , Dados de Sequência Molecular
19.
Nucleic Acids Res ; 37(20): 6942-9, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19755501

RESUMO

Accurate aminoacylation of tRNAs by the aminoacyl-tRNA synthetases (aaRSs) plays a critical role in protein translation. However, some of the aaRSs are missing in many microorganisms. Helicobacter pylori does not have a glutaminyl-tRNA synthetase (GlnRS) but has two divergent glutamyl-tRNA synthetases: GluRS1 and GluRS2. Like a canonical GluRS, GluRS1 aminoacylates tRNA(Glu1) and tRNA(Glu2). In contrast, GluRS2 only misacylates tRNA(Gln) to form Glu-tRNA(Gln). It is not clear how GluRS2 achieves specific recognition of tRNA(Gln) while rejecting the two H. pylori tRNA(Glu) isoacceptors. Here, we show that GluRS2 recognizes major identity elements clustered in the tRNA(Gln) acceptor stem. Mutations in the tRNA anticodon or at the discriminator base had little to no impact on enzyme specificity and activity.


Assuntos
Glutamato-tRNA Ligase/metabolismo , Helicobacter pylori/enzimologia , RNA de Transferência de Glutamina/metabolismo , Anticódon , Mutagênese , RNA de Transferência de Glutamina/química , RNA de Transferência de Glutamina/genética , Especificidade por Substrato
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