RESUMEN
Brucella spp. are pathogenic intracellular Gram-negative bacteria adapted to life within cells of several mammals, including humans. These bacteria are the causative agent of brucellosis, one of the zoonotic infections with the highest incidence in the world and for which a human vaccine is still unavailable. Current therapeutic treatments against brucellosis are based on the combination of two or more antibiotics for prolonged periods, which may lead to antibiotic resistance in the population. Riboflavin (vitamin B2) is biosynthesized by microorganisms and plants but mammals, including humans, must obtain it from dietary sources. Owing to the absence of the riboflavin biosynthetic enzymes in animals, this pathway is nowadays regarded as a rich resource of targets for the development of new antimicrobial agents. In this work, we describe a high-throughput screening approach to identify inhibitors of the enzymatic activity of riboflavin synthase, the last enzyme in this pathway. We also provide evidence for their subsequent validation as potential drug candidates in an in vitro brucellosis infection model. From an initial set of 44 000 highly diverse low molecular weight compounds with drug-like properties, we were able to identify ten molecules with 50% inhibitory concentrations in the low micromolar range. Further Brucella culture and intramacrophagic replication experiments showed that the most effective bactericidal compounds share a 2-Phenylamidazo[2,1-b][1,3]benzothiazole chemical scaffold. Altogether, these findings set up the basis for the subsequent lead optimization process and represent a promising advancement in the pursuit of novel and effective antimicrobial compounds against brucellosis.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Brucella abortus/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Riboflavina Sintasa/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Antibacterianos/química , Proteínas Bacterianas/metabolismo , Brucella abortus/enzimología , Línea Celular , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento/métodos , Ratones , Unión Proteica , Riboflavina Sintasa/metabolismo , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Coenzyme F420 is a redox cofactor found in methanogens and in various actinobacteria. Despite the major biological importance of this cofactor, the biosynthesis of its deazaflavin core (8-hydroxy-5-deazaflavin, F(o)) is still poorly understood. F(o) synthase, the enzyme involved, is an unusual multidomain radical SAM enzyme that uses two separate 5'-deoxyadenosyl radicals to catalyze F(o) formation. In this paper, we report a detailed mechanistic study on this complex enzyme that led us to identify (1) the hydrogen atoms abstracted from the substrate by the two radical SAM domains, (2) the second tyrosine-derived product, (3) the reaction product of the CofH-catalyzed reaction, (4) the demonstration that this product is a substrate for CofG, and (5) a stereochemical study that is consistent with the formation of a p-hydroxybenzyl radical at the CofH active site. These results enable us to propose a mechanism for F(o) synthase and uncover a new catalytic motif in radical SAM enzymology involving the use of two 5'-deoxyadenosyl radicals to mediate the formation of a complex heterocycle.
Asunto(s)
Actinobacteria/enzimología , Radicales Libres/metabolismo , Riboflavina Sintasa/metabolismo , Riboflavina/análogos & derivados , Actinobacteria/química , Actinobacteria/metabolismo , Vías Biosintéticas , Radicales Libres/química , Riboflavina/química , Riboflavina/metabolismo , Tirosina/química , Tirosina/metabolismoRESUMEN
Isotope-labeled flavins are crucial reporters for many biophysical studies of flavoproteins. A purine-deficient Escherichia coli strain engineered for expression of the ribAGH genes of Bacillus subtilis converts isotope-labeled purine supplements into the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, with yields up to 40%. The fermentation products can subsequently be converted into isotope-labeled riboflavin and the cognate flavocoenzymes, FMN and FAD, by in vitro biotransformation with better than 90% yield. Using this approach, more than 100 single or multiple (13)C-, (15)N-, (17)O-, and (18)O-labeled isotopologues of these cofactors and ligands become easily accessible, enabling advanced ligand-based spectroscopy of flavoproteins and lumazine receptor proteins at unprecedented resolution.
Asunto(s)
Bacillus subtilis/química , Escherichia coli/química , Escherichia coli/enzimología , Flavoproteínas/química , Marcaje Isotópico/métodos , Pteridinas/química , Pteridinas/síntesis química , Purinas/química , Riboflavina Sintasa/química , Riboflavina/química , Biotransformación , Ligandos , Riboflavina Sintasa/metabolismoRESUMEN
Riboflavin (vitamin B2) is the precursor of flavin mononucleotide and flavin adenine dinucleotide-essential cofactors for a wide variety of enzymes involving in numerous metabolic processes. In this study, a partial-length cDNA encoding bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone-4-phosphate synthase (LcRIBA), 2 full-length cDNAs encoding lumazine synthase (LcLS1 and LcLS2), and a full-length cDNA encoding riboflavin synthase (LcRS) were isolated from Lycium chinense, an important traditional medicinal plant. Sequence analyses showed that these genes exhibited high identities with their orthologous genes as well as having the same common features related to plant riboflavin biosynthetic genes. LcRIBA, like other plant RIBAs, contained a DHBPS region in its N terminus and a GCHII region in its C-terminal part. LcLSs and LcRS carried an N-terminal extension found in plant riboflavin biosynthetic genes unlike the orthologous microbial genes. Quantitative real-time polymerase chain reaction analysis showed that 4 riboflavin biosynthetic genes were constitutively expressed in all organs examined of L. chinense plants with the highest expression levels found in the leaves or red fruits. LcRIBA, which catalyzes 2 initial reactions in riboflavin biosynthetic pathway, was the highest transcript in the leaves, and hence, the richest content of riboflavin was detected in this organ. Our study might provide the basis for investigating the contribution of riboflavin in diverse biological activities of L. chinense and may facilitate the metabolic engineering of vitamin B2 in crop plants.
Asunto(s)
ADN Complementario/genética , GTP Ciclohidrolasa/genética , Lycium/genética , Complejos Multienzimáticos/genética , Riboflavina Sintasa/genética , Riboflavina/genética , Riboflavina/metabolismo , Secuencia de Aminoácidos , Biodiversidad , GTP Ciclohidrolasa/metabolismo , Genes de Plantas/genética , Lycium/metabolismo , Complejos Multienzimáticos/metabolismo , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Plantas Medicinales/genética , Plantas Medicinales/metabolismo , Riboflavina Sintasa/metabolismo , Alineación de Secuencia , Fosfatos de Azúcar/metabolismoRESUMEN
Nitrofurantoin has been used for decades for the treatment of urinary tract infections (UTIs), but clinically significant resistance in Escherichia coli is uncommon. Nitrofurantoin concentrations in the gastrointestinal tract tend to be low, which might facilitate selection of nitrofurantoin-resistant (NIT-R) strains in the gut flora. We subjected two nitrofurantoin-susceptible intestinal E. coli strains (ST540-p and ST2747-p) to increasing nitrofurantoin concentrations under aerobic and anaerobic conditions. Whole-genome sequencing was performed for both susceptible isolates and selected mutants that exhibited the highest nitrofurantoin resistance levels aerobically (ST540-a and ST2747-a) and anaerobically (ST540-an and ST2747-an). ST540-a/ST540-an and ST2747-a (aerobic MICs of >64 µg/ml) harbored mutations in the known nitrofurantoin resistance determinants nfsA and/or nfsB, which encode oxygen-insensitive nitroreductases. ST2747-an showed reduced nitrofurantoin susceptibility (aerobic MIC of 32 µg/ml) and exhibited remarkable growth deficits but did not harbor nfsA/nfsB mutations. We identified a 12-nucleotide deletion in ribE, encoding lumazine synthase, an essential enzyme involved in the biosynthesis of flavin mononucleotide (FMN), which is an important cofactor for NfsA and NfsB. Complementing ST2747-an with a functional wild-type lumazine synthase restored nitrofurantoin susceptibility. Six NIT-R E. coli isolates (NRCI-1 to NRCI-6) from stools of UTI patients treated with nitrofurantoin, cefuroxime, or a fluoroquinolone harbored mutations in nfsA and/or nfsB but not ribE. Sequencing of the ribE gene in six intestinal and three urinary E. coli strains showing reduced nitrofurantoin susceptibility (MICs of 16 to 48 µg/ml) also did not identify any relevant mutations. NRCI-1, NRCI-2, and NRCI-5 exhibited up to 4-fold higher anaerobic MICs, compared to the mutants generated in vitro, presumably because of additional mutations in oxygen-sensitive nitroreductases.
Asunto(s)
Secuencia de Bases , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Complejos Multienzimáticos/genética , Riboflavina Sintasa/genética , Eliminación de Secuencia , Aerobiosis , Anaerobiosis , Antibacterianos/farmacología , Cefuroxima/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fluoroquinolonas/farmacología , Prueba de Complementación Genética , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Complejos Multienzimáticos/metabolismo , Nitrofurantoína/farmacología , Nitrorreductasas/genética , Nitrorreductasas/metabolismo , Riboflavina Sintasa/metabolismo , Infecciones Urinarias/tratamiento farmacológico , Infecciones Urinarias/microbiologíaRESUMEN
Riboflavin synthase (RS) catalyzes the last step of riboflavin biosynthesis in microorganisms and plants, which corresponds to the dismutation of two molecules of 6,7-dimethyl-8-ribityllumazine to yield one molecule of riboflavin and one molecule of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Owing to the absence of this enzyme in animals and the fact that most pathogenic bacteria show a strict dependence on riboflavin biosynthesis, RS has been proposed as a potential target for antimicrobial drug development. Eubacterial, fungal and plant RSs assemble as homotrimers lacking C3 symmetry. Each monomer can bind two substrate molecules, yet there is only one active site for the whole enzyme, which is located at the interface between two neighbouring chains. This work reports the crystallographic structure of RS from the pathogenic bacterium Brucella abortus (the aetiological agent of the disease brucellosis) in its apo form, in complex with riboflavin and in complex with two different product analogues, being the first time that the structure of an intact RS trimer with bound ligands has been solved. These crystal models support the hypothesis of enhanced flexibility in the particle and also highlight the role of the ligands in assembling the unique active site. Kinetic and binding studies were also performed to complement these findings. The structural and biochemical information generated may be useful for the rational design of novel RS inhibitors with antimicrobial activity.
Asunto(s)
Brucella abortus/enzimología , Riboflavina Sintasa/química , Riboflavina Sintasa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Riboflavina/química , Riboflavina Sintasa/genética , Homología de Secuencia de AminoácidoRESUMEN
Riboflavin (vitamin B2) is an essential nutrition component serving as a precursor of coenzymes FMN and FAD that are involved mostly in reactions of oxidative metabolism. Riboflavin is produced in commercial scale and is used in feed and food industries, and in medicine. The yeast Candida famata (Candida flareri) belongs to the group of so called "flavinogenic yeasts" which overproduce riboflavin under iron limitation. Three genes SEF1, RIB1 and RIB7 coding for a putative transcription factor, GTP cyclohydrolase II and riboflavin synthase, respectively were simultaneously overexpressed in the background of a non-reverting riboflavin producing mutant AF-4, obtained earlier in our laboratory using methods of classical selection (Dmytruk et al. (2011), Metabolic Engineering 13, 82-88). Cultivation conditions of the constructed strain were optimized for shake-flasks and bioreactor cultivations. The constructed strain accumulated up to 16.4g/L of riboflavin in optimized medium in a 7L laboratory bioreactor during fed-batch fermentation.
Asunto(s)
Candida/crecimiento & desarrollo , Candida/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hierro/metabolismo , Riboflavina/biosíntesis , Técnicas de Cultivo Celular por Lotes , Reactores Biológicos , Fermentación , GTP Ciclohidrolasa/genética , GTP Ciclohidrolasa/metabolismo , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Ingeniería Metabólica , Mutación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Riboflavina Sintasa/genética , Riboflavina Sintasa/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The xylene ring of riboflavin (vitamin B2 ) is assembled from two molecules of 3,4-dihydroxy-2-butanone 4-phosphate by a mechanistically complex process that is jointly catalyzed by lumazine synthase and riboflavin synthase. In Bacillaceae, these enzymes form a structurally unique complex comprising an icosahedral shell of 60 lumazine synthase subunits and a core of three riboflavin synthase subunits, whereas many other bacteria have empty lumazine synthase capsids, fungi, Archaea and some eubacteria have pentameric lumazine synthases, and the riboflavin synthases of Archaea are paralogs of lumazine synthase. The structures of the molecular ensembles have been studied in considerable detail by X-ray crystallography, X-ray small-angle scattering and electron microscopy. However, certain mechanistic aspects remain unknown. Surprisingly, the quaternary structure of the icosahedral ß subunit capsids undergoes drastic changes, resulting in formation of large, quasi-spherical capsids; this process is modulated by sequence mutations. The occurrence of large shells consisting of 180 or more lumazine synthase subunits has recently generated interest for protein engineering topics, particularly the construction of encapsulation systems.
Asunto(s)
Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Riboflavina Sintasa/química , Riboflavina Sintasa/metabolismo , Archaea/enzimología , Bacterias/enzimología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Hongos/enzimología , Plantas/enzimología , Pteridinas/metabolismo , Riboflavina/biosíntesis , Schizosaccharomyces/química , Schizosaccharomyces/metabolismoRESUMEN
Riboflavin (vitamin B2) is the universal precursor of the coenzymes flavin mononucleotide and flavin adenine dinucleotide--cofactors that are essential for the activity of a wide variety of metabolic enzymes in animals, plants, and microbes. Using the RACE PCR approach, cDNAs encoding lumazine synthase (McLS) and riboflavin synthase (McRS), which catalyze the last two steps in the riboflavin biosynthetic pathway, were cloned from bitter melon (Momordica charantia), a popular vegetable crop in Asia. Amino acid sequence alignments indicated that McLS and McRS share high sequence identity with other orthologous genes and carry an N-terminal extension, which is reported to be a plastid-targeting sequence. Organ expression analysis using quantitative real-time RT PCR showed that McLS and McRS were constitutively expressed in M. charantia, with the strongest expression levels observed during the last stage of fruit ripening (stage 6). This correlated with the highest level of riboflavin content, which was detected during ripening stage 6 by HPLC analysis. McLS and McRS were highly expressed in the young leaves and flowers, whereas roots exhibited the highest accumulation of riboflavin. The cloning and characterization of McLS and McRS from M. charantia may aid the metabolic engineering of vitamin B2 in crops.
Asunto(s)
Momordica charantia/genética , Complejos Multienzimáticos/genética , Riboflavina Sintasa/genética , Riboflavina/metabolismo , Regiones no Traducidas 3' , Secuencia de Aminoácidos , Clonación Molecular , ADN Complementario , Frutas/metabolismo , Frutas/fisiología , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Momordica charantia/enzimología , Momordica charantia/fisiología , Complejos Multienzimáticos/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Riboflavina Sintasa/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Cofactors play key roles in metabolic pathways. Among them F(420) has proved to be a very attractive target for the selective inhibition of archaea and actinobacteria. Its biosynthesis, in a unique manner, involves a key enzyme, F(0)-synthase. This enzyme is a large monomer in actinobacteria, while it is constituted of two subunits in archaea and cyanobacteria. We report here the purification of both types of F(0)-synthase and their in vitro activities. Our study allows us to establish that F(0)-synthase, from both types, uses 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and tyrosine as substrates but not 4-hydroxylphenylpyruvate as previously suggested. Furthermore, our data support the fact that F(0)-synthase generates two 5'-deoxyadenosyl radicals for catalysis which is unprecedented in reaction catalyzed by radical SAM enzymes.
Asunto(s)
Actinomycetales/enzimología , Methanococcus/enzimología , Nostoc/enzimología , Riboflavina Sintasa/metabolismo , Riboflavina/análogos & derivados , Tirosina/metabolismo , Actinomycetales/química , Actinomycetales/metabolismo , Methanococcus/química , Methanococcus/metabolismo , Nostoc/química , Nostoc/metabolismo , Estructura Terciaria de Proteína , Riboflavina/química , Riboflavina/metabolismo , Riboflavina Sintasa/química , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismoRESUMEN
Streptomyces davawensis is the only organism known to synthesize the antibiotic roseoflavin, a riboflavin (vitamin B2) analog. Roseoflavin is converted to roseoflavin mononucleotide (RoFMN) and roseoflavin adenine dinucleotide in the cytoplasm of target cells. (Ribo-)Flavin mononucleotide (FMN) riboswitches are genetic elements, which in many bacteria control genes responsible for the biosynthesis and transport of riboflavin. Streptomyces davawensis is roseoflavin resistant, and the closely related bacterium Streptomyces coelicolor is roseoflavin sensitive. The two bacteria served as models to investigate roseoflavin resistance of S. davawensis and to analyze the mode of action of roseoflavin in S. coelicolor. Our experiments demonstrate that the ribB FMN riboswitch of S. davawensis (in contrast to the corresponding riboswitch of S. coelicolor) is able to discriminate between the two very similar flavins FMN and RoFMN and shows opposite responses to the latter ligands.
Asunto(s)
Antibacterianos/farmacología , Regulación Fúngica de la Expresión Génica , Riboswitch , Streptomyces/genética , Aptámeros de Nucleótidos/metabolismo , Citoplasma/metabolismo , Farmacorresistencia Fúngica , Mononucleótido de Flavina/metabolismo , Flavina-Adenina Dinucleótido/análogos & derivados , Flavina-Adenina Dinucleótido/metabolismo , Genoma Fúngico , Ligandos , Mutación Puntual , Biosíntesis de Proteínas , Riboflavina/análogos & derivados , Riboflavina/metabolismo , Riboflavina/farmacología , Riboflavina Sintasa/metabolismo , Streptomyces/efectos de los fármacos , Streptomyces/enzimología , Streptomyces coelicolor/efectos de los fármacos , Streptomyces coelicolor/enzimología , Streptomyces coelicolor/genéticaRESUMEN
The biosynthesis of one riboflavin (vitamin B(2)) molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate as substrates. In the final step, the tricyclic isoalloxazine chromophore, which is the hallmark of flavocoenzymes, arises from a highly unusual dismutation of bicyclic 6,7-dimethyl-8-ribityllumazine that is catalyzed by riboflavin synthase but can also proceed without catalysis. The reaction proceeds via a pentacyclic adduct of two 6,7-dimethyl-8-ribityllumazine molecules, whose cleavage into riboflavin and a pyrimidine derivative (by a sequence of two elimination steps) is mechanistically straightforward. Recently, the formation of the pentacyclic adduct has been proposed to involve a hydride transfer step followed by a [4+2] cycloaddition. Surprisingly, two different classes of riboflavin synthases utilize different diastereomers of the pentacyclic adduct, but the newly generated chiral centers are lost upon the intermediates' subsequent fragmentation.
Asunto(s)
Riboflavina/biosíntesis , Secuencia de Aminoácidos , Archaea/enzimología , Bacterias/enzimología , Hongos/enzimología , Modelos Moleculares , Datos de Secuencia Molecular , Riboflavina/química , Riboflavina/metabolismo , Riboflavina Sintasa/metabolismo , Alineación de Secuencia , Xilenos/química , Xilenos/metabolismoRESUMEN
DNA photolyases use two noncovalently bound chromophores to catalyze photoreactivation, the blue light-dependent repair of DNA that has been damaged by ultraviolet light. FAD is the catalytic chromophore for all photolyases and is essential for photoreactivation. The identity of the second chromophore is often 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO). Under standard light conditions, the second chromophore is considered nonessential for photoreactivation because DNA photolyase bound to only FAD is sufficient to catalyze the repair of UV-damaged DNA. phr1 is a photoreactivation-deficient strain of Chlamydomonas. In this work, the PHR1 gene of Chlamydomonas was cloned through molecular mapping and shown to encode a protein similar to known FO synthases. Additional results revealed that the phr1 strain was deficient in an FO-like molecule and that this deficiency, as well as the phr1 photoreactivation deficiency, could be rescued by transformation with DNA constructs containing the PHR1 gene. Furthermore, expression of a PHR1 cDNA in Escherichia coli produced a protein that generated a molecule with characteristics similar to FO. Together, these results indicate that the Chlamydomonas PHR1 gene encodes an FO synthase and that optimal photoreactivation in Chlamydomonas requires FO, a molecule known to serve as a second chromophore for DNA photolyases.
Asunto(s)
Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Desoxirribodipirimidina Fotoliasa/metabolismo , Riboflavina/análogos & derivados , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Exones , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Prueba de Complementación Genética , Intrones , Luz , Datos de Secuencia Molecular , Riboflavina/química , Riboflavina/metabolismo , Riboflavina Sintasa/genética , Riboflavina Sintasa/metabolismoRESUMEN
Riboflavin synthase catalyzes the transfer of a four-carbon fragment between two molecules of the substrate, 6,7-dimethyl-8-ribityllumazine, resulting in the formation of riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Earlier, a pentacyclic adduct formed from two substrate molecules was shown to be a catalytically competent intermediate, but the mechanism of its formation is still poorly understood. The present study shows that the recombinant N-terminal domain of riboflavin synthase from Escherichia coli interacts specifically with the exomethylene-type anion of 6,7-dimethyl-8-ribityllumazine but not with any of the tricyclic adduct-type anions that dominate the complex anion equilibrium in aqueous solution. Whereas these findings can be implemented into previously published mechanistic hypotheses, we also present a novel, hypothetical reaction sequence that starts with the transfer of a hydride ion from the 6,7-dimethyl-8-ribityllumazine exomethylene anion to an electroneutral 6,7-dimethyl-8-ribityllumazine molecule. The pair of dehydrolumazine and dihydrolumazine molecules resulting from this hydride transfer is proposed to undergo a 4 + 2 cycloaddition, affording the experimentally documented pentacyclic intermediate. In contrast to earlier mechanistic concepts requiring the participation of a nucleophilic agent, which is not supported by structural and mutagenesis data, the novel concept has no such requirement. Moreover, it requires fewer reaction steps and is consistent with all experimental data.
Asunto(s)
Pteridinas/química , Riboflavina Sintasa/química , Aniones/química , Sitios de Unión , Biocatálisis , Estructura Molecular , Riboflavina/síntesis química , Riboflavina/química , Riboflavina Sintasa/metabolismo , Estereoisomerismo , Uridina/análogos & derivados , Uridina/síntesis química , Uridina/químicaRESUMEN
Riboflavin (vitamin B2) is the direct precursor of redox enzyme cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are essential for multiple cell physiology. The riboflavin biosynthetic pathway is regarded as a rich resource for therapeutic targets for broad spectrum antibiotics. Enzymatic pathways, regulatory factors of the riboflavin biosynthesis, and relevant drug discovery are summarized in this review. The novel riboswitch regulatory mechanism of riboflavin metabolism is also described. A compendium of chemical modulators of riboflavin biosynthesis and regulatory networks is listed and such demonstrates the promise of riboflavin biosynthesis and regulatory mechanisms as potential therapeutic targets for novel antibiotic drug discovery.
Asunto(s)
Antiinfecciosos/farmacología , Riboflavina Sintasa/metabolismo , Riboflavina/biosíntesis , Secuencia de Aminoácidos , Antiinfecciosos/química , Antituberculosos/química , Antituberculosos/farmacología , Datos de Secuencia Molecular , Riboflavina/metabolismo , Riboflavina Sintasa/antagonistas & inhibidores , Riboflavina Sintasa/química , Alineación de SecuenciaRESUMEN
The pathway of riboflavin (vitamin B2) biosynthesis is significantly different in archaea, eubacteria, fungi and plants. Specifically, the first committed intermediate, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate, can either undergo hydrolytic cleavage of the position 2 amino group by a deaminase (in plants and most eubacteria) or reduction of the ribose side chain by a reductase (in fungi and archaea). We compare 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate synthases from the yeast Candida glabrata, the archaeaon Methanocaldococcus jannaschii and the eubacterium Aquifex aeolicus. All three enzymes convert 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate into 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate, as shown by 13C-NMR spectroscopy using [2,1',2',3',4',5'-13C6]2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate as substrate. The beta anomer was found to be the authentic substrate, and the alpha anomer could serve as substrate subsequent to spontaneous anomerisation. The M. jannaschii and C. glabrata enzymes were shown to be A-type reductases catalysing the transfer of deuterium from the 4(R) position of NADPH to the 1' (S) position of the substrate. These results are in agreement with the known three-dimensional structure of the M. jannaschii enzyme.
Asunto(s)
Archaea/enzimología , Hongos/enzimología , Riboflavina Sintasa/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Cinética , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Riboflavina Sintasa/química , Riboflavina Sintasa/genética , Homología de Secuencia de Aminoácido , Estereoisomerismo , UltracentrifugaciónRESUMEN
The intensely fluorescent lumazine protein is believed to be involved in the bioluminescence of certain marine bacteria. The sequence of the catalytically inactive protein resembles that of the enzyme riboflavin synthase. Its non-covalently bound fluorophore, 6,7-dimethyl-8-ribityllumazine, is the substrate of this enzyme and also the committed precursor of vitamin B(2). An extensive crystallization screen was performed using numerous single-site mutants of the lumazine protein from Photobacterium leiognathi in complex with its fluorophore and with riboflavin, respectively. Only the L49N mutant in complex with riboflavin yielded suitable crystals, allowing X-ray structure determination to a resolution of 2.5 A. The monomeric protein folds into two closely similar domains that are structurally related by pseudo-C(2) symmetry, whereby the entire domain topology resembles that of riboflavin synthase. Riboflavin is bound to a shallow cavity in the N-terminal domain of lumazine protein, whereas the C-terminal domain lacks a ligand.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Luminiscentes/química , Photobacterium/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Evolución Molecular , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Óptica y Fotónica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Riboflavina/metabolismo , Riboflavina Sintasa/química , Riboflavina Sintasa/metabolismo , Electricidad EstáticaRESUMEN
The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate as substrates. GTP is hydrolytically opened, converted into 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction and dephosphorylation. Condensation with 3,4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate leads to 6,7-dimethyl-8-ribityllumazine. The final step in the biosynthesis of the vitamin involves the dismutation of 6,7-dimethyl-8-ribityllumazine catalyzed by riboflavin synthase. The mechanistically unusual reaction involves the transfer of a four-carbon fragment between two identical substrate molecules. The second product, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, is recycled in the biosynthetic pathway by 6,7-dimethyl-8-ribityllumazine synthase. This article will review structures and reaction mechanisms of riboflavin synthases and related proteins up to 2007 and 122 references are cited.
Asunto(s)
Riboflavina Sintasa/metabolismo , Riboflavina/biosíntesis , Secuencia de Aminoácidos , Archaea/metabolismo , Bacterias/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Pliegue de Proteína , Riboflavina/química , Riboflavina Sintasa/química , Homología de Secuencia de AminoácidoRESUMEN
Lumazine synthase and riboflavin synthase catalyze the last two steps in the biosynthesis of riboflavin. To obtain structural and mechanistic probes of these two enzymes, as well as inhibitors of potential value as antibiotics, a sulfur analogue of the pyrimidine substrate of the lumazine synthase-catalyzed reaction and product of the riboflavin synthase-catalyzed reaction was designed. Facile syntheses of the S-nucleoside 5-amino-6-(D-ribitylthio)pyrimidine-2,4(1H,3H)-dione hydrochloride (15) and its nitro precursor 5-nitro-6-(D-ribitylthio)pyrimidine-2,4(1H,3H)-dione (14) are described. These compounds were tested against lumazine synthase and riboflavin synthase obtained from a variety of microorganisms. Compounds 14 and 15 were found to be inhibitors of both riboflavin synthase and lumazine synthase. Compound 14 is an inhibitor of Bacillus subtilis lumazine synthase (Ki 26 microM), Schizosaccharomyces pombe lumazine synthase (Ki 2.0 microM), Mycobacterium tuberculosis lumazine synthase (Ki 11 microM), Escherichia coli riboflavin synthase (Ki 2.7 microM), and Mycobacterium tuberculosis riboflavin synthase (Ki 0.56 muM), while compound 15 is an inhibitor of B. subtilis lumazine synthase (Ki 2.6 microM), S. pombe lumazine synthase (Ki 0.16 microM), M. tuberculosis lumazine synthase (Ki 31 microM), E. coli riboflavin synthase (Ki 47 microM), and M. tuberculosis riboflavin synthase (Ki 2.5 microM).
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Complejos Multienzimáticos/antagonistas & inhibidores , Nucleósidos de Pirimidina/síntesis química , Nucleósidos de Pirimidina/farmacología , Riboflavina Sintasa/antagonistas & inhibidores , Bacillus subtilis/enzimología , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Escherichia coli/enzimología , Modelos Moleculares , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Mycobacterium tuberculosis/enzimología , Nucleósidos de Pirimidina/metabolismo , Riboflavina Sintasa/química , Riboflavina Sintasa/metabolismo , Especificidad por SustratoRESUMEN
Mycobacteria can tolerate relatively high concentrations of triphenylmethane dyes such as malachite green and methyl violet. To identify mycobacterial genes involved in the decolorization of malachite green, a transposon mutant library of Mycobacterium smegmatis mc2 155 was screened for mutants unable to decolorize this dye. One of the genes identified was MSMEG_5126, an orthologue of Mycobacterium bovis fbiC encoding a 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO) synthase, which is essential for the biosynthesis of the electron carrier coenzyme F420. The other gene identified was MSMEG_2392, encoding an alanine-rich protein with a DUF121 domain. The minimum inhibitory concentrations (MICs) for malachite green and methyl violet of the six fbiC mutants and two MSMEG_2392 mutants were one-third and one-fifth, respectively, of the MIC of the parent strain M. smegmatis mc2 155. Representative fbiC and MSMEG_2392 mutant strains were also sensitive to oxidative stress caused by the redox-cycling agents plumbagin and menadione, and the sensitivity was reversed in the complemented strains. HPLC analysis of representative fbiC and MSMEG_2392 strains revealed that, while the fbiC mutant lacked both coenzyme F420 and FO, the MSMEG_2392 mutant contained FO but not coenzyme F420. These results indicate that MSMEG_2392 is involved in the biosynthesis of coenzyme F420.