RESUMEN
Riboswitches are structured non-coding RNAs often located upstream of essential genes in bacterial messenger RNAs. Such RNAs regulate expression of downstream genes by recognizing a specific cellular effector. Although nearly 50 riboswitch classes are known, only a handful recognize multiple effectors. Here, we report the 2.60-Å resolution co-crystal structure of a class I type I preQ1-sensing riboswitch that reveals two effectors stacked atop one another in a single binding pocket. These effectors bind with positive cooperativity in vitro and both molecules are necessary for gene regulation in bacterial cells. Stacked effector recognition appears to be a hallmark of the largest subgroup of preQ1 riboswitches, including those from pathogens such as Neisseria gonorrhoeae. We postulate that binding to stacked effectors arose in the RNA World to closely position two substrates for RNA-mediated catalysis. These findings expand known effector recognition capabilities of riboswitches and have implications for antimicrobial development.
Asunto(s)
Neisseria gonorrhoeae/genética , Nucleósido Q/química , Pirimidinonas/química , Pirroles/química , ARN Bacteriano/química , ARN Mensajero/química , Riboswitch , Emparejamiento Base , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Neisseria gonorrhoeae/metabolismo , Conformación de Ácido Nucleico , Nucleósido Q/biosíntesis , Pirimidinonas/metabolismo , Pirroles/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Mitochondria generate most cellular energy via oxidative phosphorylation. Twenty-two species of mitochondrial (mt-)tRNAs encoded in mtDNA translate essential subunits of the respiratory chain complexes. mt-tRNAs contain post-transcriptional modifications introduced by nuclear-encoded tRNA-modifying enzymes. They are required for deciphering genetic code accurately, as well as stabilizing tRNA. Loss of tRNA modifications frequently results in severe pathological consequences. Here, we perform a comprehensive analysis of post-transcriptional modifications of all human mt-tRNAs, including 14 previously-uncharacterized species. In total, we find 18 kinds of RNA modifications at 137 positions (8.7% in 1575 nucleobases) in 22 species of human mt-tRNAs. An up-to-date list of 34 genes responsible for mt-tRNA modifications are provided. We identify two genes required for queuosine (Q) formation in mt-tRNAs. Our results provide insight into the molecular mechanisms underlying the decoding system and could help to elucidate the molecular pathogenesis of human mitochondrial diseases caused by aberrant tRNA modifications.
Asunto(s)
Procesamiento Postranscripcional del ARN , ARN Mitocondrial/química , ARN de Transferencia/química , Femenino , Código Genético , Células HEK293 , Células HeLa , Humanos , Espectrometría de Masas , Mitocondrias/metabolismo , Mitocondrias/patología , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/patología , Estructura Molecular , Nucleósido Q/biosíntesis , Nucleósido Q/química , Fosforilación Oxidativa , Placenta , Embarazo , ARN Mitocondrial/aislamiento & purificación , ARN Mitocondrial/metabolismo , ARN de Transferencia/aislamiento & purificación , ARN de Transferencia/metabolismo , RNA-SeqRESUMEN
Covering: up to the end of 2017 The human body is composed of an equal number of human and microbial cells. While the microbial community inhabiting the human gastrointestinal tract plays an essential role in host health, these organisms have also been connected to various diseases. Yet, the gut microbial functions that modulate host biology are not well established. In this review, we describe metabolic functions of the human gut microbiota that involve metalloenzymes. These activities enable gut microbial colonization, mediate interactions with the host, and impact human health and disease. We highlight cases in which enzyme characterization has advanced our understanding of the gut microbiota and examples that illustrate the diverse ways in which metalloenzymes facilitate both essential and unique functions of this community. Finally, we analyze Human Microbiome Project sequencing datasets to assess the distribution of a prominent family of metalloenzymes in human-associated microbial communities, guiding future enzyme characterization efforts.
Asunto(s)
Enzimas/metabolismo , Microbioma Gastrointestinal/fisiología , Interacciones Huésped-Patógeno/fisiología , Amoníaco/metabolismo , Cresoles/metabolismo , Enzimas/química , Interacciones Huésped-Patógeno/inmunología , Humanos , Sulfuro de Hidrógeno/metabolismo , Metales/química , Metales/metabolismo , Metilaminas/metabolismo , Nucleósido Q/biosíntesis , Polisacáridos/metabolismo , Vitaminas/biosíntesis , Xenobióticos/farmacocinéticaRESUMEN
QueF enzymes catalyze the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of the nitrile group of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine (preQ1) in the biosynthetic pathway to the tRNA modified nucleoside queuosine. The QueF-catalyzed reaction includes formation of a covalent thioimide intermediate with a conserved active site cysteine that is prone to oxidation in vivo. Here, we report the crystal structure of a mutant of Bacillus subtilis QueF, which reveals an unanticipated intramolecular disulfide formed between the catalytic Cys55 and a conserved Cys99 located near the active site. This structure is more symmetric than the substrate-bound structure and exhibits major rearrangement of the loops responsible for substrate binding. Mutation of Cys99 to Ala/Ser does not compromise enzyme activity, indicating that the disulfide does not play a catalytic role. Peroxide-induced inactivation of the wild-type enzyme is reversible with thioredoxin, while such inactivation of the Cys99Ala/Ser mutants is irreversible, consistent with protection of Cys55 from irreversible oxidation by disulfide formation with Cys99. Conservation of the cysteine pair, and the reported in vivo interaction of QueF with the thioredoxin-like hydroperoxide reductase AhpC in Escherichia coli suggest that regulation by the thioredoxin disulfide-thiol exchange system may constitute a general mechanism for protection of QueF from oxidative stress in vivo.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Disulfuros/metabolismo , Nucleósido Q/biosíntesis , Proteínas Bacterianas/química , Biocatálisis , Vías Biosintéticas , Secuencia Conservada , Cristalografía por Rayos X , Cisteína/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oxidación-Reducción , Filogenia , Factores de TiempoRESUMEN
Queuosine (Q) is a complex modification of the wobble base in tRNAs with GUN anticodons. The full Q biosynthesis pathway has been elucidated in Escherichia coli. FolE, QueD, QueE and QueC are involved in the conversion of guanosine triphosphate (GTP) to 7-cyano-7-deazaguanine (preQ0), an intermediate of increasing interest for its central role in tRNA and DNA modification and secondary metabolism. QueF then reduces preQ0 to 7-aminomethyl-7-deazaguanine (preQ1). PreQ1 is inserted into tRNAs by tRNA guanine(34) transglycosylase (TGT). The inserted base preQ1 is finally matured to Q by two additional steps involving QueA and QueG or QueH. Most Eubacteria harbor the full set of Q synthesis genes and are predicted to synthesize Q de novo. However, some bacteria only encode enzymes involved in the second half of the pathway downstream of preQ0 synthesis, including the signature enzyme TGT. Different patterns of distribution of the queF, tgt, queA and queG or queH genes are observed, suggesting preQ0, preQ1 or even the queuine base being salvaged in specific organisms. Such salvage pathways require the existence of specific 7-deazapurine transporters that have yet to be identified. The COG1738 family was identified as a candidate for a missing preQ0/preQ1 transporter in prokaryotes, by comparative genomics analyses. The existence of Q precursor salvage was confirmed for the first time in bacteria, in vivo, through an indirect assay. The involvement of the COG1738 in salvage of a Q precursor was experimentally validated in Escherichia coli, where it was shown that the COG1738 family member YhhQ is essential for preQ0 transport.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Guanina/análogos & derivados , Proteínas de Transporte de Membrana/metabolismo , Transporte Biológico , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Guanina/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Nucleósido Q/biosíntesis , Nucleósido Q/química , ARN de Transferencia de AspárticoRESUMEN
Antimicrobial peptides are an important component of the molecular arsenal employed by hosts against bacteria. Many bacteria in turn possess pathways that provide protection against these compounds. In Escherichia coli and related bacteria, the PhoQ/PhoP signalling system is a key regulator of this antimicrobial peptide defence. Here we show that treating E. coli with sublethal concentrations of antimicrobial peptides causes cells to filament, and that this division block is controlled by the PhoQ/PhoP system. The filamentation results from increased expression of QueE, an enzyme that is part of a tRNA modification pathway but that, as we show here, also affects cell division. We also find that a functional YFP-QueE fusion localizes to the division septum in filamentous cells, suggesting QueE blocks septation through interaction with the divisome. Regulation of septation by PhoQ/PhoP may protect cells from antimicrobial peptide-induced stress or other conditions associated with high-level stimulation of this signalling system.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Escherichia coli/metabolismo , Transducción de Señal/efectos de los fármacos , Vías Biosintéticas/efectos de los fármacos , División Celular/efectos de los fármacos , División Celular/genética , Segregación Cromosómica/efectos de los fármacos , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/metabolismo , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Modelos Biológicos , Nucleósido Q/biosíntesis , Transcripción Genética/efectos de los fármacosRESUMEN
Queuosine (Q) is a hypermodified RNA base that replaces guanine in the wobble positions of 5'-GUN-3' tRNA molecules. Q is exclusively made by bacteria, and the corresponding queuine base is a micronutrient salvaged by eukaryotic species. The final step in Q biosynthesis is the reduction of the epoxide precursor, epoxyqueuosine, to yield the Q cyclopentene ring. The epoxyqueuosine reductase responsible, QueG, shares distant homology with the cobalamin-dependent reductive dehalogenase (RdhA), however the role played by cobalamin in QueG catalysis has remained elusive. We report the solution and structural characterization of Streptococcus thermophilus QueG, revealing the enzyme harbors a redox chain consisting of two [4Fe-4S] clusters and a cob(II)alamin in the base-off form, similar to RdhAs. In contrast to the shared redox chain architecture, the QueG active site shares little homology with RdhA, with the notable exception of a conserved Tyr that is proposed to function as a proton donor during reductive dehalogenation. Docking of an epoxyqueuosine substrate suggests the QueG active site places the substrate cyclopentane moiety in close proximity of the cobalt. Both the Tyr and a conserved Asp are implicated as proton donors to the epoxide leaving group. This suggests that, in contrast to the unusual carbon-halogen bond chemistry catalyzed by RdhAs, QueG acts via Co-C bond formation. Our study establishes the common features of Class III cobalamin-dependent enzymes, and reveals an unexpected diversity in the reductive chemistry catalyzed by these enzymes.
Asunto(s)
Nucleósido Q/análogos & derivados , Nucleósido Q/biosíntesis , Oxidorreductasas/química , ARN de Transferencia/química , Streptococcus thermophilus/enzimología , Vitamina B 12/química , Secuencia de Aminoácidos , Catálisis , Cobalto/química , Cristalografía por Rayos X , Halogenación , Datos de Secuencia Molecular , Nucleósido Q/química , Oxidación-Reducción , Oxidorreductasas/genética , Estructura Secundaria de Proteína , SolucionesRESUMEN
Queuosine is a hypermodified nucleoside present in the wobble position of tRNAs with a 5'-GUN-3' sequence in their anticodon (His, Asp, Asn, and Tyr). The 7-deazapurine core of the base is synthesized de novo in prokaryotes from guanosine 5'-triphosphate in a series of eight sequential enzymatic transformations, the final three occurring on tRNA. Epoxyqueuosine reductase (QueG) catalyzes the final step in the pathway, which entails the two-electron reduction of epoxyqueuosine to form queuosine. Biochemical analyses reveal that this enzyme requires cobalamin and two [4Fe-4S] clusters for catalysis. Spectroscopic studies show that the cobalamin appears to bind in a base-off conformation, whereby the dimethylbenzimidazole moiety of the cofactor is removed from the coordination sphere of the cobalt but not replaced by an imidazole side chain, which is a hallmark of many cobalamin-dependent enzymes. The bioinformatically identified residues are shown to have a role in modulating the primary coordination sphere of cobalamin. These studies provide the first demonstration of the cofactor requirements for QueG.
Asunto(s)
Bacillus subtilis , Proteínas Bacterianas , Proteínas Hierro-Azufre , Nucleósido Q , Oxidorreductasas , Vitamina B 12 , Bacillus subtilis/química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catálisis , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Nucleósido Q/biosíntesis , Nucleósido Q/química , Nucleósido Q/genética , Oxidorreductasas/química , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Vitamina B 12/química , Vitamina B 12/genética , Vitamina B 12/metabolismoRESUMEN
Rhizobia are symbiotic soil bacteria able to intracellularly colonize legume nodule cells and form nitrogen-fixing symbiosomes therein. How the plant cell cytoskeleton reorganizes in response to rhizobium colonization has remained poorly understood especially because of the lack of an in vitro infection assay. Here, we report on the use of the heterologous HeLa cell model to experimentally tackle this question. We observed that the model rhizobium Sinorhizobium meliloti, and other rhizobia as well, were able to trigger a major reorganization of actin cytoskeleton of cultured HeLa cells in vitro. Cell deformation was associated with an inhibition of the three major small RhoGTPases Cdc42, RhoA and Rac1. Bacterial entry, cytoskeleton rearrangements and modulation of RhoGTPase activity required an intact S. meliloti biosynthetic pathway for queuosine, a hypermodifed nucleoside regulating protein translation through tRNA, and possibly mRNA, modification. We showed that an intact bacterial queuosine biosynthetic pathway was also required for effective nitrogen-fixing symbiosis of S. meliloti with its host plant Medicago truncatula, thus indicating that one or several key symbiotic functions of S. meliloti are under queuosine control. We discuss whether the symbiotic defect of que mutants may originate, at least in part, from an altered capacity to modify plant cell actin cytoskeleton.
Asunto(s)
Citoesqueleto/metabolismo , Medicago truncatula/microbiología , Nucleósido Q/biosíntesis , Sinorhizobium meliloti/metabolismo , Simbiosis , Vías Biosintéticas , GTP Fosfohidrolasas/metabolismo , Células HeLa , Humanos , Mutación , Nucleósido Q/genética , Sinorhizobium meliloti/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
The expression and characterization of a nitrile reductase from Escherichia coli K-12 (EcoNR), a newly discovered enzyme class, is described. This enzyme has a potential application for an alternative nitrile reduction pathway. The enzyme activity towards its natural substrate, preQ(0), was demonstrated and optimal working conditions were found to be at 37°C and at pH 7 with Tris buffer.
Asunto(s)
Escherichia coli K12/enzimología , Proteínas de Escherichia coli/metabolismo , Oxidorreductasas/metabolismo , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Secuencia de Bases , Dominio Catalítico , Estabilidad de Enzimas , Escherichia coli K12/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Genes Bacterianos , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , NADP/metabolismo , Resonancia Magnética Nuclear Biomolecular , Nucleósido Q/biosíntesis , Oxidorreductasas/química , Oxidorreductasas/genética , Conformación Proteica , Pirimidinonas/metabolismo , Pirroles/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por Sustrato , TemperaturaRESUMEN
The radical S-adenosyl-l-methionine (SAM) superfamily is a widely distributed group of iron-sulfur containing proteins that exploit the reactivity of the high energy intermediate, 5'-deoxyadenosyl radical, which is produced by the reductive cleavage of SAM, to carry-out complex radical-mediated transformations. The reactions catalyzed by radical SAM enzymes range from simple group migrations to complex reactions in protein and RNA modification. This review will highlight three radical SAM enzymes that catalyze reactions involving modified guanosines in the biosynthesis pathways of the hypermodified tRNA base wybutosine; secondary metabolites of 7-deazapurine structure, including the hypermodified tRNA base queuosine; and the redox cofactor F(420). This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.
Asunto(s)
Proteínas Bacterianas/metabolismo , Productos Biológicos/metabolismo , Proteínas Hierro-Azufre/metabolismo , Nucleósido Q/biosíntesis , Nucleósidos/biosíntesis , Riboflavina/análogos & derivados , S-Adenosilmetionina/metabolismo , Proteínas Bacterianas/química , Biocatálisis , Productos Biológicos/química , Radicales Libres/química , Radicales Libres/metabolismo , Proteínas Hierro-Azufre/química , Nucleósido Q/química , Nucleósidos/química , Oxidación-Reducción , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Riboflavina/biosíntesis , Riboflavina/química , S-Adenosilmetionina/químicaRESUMEN
Pyrrolopyrimidine containing compounds, also known as 7-deazapurines, are a collection of purine-based metabolites that have been isolated from a variety of biological sources and have diverse functions which range from secondary metabolism to RNA modification. To date, nearly 35 compounds with the common 7-deazapurine core structure have been described. This article will illustrate the structural diversity of these compounds and review the current state of knowledge on the biosynthetic pathways that give rise to them.
Asunto(s)
Pirimidinas/biosíntesis , Enzimas/metabolismo , Guanosina/análogos & derivados , Guanosina/biosíntesis , Nucleósido Q/biosíntesis , Purinas/biosíntesis , Pirroles , ARN de Transferencia/metabolismoRESUMEN
Transfer RNA is one of the most richly modified biological molecules. Biosynthetic pathways that introduce these modifications are underexplored, largely because their absence does not lead to obvious phenotypes under normal growth conditions. Queuosine (Q) is a hypermodified base found in the wobble positions of tRNA Asp, Asn, His, and Tyr from bacteria to mankind. Using liquid chromatography MS methods, we have screened 1,755 single gene knockouts of Escherichia coli and have identified the key final step in the biosynthesis of Q. The protein is homologous to B(12)-dependent iron-sulfur proteins involved in halorespiration. The recombinant Bacillus subtilis epoxyqueuosine (oQ) reductase catalyzes the conversion of oQ to Q in a synthetic substrate, as well as undermodified RNA isolated from an oQ reductase knockout strain. The activity requires inclusion of a reductant and a redox mediator. Finally, exogenously supplied cobalamin stimulates the activity. This work provides the framework for studies of the biosynthesis of other modified RNA components, where lack of accessible phenotype or obvious gene clustering has impeded discovery. Moreover, discovery of the elusive oQ reductase protein completes the biosynthetic pathway of Q.
Asunto(s)
Bacillus subtilis/enzimología , Nucleósido Q/análogos & derivados , Nucleósido Q/biosíntesis , Oxidorreductasas/aislamiento & purificación , Oxidorreductasas/metabolismo , ARN de Transferencia/química , Cromatografía Liquida , Clonación Molecular , Biología Computacional , Cartilla de ADN/genética , Escherichia coli , Técnicas de Inactivación de Genes , Espectrometría de Masas , Estructura Molecular , Nucleósido Q/metabolismo , Vitamina B 12RESUMEN
Queuosine (Q) and archaeosine (G(+)) are hypermodified ribonucleosides found in tRNA. Q is present in the anticodon region of tRNA(GUN) in Eukarya and Bacteria, while G(+) is found at position 15 in the D-loop of archaeal tRNA. Prokaryotes produce these 7-deazaguanosine derivatives de novo from GTP through the 7-cyano-7-deazaguanine (pre-Q(0)) intermediate, but mammals import the free base, queuine, obtained from the diet or the intestinal flora. By combining the results of comparative genomic analysis with those of genetic studies, we show that the first enzyme of the folate pathway, GTP cyclohydrolase I (GCYH-I), encoded in Escherichia coli by folE, is also the first enzyme of pre-Q(0) biosynthesis in both prokaryotic kingdoms. Indeed, tRNA extracted from an E. coli DeltafolE strain is devoid of Q and the deficiency is complemented by expressing GCYH-I-encoding genes from different bacterial or archaeal origins. In a similar fashion, tRNA extracted from a Haloferax volcanii strain carrying a deletion of the GCYH-I-encoding gene contains only traces of G(+). These results link the production of a tRNA-modified base to primary metabolism and further clarify the biosynthetic pathway for these complex modified nucleosides.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , GTP Ciclohidrolasa/metabolismo , Guanosina/análogos & derivados , ARN de Transferencia/biosíntesis , Análisis por Conglomerados , Hibridación Genómica Comparativa , Biología Computacional , Escherichia coli/enzimología , Proteínas de Escherichia coli/genética , GTP Ciclohidrolasa/genética , Guanosina/biosíntesis , Haloferax volcanii/enzimología , Haloferax volcanii/genética , Nucleósido Q/biosíntesis , Filogenia , ARN Bacteriano/biosíntesisRESUMEN
QueD (previously named ykvK) is one of several enzymes involved in the biosynthesis of the hypermodified nucleoside queuosine. Queuosine is incorporated into tRNA at position 34 of four tRNAs: tRNA(His), tRNA(Asp), tRNA(Asn) and tRNA(Tyr). The crystallization and preliminary X-ray crystallographic studies of queD are described here. The recombinant protein from Bacillus subtilis was overproduced in Escherichia coli and crystallized using the hanging-drop vapor-diffusion method from 25% PEG 600, 100 mM NaCl and sodium citrate buffer pH 5.5 at 291 K. The crystals diffract to 3.6 A resolution and belong to the cubic space group F4(1)32, with unit-cell parameter a = 240.88 A.
Asunto(s)
Bacillus subtilis/enzimología , Nucleósido Q/biosíntesis , Bacillus subtilis/metabolismo , Secuencia de Bases , Cristalización , Cristalografía por Rayos X , Cartilla de ADN , Conformación ProteicaRESUMEN
The maturation of transfer RNA (tRNA) involves extensive chemical modification of the constituent nucleosides and results in the introduction of significant chemical diversity to tRNA. Many of the pathways to these modified nucleosides are characterized by chemically complex transformations, some of which are unprecedented in other areas of biology. To illustrate the scope of the field, recent progress in understanding the enzymology leading to the formation of two distinct classes of modified nucleosides, the thiouridines and queuosine, a 7-deazaguanosine, is reviewed. In particular, recent data validating the involvement of several proposed intermediates in the formation of thiouridines are discussed, including two key enzyme intermediates and the activated tRNA intermediate. The discovery and mechanistic characterization of a new enzyme activity in the queuosine pathway is discussed.
Asunto(s)
Enzimas/metabolismo , Procesamiento Postranscripcional del ARN , ARN de Transferencia/metabolismo , Nucleósido Q/biosíntesis , Azufre/metabolismo , Tiouridina/metabolismoRESUMEN
The enzyme QueF was recently identified as an enzyme involved in the biosynthesis of queuosine, a 7-deazaguanosine modified nucleoside found in bacterial and eukaryotic tRNA. QueF exhibits sequence homology to the type I GTP cyclohydrolases characterized by FolE, but contrary to the predictions based on sequence analysis the enzyme in fact catalyzes a mechanistically unrelated reaction, the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine (preQ1), a late step in the queuosine pathway. The reduction of a nitrile is unprecedented in biology, and we report here characterization and mechanistic studies of the enzyme from Bacillus subtilis. The recombinant enzyme exhibits optimal activity at pH 7.5 and moderate ionic strength, and is not dependent on metal ions for catalytic activity. Steady-state kinetic analysis provided a kcat = 0.66 +/- 0.04 min-1, KM (preQ0) = 0.237 +/- 0.045 microM, and KM (NADPH) = 19.2 +/- 1.1 microM. Based on sequence analysis and homology modeling we predicted previously that Cys55 would be present in the active site and in proximity to the nitrile group of preQ0. Consistent with that prediction we observed that the enzyme was inactivated when preincubated with iodoacetamide, and protected from inactivation when preQ0 was present. Furthermore, titrations of the enzyme with preQ0 in the absence of NADPH were accompanied by the appearance of a new absorption band at 376 nm in the UV-vis spectrum consistent with the formation of an alpha,beta-unsaturated thioimide. Site-directed mutagenesis of Cys55 to Ala or Ser resulted in loss of catalytic activity and no absorption at 376 nm upon addition of preQ0. Based on our data we propose a chemical mechanism for the enzyme-catalyzed reaction, and a chemical rationale for the observation of covalent catalysis.
Asunto(s)
Bacillus subtilis/enzimología , Nitrilos/metabolismo , Nucleósido Q/biosíntesis , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Activación Enzimática/efectos de los fármacos , Concentración de Iones de Hidrógeno , Cinética , Modelos Biológicos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , NADP/metabolismo , Sales (Química)/farmacología , Especificidad por Sustrato , VolumetríaRESUMEN
The genome sequences of several pseudomonads have revealed a gene cluster containing genes for a two-component heavy metal histidine sensor kinase and response regulator upstream of cinA and cinQ, which we show herein to encode a copper-containing azurin-like protein and a pre-Q(0) reductase, respectively. In the presence of copper, Pseudomonas putida KT2440 produces the CinA and CinQ proteins from a bicistronic mRNA. UV-visible spectra of CinA show features at 439, 581, and 719 nm, which is typical of the plastocyanin family of proteins. The redox potential of the protein was shown to be 456 +/- 4 mV by voltametric titrations. Surprisingly, CinQ is a pyridine nucleotide-dependent nitrile oxidoreductase that catalyzes the conversion of pre-Q(0) to pre-Q(1) in the nucleoside queuosine biosynthetic pathway. Gene disruptions of cinA and cinQ did not lead to a significant increase in the copper sensitivity of P. putida KT2440 under the conditions tested. Possible roles of CinA and CinQ to help pseudomonads adapt and survive under prolonged copper stress are discussed.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cobre/farmacología , NADH NADPH Oxidorreductasas/metabolismo , Operón , Pseudomonas putida/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Metionina/metabolismo , Estructura Molecular , Mutación , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/aislamiento & purificación , Nitrilos/metabolismo , Nucleósido Q/biosíntesis , Nucleósido Q/química , Pseudomonas putida/enzimología , Pseudomonas putida/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrofotometría UltravioletaRESUMEN
Queuosine (Q), one of the most complex modifications occurring at the wobble position of tRNAs with GUN anticodons, is implicated in a number of biological activities, including accuracy of decoding, virulence, and cellular differentiation. Despite these important implications, its biosynthetic pathway has remained unresolved. Earlier, we observed that a naturally occurring strain of Escherichia coli B105 lacked Q modification in the tRNAs. In the present study, we developed a genetic screen to map the defect in E. coli B105 to a single gene, queC (renamed from ybaX), predicted to code for a 231-amino-acid-long protein with a pI of 5.6. As analyzed by mobility of tRNA(Tyr) on acid urea gels and two-dimensional thin-layer chromatography of the modified nucleosides, expression of QueC from a plasmid-borne copy confers a Q+ phenotype to E. coli B105. Further, analyses of tRNA(Tyr) from E. coli JE10651 (queA mutant), its derivative generated by deletion of chromosomal queC (queA deltaqueC), and E. coli JE7325, deficient in converting preQ0 to preQ1, have provided the first genetic evidence for the involvement of QueC at a step leading to production of preQ0, the first known intermediate in the generally accepted pathway that utilizes GTP as the starting molecule. In addition, we discuss the possibilities of collaboration of QueC with other cellular proteins in the production of preQ0.
Asunto(s)
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Nucleósido Q/biosíntesis , Secuencia de Aminoácidos , Anticodón/genética , Elementos Transponibles de ADN , Prueba de Complementación Genética , Pruebas Genéticas/métodos , Datos de Secuencia Molecular , Mutación , Fenotipo , ARN de Transferencia de Tirosina/química , ARN de Transferencia de Tirosina/genética , Transducción GenéticaRESUMEN
The enzyme YkvM from Bacillus subtilis was identified previously along with three other enzymes (YkvJKL) in a bioinformatics search for enzymes involved in the biosynthesis of queuosine, a 7-deazaguanine modified nucleoside found in tRNA(GUN) of Bacteria and Eukarya. Genetic analysis of ykvJKLM mutants in Acinetobacter confirmed that each was essential for queuosine biosynthesis, and the genes were renamed queCDEF. QueF exhibits significant homology to the type I GTP cyclohydrolases characterized by FolE. Given that GTP is the precursor to queuosine and that a cyclohydrolase-like reaction was postulated as the initial step in queuosine biosynthesis, QueF was proposed to be the putative cyclohydrolase-like enzyme responsible for this reaction. We have cloned the queF genes from B. subtilis and Escherichia coli and characterized the recombinant enzymes. Contrary to the predictions based on sequence analysis, we discovered that the enzymes, in fact, catalyze a mechanistically unrelated reaction, the NADPH-dependent reduction of 7-cyano-7-deazaguanineto7-aminomethyl-7-deazaguanine, a late step in the biosynthesis of queuosine. We report here in vitro and in vivo studies that demonstrate this catalytic activity, as well as preliminary biochemical and bioinformatics analysis that provide insight into the structure of this family of enzymes.