RESUMEN
AIMS: The objective of the study was to produce and characterize the cinnamoyl esterase EstA from the anaerobic fungus Piromyces equi for potential industrial applications. METHODS AND RESULTS: The catalytic domain EstA was produced in Trichoderma reesei. Because the two fungi displayed different genome features, including different codon usage and GC content, a synthetic gene was designed and expressed, leading to the production of the corresponding protein at around 33 mg per litre in the T. reesei culture medium. After the recombinant protein was purified, biochemical characterization showed that EstA presents peak activity at pH 6.5 and at 50-60 degrees C. Furthermore, EstA remained stable at pH 6-8 and below 50 degrees C. EstA was compared to cinnamoyl esterases FaeA and FaeB from Aspergillus niger in terms of ferulic acid (FA) release from wheat bran (WB), maize bran (MB) and sugar beet pulp (SBP). CONCLUSION: The synthetic gene was successfully cloned and overexpressed in T. reesei. EstA from P. equi was demonstrated to efficiently release FA from various natural substrates. SIGNIFICANCE AND IMPACT OF THE STUDY: Recombinant EstA produced in an industrial enzyme producer, T. reesei, was biochemically characterized, and its capacity to release an aromatic compound (FA) for biotechnological applications was demonstrated.
Asunto(s)
Hidrolasas de Éster Carboxílico/metabolismo , Proteínas Fúngicas/metabolismo , Microbiología Industrial , Piromyces/enzimología , Trichoderma/metabolismo , Aspergillus niger/enzimología , Hidrolasas de Éster Carboxílico/genética , Clonación Molecular , Ácidos Cumáricos/metabolismo , Proteínas Fúngicas/genética , Concentración de Iones de Hidrógeno , Piromyces/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Temperatura , Trichoderma/genéticaRESUMEN
An innovative "biodrug" concept, based on the oral administration of living recombinant microorganisms, has recently emerged for the prevention or treatment of various diseases. An engineered Saccharomyces cerevisiae strain expressing plant P450 73A1 (cinnamate-4-hydroxylase [CA4H] activity) was used, and its survival and ability to convert trans-cinnamic acid (CIN) into p-coumaric acid (COU) were investigated in vivo. In rats, the recombinant yeast was resistant to gastric and small intestinal secretions but was more sensitive to the conditions found in the large intestine. After oral administration of yeast and CIN, the CA4H activity was shown in vivo, with COU being found throughout the rat's digestive tract and in its urine. The bioconversion reaction occurred very fast, with most of the COU being produced within the first 5 min. The gastrointestinal sac technique demonstrated that the recombinant yeast was able to convert CIN into COU (conversion rate ranging from 2 to 5%) in all the organs of the rat's digestive tract: stomach, duodenum, jejunum, ileum, cecum, and colon. These results promise new opportunities for the development of drug delivery systems based on engineered yeasts catalyzing a bioconversion reaction directly in the digestive tract.
Asunto(s)
Tracto Gastrointestinal/microbiología , Expresión Génica , Probióticos , Saccharomyces cerevisiae/genética , Transcinamato 4-Monooxigenasa/biosíntesis , Animales , Biotransformación , Cinamatos/metabolismo , Ácidos Cumáricos/metabolismo , Tracto Gastrointestinal/química , Helianthus/genética , Técnicas In Vitro , Masculino , Viabilidad Microbiana , Modelos Animales , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Propionatos , Ratas , Ratas Wistar , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Transcinamato 4-Monooxigenasa/genética , Orina/químicaRESUMEN
AIMS: A molecular tool for extensive detection of prokaryotic alkane hydroxylase genes (alkB) was developed. AlkB genotypes involved in the degradation of short-chain alkanes were quantified in environmental samples in order to assess their occurrence and ecological importance. METHODS AND RESULTS: Four primer pairs specific for distinct clusters of alkane hydroxylase genes were designed, allowing amplification of alkB-related genes from all tested alkane-degrading strains and from six of seven microcosms. For the primer pair detecting alkB genes related to the Pseudomonas putida GPo1 alkB gene and the one targeting alkB genes of Gram-positive strains, both involved in short-chain alkane degradation (Asunto(s)
Citocromo P-450 CYP4A/genética
, Ecología
, Microbiología Ambiental
, Contaminación Ambiental
, Alcanos/metabolismo
, Animales
, Biodegradación Ambiental
, Citocromo P-450 CYP4A/metabolismo
, Cartilla de ADN/genética
, Monitoreo del Ambiente/métodos
, Agua Dulce
, Genotipo
, Microscopía Fluorescente
, Reacción en Cadena de la Polimerasa/métodos
, Microbiología del Suelo
, Microbiología del Agua
RESUMEN
The use of genetically engineered microorganisms such as bacteria or yeasts as live vehicles to carry out bioconversion directly in the digestive environment is an important challenge for the development of innovative biodrugs. A system that mimics the human gastrointestinal tract was combined with a computer simulation to evaluate the survival rate and cinnamate 4-hydroxylase activity of a recombinant model of Saccharomyces cerevisiae expressing the plant P450 73A1. The yeasts showed a high level of resistance to gastric and small intestinal secretions (survival rate after 4 h of digestion, 95.6% +/- 10.1% [n = 4]) but were more sensitive to the colonic conditions (survival rate after 4 h of incubation, 35.9% +/- 2.7% [n = 3]). For the first time, the ability of recombinant S. cerevisiae to carry out a bioconversion reaction has been demonstrated throughout the gastrointestinal tract. In the gastric-small intestinal system, 41.0% +/- 5.8% (n = 3) of the ingested trans-cinnamic acid was converted into p-coumaric acid after 4 h of digestion, as well as 8.9% +/- 1.6% (n = 3) in the stomach, 13.8% +/- 3.3% (n = 3) in the duodenum, 11.8% +/- 3.4% (n = 3) in the jejunum, and 6.5% +/- 1.0% (n = 3) in the ileum. In the large intestinal system, cinnamate 4-hydroxylase activity was detected but was too weak to be quantified. These results suggest that S. cerevisiae may afford a useful host for the development of biodrugs and may provide an innovative system for the prevention or treatment of diseases that escape classical drug action. In particular, yeasts may provide a suitable vector for biodetoxication in the digestive environment.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Digestivo/metabolismo , Sistema Digestivo/microbiología , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Modelos Biológicos , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Simulación por Computador , Ingeniería Genética , Helianthus/enzimología , Helianthus/genética , Humanos , Técnicas In Vitro , Inactivación Metabólica , Transcinamato 4-MonooxigenasaRESUMEN
Cell engineering technology using recombinant microorganisms has created new opportunities in the development of innovative drugs. This article presents the use of living genetically engineered microorganisms, such as bacteria or yeasts, as a new delivery vehicle to the gastrointestinal tract. This 'biodrug' concept was demonstrated using recombinant Saccharomyces cerevisiae expressing the plant cytochrome P450 73A1. This enzyme provides a relevant model for potential therapeutic applications, such as 'biodetoxication' in the digestive environment. An artificial gastrointestinal tract simulating human digestion was chosen as a powerful tool to validate the biodrug concept. This approach offers a novel strategy for drug discovery and testing.
Asunto(s)
Terapia Biológica/tendencias , Terapia Genética/tendencias , Órganos Artificiales , Sistema Digestivo , Humanos , Lactobacillaceae , Saccharomyces cerevisiaeRESUMEN
Cell growth inhibition by several d-amino acids can be explained by an in vivo production of d-aminoacyl-tRNA molecules. Escherichia coli and yeast cells express an enzyme, d-Tyr-tRNA(Tyr) deacylase, capable of recycling such d-aminoacyl-tRNA molecules into free tRNA and d-amino acid. Accordingly, upon inactivation of the genes of the above deacylases, the toxicity of d-amino acids increases. Orthologs of the deacylase are found in many cells. In this study, the crystallographic structure of dimeric E. coli d-Tyr-tRNA(Tyr) deacylase at 1.55 A resolution is reported. The structure corresponds to a beta-barrel closed on one side by a beta-sheet lid. This barrel results from the assembly of the two subunits. Analysis of the structure in relation with sequence homologies in the orthologous family suggests the location of the active sites at the carboxy end of the beta-strands. The solved structure markedly differs from those of all other documented tRNA-dependent hydrolases.
Asunto(s)
Aminoaciltransferasas/química , Aminoaciltransferasas/clasificación , Secuencia de Aminoácidos , Sitios de Unión , División Celular , Cristalografía por Rayos X , Dimerización , Escherichia coli/enzimología , Iones , Ligandos , Modelos Biológicos , Modelos Moleculares , Modelos Estadísticos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , ARN de Transferencia/metabolismo , Espectrofotometría Atómica , Zinc/químicaRESUMEN
D-cysteine, a powerful inhibitor of Escherichia coli growth, is decomposed in vitro into pyruvate, H2S, and NH3 by D-cysteine desulfhydrase. To assess the role of this reaction in the adaptation of the bacterium to growth on D-cysteine, the gene of the desulfhydrase was cloned. It corresponds to the open reading frame yedO at 43.03 min on the genetic map of E. coli. The amino acid sequence deduced from this gene is homologous to those of several 1-aminocyclopropane-carboxylate deaminases. However, the E. coli desulfhydrase does not use 1-aminocyclopropane-1-carboxylate as substrate. Various mutants in which the yedO gene was inactivated or overexpressed were constructed. They exhibited hypersensitivity or resistance, respectively, to the presence of d-cysteine in the culture medium. Growth protection against D-cysteine in minimal medium was conferred by the simultaneous addition of isoleucine, leucine, and valine. In agreement with this behavior, D-cysteine inhibited the activity of threonine deaminase, a key enzyme of the isoleucine, leucine, and valine pathway. Finally, in the presence of the intact yedO gene, E. coli growth was improved by addition of D-cysteine as the sole sulfur source. In agreement with a role of the desulfhydrase in sulfur metabolism, yedO expression was induced under conditions of sulfate limitation.
Asunto(s)
Adaptación Fisiológica , Cistationina gamma-Liasa/metabolismo , Cisteína/toxicidad , Escherichia coli/efectos de los fármacos , Secuencia de Bases , Cromosomas Bacterianos , Cistationina gamma-Liasa/química , Cistationina gamma-Liasa/genética , Cisteína/metabolismo , Cartilla de ADN , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/fisiología , Genes Bacterianos , Filogenia , Especificidad por SustratoRESUMEN
Protein synthesis involves two methionine-isoaccepting tRNAs, an initiator and an elongator. In eubacteria, mitochondria, and chloroplasts, the addition of a formyl group gives its full functional identity to initiator Met-tRNA(Met). In Escherichia coli, it has been shown that the specific action of methionyl-tRNA transformylase on Met-tRNA(f)(Met) mainly involves a set of nucleotides in the acceptor stem, particularly a C(1)A(72) mismatch. In animal mitochondria, only one tRNA(Met) species has yet been described. It is admitted that this species can engage itself either in initiation or elongation of translation, depending on the presence or absence of a formyl group. In the present study, we searched for the identity elements of tRNA(Met) that govern its formylation by bovine mitochondrial transformylase. The main conclusion is that the mitochondrial formylase preferentially recognizes the methionyl moiety of its tRNA substrate. Moreover, the relatively small importance of the tRNA acceptor stem in the recognition process accounts for the protection against formylation of the mitochondrial tRNAs that share with tRNA(Met) an A(1)U(72) motif.
Asunto(s)
Transferasas de Hidroximetilo y Formilo/metabolismo , Mitocondrias/enzimología , ARN de Transferencia/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Catálisis , Bovinos , Hidrólisis , Transferasas de Hidroximetilo y Formilo/química , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica , ARN de Transferencia/química , Homología de Secuencia de AminoácidoRESUMEN
Lysyl-tRNA synthesis is catalyzed by two unrelated families of aminoacyl-tRNA synthetases. In most bacteria and all eukarya, the known lysyl-tRNA synthetases (LysRSs) are subclass IIb-type aminoacyl-tRNA synthetases, whereas many archaea and a scattering of bacteria contain an unrelated class I-type LysRS. Examination of the recognition of partially modified tRNA(Lys) anticodon variants by a bacterial (from Borrelia burgdorferi) and an archaeal (from Methanococcus maripaludis) class I lysyl-tRNA synthetase revealed differences in the pattern of anticodon recognition between the two enzymes. U35 and U36 were both important for recognition by the B. burgdorferi enzyme, whereas only U36 played a role in recognition by M. maripaludis LysRS. Examination of the phylogenetic distribution of class I LysRSs suggested a correlation between recognition of U35 and U36 and the presence of asparaginyl-tRNA synthetase (AsnRS), which also recognizes U35 and U36 in the anticodon of tRNA(Asn). However, the class II LysRS of Helicobacter pylori, an organism that lacks AsnRS, also recognizes both U35 and U36, indicating that the presence of AsnRS has solely influenced the phylogenetic distribution of class I LysRSs. These data suggest that competition between unrelated aminoacyl-tRNA synthetases for overlapping anticodon sequences is a determinant of the phylogenetic distribution of extant synthetase families. Such patterns of competition also provide a basis for the two separate horizontal gene transfer events hypothesized in the evolution of the class I lysyl-tRNA synthetases.
Asunto(s)
Anticodón/metabolismo , Lisina-ARNt Ligasa/metabolismo , Grupo Borrelia Burgdorferi/enzimología , Helicobacter pylori/enzimología , Lisina-ARNt Ligasa/clasificación , Conformación de Ácido Nucleico , Filogenia , ARN de Transferencia de Lisina/química , ARN de Transferencia de Lisina/metabolismoRESUMEN
Lysyl-tRNA synthetase is a member of the class II aminoacyl-tRNA synthetases and catalyses the specific aminoacylation of tRNA(Lys). The crystal structure of the constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli has been determined to 2.7 A resolution in the unliganded form and in a complex with the lysine substrate. A comparison between the unliganded and lysine-bound structures reveals major conformational changes upon lysine binding. The lysine substrate is involved in a network of hydrogen bonds. Two of these interactions, one between the alpha-amino group and the carbonyl oxygen of Gly 216 and the other between the carboxylate group and the side chain of Arg 262, trigger a subtle and complicated reorganization of the active site, involving the ordering of two loops (residues 215-217 and 444-455), a change in conformation of residues 393-409, and a rotation of a 4-helix bundle domain (located between motif 2 and 3) by 10 degrees. The result of these changes is a closing up of the active site upon lysine binding.
Asunto(s)
Lisina-ARNt Ligasa/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Escherichia coli/enzimología , Isoenzimas/química , Isoenzimas/metabolismo , Lisina/química , Lisina/metabolismo , Lisina-ARNt Ligasa/metabolismo , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Especificidad por SustratoRESUMEN
In Escherichia coli, tyrosyl-tRNA synthetase is known to esterify tRNA(Tyr) with tyrosine. Resulting d-Tyr-tRNA(Tyr) can be hydrolyzed by a d-Tyr-tRNA(Tyr) deacylase. By monitoring E. coli growth in liquid medium, we systematically searched for other d-amino acids, the toxicity of which might be exacerbated by the inactivation of the gene encoding d-Tyr-tRNA(Tyr) deacylase. In addition to the already documented case of d-tyrosine, positive responses were obtained with d-tryptophan, d-aspartate, d-serine, and d-glutamine. In agreement with this observation, production of d-Asp-tRNA(Asp) and d-Trp-tRNA(Trp) by aspartyl-tRNA synthetase and tryptophanyl-tRNA synthetase, respectively, was established in vitro. Furthermore, the two d-aminoacylated tRNAs behaved as substrates of purified E. coli d-Tyr-tRNA(Tyr) deacylase. These results indicate that an unexpected high number of d-amino acids can impair the bacterium growth through the accumulation of d-aminoacyl-tRNA molecules and that d-Tyr-tRNA(Tyr) deacylase has a specificity broad enough to recycle any of these molecules. The same strategy of screening was applied using Saccharomyces cerevisiae, the tyrosyl-tRNA synthetase of which also produces d-Tyr-tRNA(Tyr), and which, like E. coli, possesses a d-Tyr-tRNA(Tyr) deacylase activity. In this case, inhibition of growth by the various 19 d-amino acids was followed on solid medium. Two isogenic strains containing or not the deacylase were compared. Toxic effects of d-tyrosine and d-leucine were reinforced upon deprivation of the deacylase. This observation suggests that, in yeast, at least two d-amino acids succeed in being transferred onto tRNAs and that, like in E. coli, the resulting two d-aminoacyl-tRNAs are substrates of a same d-aminoacyl-tRNA deacylase.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Escherichia coli/genética , ARN de Transferencia Aminoácido-Específico/metabolismo , ARN de Transferencia de Aspártico/metabolismo , Saccharomyces cerevisiae/metabolismo , Triptófano-ARNt Ligasa/metabolismo , Triptófano/metabolismo , Tirosina-ARNt Ligasa/metabolismo , Aspartato-ARNt Ligasa/metabolismo , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Genotipo , Plásmidos , Saccharomyces cerevisiae/genética , Estereoisomerismo , Especificidad por SustratoRESUMEN
Bromomethyl ketone derivatives of L-valine (VBMK), L-isoleucine (IBMK), L-norleucine (NleBMK) and L-phenylalanine (FBMK) were synthesized. These reagents were used for qualitative comparative labeling of Escherichia coli valyl-tRNA synthetase (ValRS), an enzyme with Val/Ile editing activity, in order to identify the binding sites for L-valine or noncognate amino acids. Labeling of E. coli ValRS with the substrate analog valyl-bromomethyl ketone (VBMK) resulted in a complete loss of valine-dependent isotopic [32P]PPi-ATP exchange activity. L-Valine protected the enzyme against inactivation. Noncognate amino acids analogs isoleucyl-, norleucyl- and phenylalanyl-bromomethyl ketones (IBMK, NleBMK and FBMK) were also capable of abolishing the activity of ValRS, FBMK being less efficient in inactivating the synthetase. Matrix-assisted laser desorption-ionization mass spectrometry designated cysteines 424 and 829 as the target residues of the substrate analog VBMK on E. coli ValRS, whereas, altogether, IBMK, NleBMK and FBMK labeled His266, Cys275, His282, His433 and Cys829, of which Cys275, His282 and His433 were labeled in common by all three noncognate amino-acid-derived bromomethyl ketones. With the exception of Cys829, which was most likely unspecifically labeled, the amino-acid residues labeled by the reagents derived from noncognate amino acids were distributed between two fragments 259-291 and 419-434 in the primary structure of E. coli ValRS. In fragment 419-434, Cys424 was specifically labeled by the substrate analog VBMK, while His433 was labeled in common by all the used bromomethyl ketone derivatives of noncognate amino acids, suggesting that the synthetic site where aminoacyl adenylate formation takes place on E. coli ValRS is built up of two subsites. One subsite containing Cys424 might represent the catalytic locus of the active center where specific L-valine activation takes place. The second subsite containing His433 might represent the binding site for noncognate amino acids. The fact that Cys275 and His282, fragment 259-291, were labeled by IBMK, NleBMK and FBMK, but not by the substrate analog VBMK, suggests that these residues might be located at or near the editing site of E. coli ValRS. Comparison of fragment 259-291 with all the available ValRS amino-acid sequences revealed that His282 is strictly conserved, with the exception of its replacement by a glycine in a subgroup corresponding to the archaebacteria. Because a nucleophile is needed in the editing site to achieve hydrolysis of an undesired product at the level of the carbonyl group thereof, it is proposed that the conserved His282 of E. coli ValRS is involved in editing.
Asunto(s)
Escherichia coli/enzimología , Valina-ARNt Ligasa/química , Valina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dietil Pirocarbonato/química , Escherichia coli/química , Escherichia coli/metabolismo , Etilmaleimida/química , Isoleucina/metabolismo , Cetonas/metabolismo , Cinética , Datos de Secuencia Molecular , Norleucina/metabolismo , Péptidos/química , Fenilalanina/metabolismo , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Thermus thermophilus/química , Thermus thermophilus/metabolismo , Factores de TiempoRESUMEN
Among elongator tRNAs, tRNA specific for histidine has the peculiarity to possess one extra nucleotide at position -1. This nucleotide is believed to be responsible for recognition by histidyl-tRNA synthetase. Here, we show that, in fact, it is the phosphate 5' to the extra nucleotide which mainly supports the efficiency of the tRNA aminoacylation reaction catalyzed by Escherichia coli histidyl-tRNA synthetase. In the case of the reaction of E. coli peptidyl-tRNA hydrolase, this atypical phosphate is dispensable. Instead, peptidyl-tRNA hydrolase recognizes the phosphate of the phosphodiester bond between residues -1 and +1 of tRNA(His). Recognition of the +1 phosphate of tRNA(His) by peptidyl-tRNA hydrolase resembles, therefore, that of the 5'-terminal phosphate of other elongator tRNAs.
Asunto(s)
Histidina-ARNt Ligasa/química , ARN de Transferencia de Histidina/química , Animales , Sitios de Unión , Escherichia coli , Histidina-ARNt Ligasa/metabolismo , Fosfatos , ARN de Transferencia de Histidina/genética , ARN de Transferencia de Histidina/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The Saccharomyces cerevisiae YDL219w (DTD1) gene, which codes for an amino acid sequence sharing 34% identity with the Escherichia coli D-Tyr-tRNA(Tyr) deacylase, was cloned, and its product was functionally characterized. Overexpression in the yeast of the DTD1 gene from a multicopy plasmid increased D-Tyr-tRNA(Tyr) deacylase activity in crude extracts by two orders of magnitude. Upon disruption of the chromosomal gene, deacylase activity was decreased by more than 90%, and the sensitivity to D-tyrosine of the growth of S. cerevisiae was exacerbated. The toxicity of D-tyrosine was also enhanced under conditions of nitrogen starvation, which stimulate the uptake of D-amino acids. In relation with these behaviors, the capacity of purified S. cerevisiae tyrosyl-tRNA synthetase to produce D-Tyr-tRNA(Tyr) could be shown. Finally, the phylogenetic distribution of genes homologous to DTD1 was examined in connection with L-tyrosine prototrophy or auxotrophy. In the auxotrophs, DTD1-like genes are systematically absent. In the prototrophs, the putative occurrence of a deacylase is variable. It possibly depends on the L-tyrosine anabolic pathway adopted by the cell.
Asunto(s)
ARN de Transferencia de Tirosina/genética , ARN de Transferencia de Tirosina/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Biblioteca de Genes , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Programas Informáticos , Tirosina/metabolismo , Tirosina-ARNt Ligasa/metabolismoRESUMEN
Subsequent to their aminoacylation, tRNAs are subject to specific maturation and/or correction processes. Aminoacylated tRNAs ready for use in translation are then specifically channelled to the ribosomal A or P sites. Structural and biochemical studies have opened the way towards furthering our understanding of these routes to the ribosome, which involve a strict distinction between initiator and elongator tRNAs.
Asunto(s)
Procesamiento Postranscripcional del ARN , ARN Bacteriano/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/metabolismo , Proteínas Bacterianas/metabolismo , Ésteres/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Factores de Elongación de Péptidos/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Biosíntesis de Proteínas , Conformación Proteica , Ribosomas/metabolismoRESUMEN
Methionyl-tRNA synthetase (MetRS) from Bacillus stearothermophilus was shown to undergo covalent methionylation by a donor methionyl-adenylate, the mixed carboxylic-phosphoric acid anhydride synthesized by the enzyme itself. Covalent reaction of methionyl-adenylate with the synthetase or other proteins proceeds through the formation of an isopeptide bond between the carboxylate of the amino acid and the epsilon-NH2 group of lysyl residues. The stoichiometries of labeling, as followed by TCA precipitation, were 2.2 +/- 0.1 and 4.3 +/- 0.1 mol of [14C]Met incorporated by 1 mol of the monomeric MS534 and the native dimeric species of B. stearo methionyl-tRNA synthetase, respectively. Matrix-assisted laser desorption-ionization mass spectrometry designated lysines-261, -295, -301 and -528 (or -534) of truncated methionyl-tRNA synthetase as the target residues for covalent binding of methionine. By analogy with the 3D structure of the monomeric M547 species of E. coli methionyl-tRNA synthetase, lysines-261, -295, and -301 would be located in the catalytic crevice of the thermostable enzyme where methionine activation and transfer take place. It is proposed that, once activated by ATP, most of the methionine molecules react with the closest reactive lysyl residues.
Asunto(s)
Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/metabolismo , Geobacillus stearothermophilus/enzimología , Metionina-ARNt Ligasa/metabolismo , Metionina/análogos & derivados , Metionina/metabolismo , Adenosina Monofosfato/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Radioisótopos de Carbono , Catálisis , Dominio Catalítico , Dimerización , Escherichia coli/enzimología , Cinética , Lisina/metabolismo , Metionina/química , Metionina-ARNt Ligasa/química , Datos de Secuencia Molecular , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The 3D structure of monomeric C-truncated Escherichia coli methionyl-tRNA synthetase, a class 1 aminoacyl-tRNA synthetase, has been solved at 2.0 A resolution. Remarkably, the polypeptide connecting the two halves of the Rossmann fold exposes two identical knuckles related by a 2-fold axis but with zinc in the distal knuckle only. Examination of available MetRS orthologs reveals four classes according to the number and zinc content of the putative knuckles. Extreme cases are exemplified by the MetRS of eucaryotic or archaeal origin, where two knuckles and two metal ions are expected, and by the mitochondrial enzymes, which are predicted to have one knuckle without metal ion.
Asunto(s)
Escherichia coli/enzimología , Metionina-ARNt Ligasa/química , Metionina-ARNt Ligasa/clasificación , Secuencia de Aminoácidos , Animales , Anticodón/metabolismo , Sitios de Unión , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Alineación de Secuencia , Electricidad Estática , Zinc/metabolismoRESUMEN
Translation initiation factor IF3, one of three factors specifically required for translation initiation in Escherichia coli, inhibits initiation on any codon other than the three canonical initiation codons, AUG, GUG, or UUG. This discrimination against initiation on non-canonical codons could be due to either direct recognition of the two last bases of the codon and their cognate bases on the anticodon or to some ability to "feel" codon-anticodon complementarity. To investigate the importance of codon-anticodon complementarity in the discriminatory role of IF3, we constructed a derivative of tRNALeuthat has all the known characteristics of an initiator tRNA except the CAU anticodon. This tRNA is efficiently formylated by methionyl-tRNAfMettransformylase and charged by leucyl-tRNA synthetase irrespective of the sequence of its anticodon. These initiator tRNALeuderivatives (called tRNALI) allow initiation at all the non-canonical codons tested, provided that the complementarity between the codon and the anticodon of the initiator tRNALeuis respected. More remarkably, the discrimination by IF3, normally observed with non-canonical codons, is neutralised if a tRNALIcarrying a complementary anticodon is used for initiation. This suggests that IF3 somehow recognises codon-anticodon complementarity, at least at the second and third position of the codon, rather than some specific bases in either the codon or the anticodon.
Asunto(s)
Codón Iniciador , Escherichia coli/genética , Iniciación de la Cadena Peptídica Traduccional , Factores de Iniciación de Péptidos/genética , Anticodón , Secuencia de Bases , Factor 3 de Iniciación Eucariótica , Regulación Bacteriana de la Expresión Génica , Genotipo , Cinética , Modelos Genéticos , Datos de Secuencia Molecular , ARN de Transferencia de Leucina/genética , ARN de Transferencia de Metionina/genéticaRESUMEN
The yihZ gene of Escherichia coli is shown to produce a deacylase activity capable of recycling misaminoacylated D-Tyr-tRNATyr. The reaction is specific and, under optimal in vitro conditions, proceeds at a rate of 6 s-1 with a Km value for the substrate equal to 1 microM. Cell growth is sensitive to interruption of the yihZ gene if D-tyrosine is added to minimal culture medium. Toxicity of exogenous D-tyrosine is exacerbated if, in addition to the disruption of yihZ, the gene of D-amino acid dehydrogenase (dadA) is also inactivated. Orthologs of the yihZ gene occur in many, but not all, bacteria. In support of the idea of a general role of the D-Tyr-tRNATyr deacylase function in the detoxification of cells, similar genes can be recognized in Saccharomyces cerevisiae, Caenorhabditis elegans, Arabidopsis thaliana, mouse, and man.
Asunto(s)
Aminoaciltransferasas/metabolismo , Escherichia coli/enzimología , ARN de Transferencia de Tirosina/metabolismo , Aminoaciltransferasas/genética , Animales , Catálisis , Clonación Molecular , ADN Bacteriano/química , Escherichia coli/genética , Biblioteca de Genes , Humanos , Masculino , Ratones , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , Mapeo RestrictivoRESUMEN
Series of substrates derivatives of peptide deformylase were systematically synthesized and studied for their capacities to undergo hydrolysis. Data analysis indicated the requirement for a hydrophobic first side chain and for at least two main chain carbonyl groups in the substrate. For instance, Fo-Met-OCH3 and Fo-Nle-OCH3 were the minimal substrates of peptide deformylase obtained in this study, while positively charged Fo-Nle-ArgNH2 was the most efficient substrate (kcat/Km = 4.5 x 10(5) M-1.s-1). On the basis of this knowledge, 3-mercapto-2-benzylpropanoylglycine (thiorphan), a known inhibitor of thermolysin, could be predicted and further shown to inhibit the deformylation reaction. The inhibition by this compound was competitive and proved to depend on the hydrophobicity at the P1' position. Spectroscopic evidence that the sulfur group of thiorphan binds next to the active site metal ion on the enzyme could be obtained. Consequently, a small thiopseudopeptide derived from Fo-Nle-OCH3 was designed and synthesized. This compound behaved as a competitive inhibitor of peptide deformylase with KI = 52 +/- 5 microM. Introduction of a positive charge to this thiopeptide via addition of an arginine at P2' improved the inhibition constant up to 2.5 +/- 0.5 microM, a value 4 orders of magnitude smaller than that of the starting inhibitors. Evidence that this inhibitor, imino[(5-methoxy-5-oxo-4-[[2-(sulfanylmethyl)hexanoyl]amino]pentyl )am ino]methanamine, binds inside the active site cavity of peptide deformylase, while keeping intact the 3D fold of the protein, was provided by NMR. A fingerprint of the interaction of the inhibitor with the residues of the enzyme was obtained.