RESUMEN
The heterologous expression of mammalian membrane proteins in lower eukaryotes is often hampered by aberrant protein localization, structure, and function, leading to enhanced degradation and, thus, low expression levels. Substantial quantities of functional membrane proteins are necessary to elucidate their structure-function relationships. Na,K-ATPases are integral, human membrane proteins that specifically interact with cholesterol and phospholipids, ensuring protein stability and enhancing ion transport activity. In this study, we present a Pichia pastoris strain which was engineered in its sterol pathway towards the synthesis of cholesterol instead of ergosterol to foster the functional expression of human membrane proteins. Western blot analyses revealed that cholesterol-producing yeast formed enhanced and stable levels of human Na,K-ATPase α3ß1 isoform. ATPase activity assays suggested that this Na,K-ATPase isoform was functionally expressed in the plasma membrane. Moreover, [(3)H]-ouabain cell surface-binding studies underscored that the Na,K-ATPase was present in high numbers at the cell surface, surpassing reported expression strains severalfold. This provides evidence that the humanized sterol composition positively influenced Na,K-ATPase α3ß1 stability, activity, and localization to the yeast plasma membrane. Prospectively, cholesterol-producing yeast will have high potential for functional expression of many mammalian membrane proteins.
Asunto(s)
Colesterol/metabolismo , Expresión Génica , Pichia/genética , Pichia/metabolismo , Isoformas de Proteínas/biosíntesis , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Vías Biosintéticas/genética , Membrana Celular/enzimología , Ergosterol/metabolismo , Humanos , Ingeniería Metabólica , Isoformas de Proteínas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
Flowing enzymes: Continuous extraction of products enhances the enzymatic productivity of sesquiterpenes. Even unnatural substrates are tolerated leading to valuable unnatural target molecules in superior yields compared with batch protocols.
RESUMEN
(13)C-NMR experiments were performed on photo-excited fully and partially (13)C-labelled LOV2 domains of the blue-light receptor phototropin. In the present paper, we report on nuclear-spin polarized tryptophan resonances that are generated by light-induced intraprotein electron transfer to the FMN cofactor. The spectra are discussed with respect to earlier data obtained from (13)C-NMR experiments on unlabelled LOV2 domains that have been reconstituted with FMN (13)C isotopologues.
Asunto(s)
Flavoproteínas/química , Triptófano/química , Isótopos de Carbono , Criptocromos , Transporte de Electrón , Mononucleótido de Flavina/química , Mononucleótido de Flavina/efectos de la radiación , Flavoproteínas/efectos de la radiación , Luz , Espectroscopía de Resonancia Magnética , Estructura Terciaria de ProteínaRESUMEN
The plant blue light receptor phototropin comprises a protein kinase domain and two FMN-binding LOV domains (LOV1 and LOV2). Blue light irradiation of recombinant LOV domains is conducive to the addition of a cysteinyl thiolate group to carbon 4a of the FMN chromophore, and spontaneous cleavage of that photoadduct completes the photocycle of the receptor. The present study is based on (13)C NMR signal modulation observed after reconstitution of LOV domains of different origins with random libraries of (13)C-labeled FMN isotopologues. Using this approach, all (13)C signals of FMN bound to LOV1 and LOV2 domains of Avena sativa and to the LOV2 domain of the fern, Adiantum capillus-veneris, could be unequivocally assigned under dark and under blue light irradiation conditions. (13)C Chemical shifts of FMN are shown to be differently modulated by complexation with the LOV domains under study, indicating slight differences in the binding interactions of FMN and the apoproteins.
Asunto(s)
Mononucleótido de Flavina/metabolismo , Flavoproteínas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Isótopos de Carbono/química , Criptocromos , ADN , Mononucleótido de Flavina/química , Flavoproteínas/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Phototropin is a blue-light receptor involved in the phototropic response of higher plants. The photoreceptor comprises a protein kinase domain and two structurally similar flavin-mononucleotide (FMN) binding domains designated LOV1 and LOV2. Blue-light irradiation of recombinant LOV2 domains induces the formation of a covalent adduct of the thiol group of a functional cysteine in the cofactor-binding pocket to C(4a) of the FMN. Cysteine-to-alanine mutants of LOV domains are unable to form that adduct but generate an FMN radical upon illumination. The recombinant C450A mutant of the LOV2 domain of Avena sativa phototropin was reconstituted with universally and site-selectively (13)C-labeled FMN and the (13)C NMR signals were unequivocally assigned. (13)C NMR spectra were acquired in darkness and under blue-light irradiation. The chemical shifts and the coupling patterns of the signals were not affected by irradiation. However, under blue-light exposure, exceptionally strong nuclear-spin polarization was developed in the resonances belonging to certain carbons of the FMN's isoalloxazine moiety. An enhancement of the NMR absorption was observed for the signals of C(5a), C(7), and C(9). NMR lines in emission were detected for the signals belonging to C(2), C(4), C(4a), C(6), C(8), and C(9a). The signal of C(10a) remained in absorption but was slightly attenuated. In contrast, the intensities of the NMR signals belonging to the carbons of the ribityl side chain of FMN were not affected by light. The observation of spin-polarized (13)C-nuclei in the NMR spectra of the mutant LOV2 domain is clear evidence for radical-pair intermediates in the reaction steps following optical sample excitation.
Asunto(s)
Avena/genética , Flavoproteínas/química , Flavoproteínas/genética , Mutación/genética , Sitios de Unión , Color , Criptocromos , Mononucleótido de Flavina/metabolismo , Flavoproteínas/metabolismo , Luz , Fotoquímica , Estructura Terciaria de ProteínaRESUMEN
The recent observation of photoinduced radical pairs comprising a flavin radical and an oxidized amino acid residue in various blue-light-sensitive proteins has highlighted the need to gain a more complete understanding of the electronic structure of flavin radicals. In particular, precise knowledge of the anisotropy of the Zeeman interaction quantified by the g-tensor is necessary for attaining an unambiguous identification of flavin radicals by electron paramagnetic resonance (EPR). In a recent study of a protein-bound neutral flavin radical, we have determined the principal values of the g-tensor using high-frequency/high magnetic field EPR performed at 360 GHz/12.8 T. However, in those experiments, the orientation of the principal axes of g could not be unambiguously established with respect to the molecular frame of the isoalloxazine moiety. In this contribution we resolve this ambiguity by pulsed electron-nuclear double resonance (ENDOR) at 95 GHz/3.5 T (W-band). At such high values of the microwave frequency and the magnetic field, the g anisotropy provides improved spectral resolution compared to an ENDOR experiment performed at conventional 9.5 GHz/0.35 mT (X-band). This enables one to utilize Zeeman magnetoselection to obtain single-crystal-like data from disordered samples in frozen solution. Experiments exploiting this orientation selection have allowed us to use the hyperfine coupling of the methyl protons at C(8alpha) of the isoalloxazine ring to determine the angle between the molecular frame and the principal axes of g. Quite surprisingly, the g-tensor in FADH* is not oriented as one would have expected for a 1,3-semibenzoquinone radical. For the latter, the X-axis of g commonly bisects the smaller angle between the two axes along the C=O bonds. In FADH*, the large spin density on N(5) and C(4a) apparently contributes to a significant (44 degrees ) reorientation of the g-tensor axes.
RESUMEN
Cyclobutane-type pyrimidine dimers generated by ultraviolet irradiation of DNA can be cleaved by DNA photolyase. The enzyme-catalysed reaction is believed to be initiated by the light-induced transfer of an electron from the anionic FADH- chromophore of the enzyme to the pyrimidine dimer. In this contribution, first infrared experiments using a novel E109A mutant of Escherichia coli DNA photolyase, which is catalytically active but unable to bind the second cofactor methenyltetrahydrofolate, are described. A stable blue-coloured form of the enzyme carrying a neutral FADH radical cofactor can be interpreted as an intermediate analogue of the light-driven DNA repair reaction and can be reduced to the enzymatically active FADH- form by red-light irradiation. Difference Fourier transform infrared (FT-IR) spectroscopy was used to monitor vibronic bands of the blue radical form and of the fully reduced FADH- form of the enzyme. Preliminary band assignments are based on experiments with 15N-labelled enzyme and on experiments with D2O as solvent. Difference FT-IR measurements were also used to observe the formation of thymidine dimers by ultraviolet irradiation and their repair by light-driven photolyase catalysis. This study provides the basis for future time-resolved FT-IR studies which are aimed at an elucidation of a detailed molecular picture of the light-driven DNA repair process.
Asunto(s)
Desoxirribodipirimidina Fotoliasa/química , Desoxirribodipirimidina Fotoliasa/metabolismo , Escherichia coli/enzimología , Flavina-Adenina Dinucleótido/análogos & derivados , Ácido Fólico/análogos & derivados , Luz , Uridina/análogos & derivados , Bacillus subtilis/genética , Catálisis/efectos de la radiación , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/aislamiento & purificación , Activación Enzimática/efectos de la radiación , Escherichia coli/genética , Flavina-Adenina Dinucleótido/química , Flavina-Adenina Dinucleótido/metabolismo , Ácido Fólico/metabolismo , Mutación/genética , Fotoquímica , Espectroscopía Infrarroja por Transformada de Fourier , Timina/química , Timina/metabolismo , Timina/efectos de la radiación , Uridina/química , Uridina/metabolismo , Uridina/efectos de la radiaciónRESUMEN
An X- (9.7 GHz and W-band (94 GHz) pulsed electron-nuclear double resonance (ENDOR) study of the flavin cofactor of Escherichia coli DNA photolyase in its neutral radical form is presented. Through proton and deuteron ENDOR measurements at T = 80 K, we detect and characterize the full anisotropy of the hyperfine coupling (hfc) tensor of the proton or deuteron bound to N(5) of the isoalloxazine ring. Scaling of the anisotropic proton hfc components by multiplication with the quotient of the magnetogyric ratio of a deuteron and a proton, chiD/chiH, reveals subtle differences compared to the respective deuteron couplings obtained by 95-GHz deuterium ENDOR spectroscopy on an H-->D buffer-exchanged sample. These differences can be attributed to the different lengths of N(5)-H and N(5)-D bonds arising from the different masses of protons and deuterons. From the R(-3) dependence of the dipolar hyperfine splitting, we estimated that the N(5)-D bond is about 2.5% shorter than the respective N(5)-H bond. That such subtle bond-length differences can be resolved by pulsed ENDOR spectroscopy suggests that this method may be favorably used to probe the geometry of hydrogen bonds between the H(5) of the paramagnetic flavin and the protein backbone. Such information is only obtained with difficulty by other types of spectroscopy.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón/métodos , Flavinas/química , Enlace de Hidrógeno , Hidrógeno/química , Nitrógeno/química , Anisotropía , Desoxirribodipirimidina Fotoliasa/química , Deuterio/química , Espectroscopía de Resonancia por Spin del Electrón/instrumentación , Electrones , Escherichia coli/enzimología , Escherichia coli/metabolismo , Modelos Químicos , Estructura Molecular , Distribución Normal , Protones , Espectrometría por Rayos X , Espectrofotometría , TemperaturaRESUMEN
The blue-light sensitive photoreceptor, phototropin, is a flavoprotein which regulates the phototropism response of higher plants. The photoinduced triplet state and the photoreactivity of the flavin-mononucleotide (FMN) cofactor in two LOV domains of Avena sativa, Adiantum capillus-veneris, and Chlamydomonas reinhardtii phototropin have been studied by time-resolved electron paramagnetic resonance (EPR) and UV-vis spectroscopy at low temperatures (T < or = 80 K). Differences in the electronic structure of the FMN as reflected by altered zero-field splitting parameters of the triplet state could be correlated with changes in the amino acid composition of the binding pocket in wild-type LOV1 and LOV2 as well as in mutant LOV domains. Even at cryogenic temperatures, time-resolved EPR experiments indicate photoreactivity of the wild-type LOV domains, which was further characterized by UV-vis spectroscopy. Wild-type LOV1 and LOV2 were found to form an adduct between the FMN cofactor and the functional cysteine with a yield of 22% and 68%, respectively. The absorption maximum of the low-temperature photoproduct of wild-type LOV2 is red-shifted by about 15 nm as compared with the FMN C(4a)-cysteinyl adduct formed at room temperature. In light of these observations, we discuss a radical-pair reaction mechanism for the primary photoreaction in LOV domains.
Asunto(s)
Flavoproteínas/química , Adiantum/química , Secuencia de Aminoácidos , Animales , Avena/química , Chlamydomonas reinhardtii/química , Criptocromos , Espectroscopía de Resonancia por Spin del Electrón , Mononucleótido de Flavina/química , Mononucleótido de Flavina/metabolismo , Flavoproteínas/metabolismo , Flavoproteínas/fisiología , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Espectrofotometría UltravioletaRESUMEN
The C terminus of transcription factor NusA from Escherichia coli comprises two repeat units, which bind during antitermination to protein N from phage lambda. To delineate the structural basis of the NusA-lambdaN interaction, we attempted to crystallize the NusA C-terminal repeats in complex with a lambdaN peptide (residues 34-47). The two NusA domains became proteolytically separated during crystallization, and crystals contained two copies of the first repeat unit in contact with a single lambdaN fragment. The NusA modules employ identical regions to contact the peptide but approach the ligand from opposite sides. In contrast to the alpha-helical conformation of the lambdaN N terminus in complex with boxB RNA, residues 34-40 of lambdaN remain extended upon interaction with NusA. Mutational analyses indicated that only one of the observed NusA-lambdaN interaction modes is biologically significant, supporting an equimolar ratio of NusA and lambdaN in antitermination complexes. Solution studies indicated that additional interactions are fostered by the second NusA repeat unit, consistent with known compensatory mutations in NusA and lambdaN. Contrary to the RNA polymerase alpha subunit, lambdaN binding does not stimulate RNA interaction of NusA. The results demonstrate that lambdaN serves as a scaffold to closely oppose NusA and the mRNA in antitermination complexes.
Asunto(s)
Bacteriófago lambda/metabolismo , Escherichia coli/metabolismo , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/metabolismo , Factores de Elongación de Péptidos/química , Factores de Elongación de Péptidos/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Calorimetría , Dicroismo Circular , Cristalografía por Rayos X , Proteínas de Escherichia coli , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Termodinámica , Factores de Elongación TranscripcionalRESUMEN
The open reading frame MJ1184 of Methanococcus jannaschii with similarity to riboflavin synthase of Methanothermobacter thermoautotrophicus was cloned into an expression vector but was poorly expressed in an Escherichia coli host strain. However, a synthetic open reading frame that was optimized for expression in E.coli directed the synthesis of abundant amounts of a protein with an apparent subunit mass of 17.5 kDa. The protein was purified to apparent homogeneity. Hydrodynamic studies indicated a relative mass of 88 kDa suggesting a homopentamer structure. The enzyme was shown to catalyze the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a rate of 24 nmol mg(-1) min(-1) at 40 degrees C. Divalent metal ions, preferably manganese or magnesium, are required for maximum activity. In contrast to pentameric archaeal type riboflavin synthases, orthologs from plants, fungi and eubacteria are trimeric proteins characterized by an internal sequence repeat with similar folding patterns. In these organisms the reaction is achieved by binding the two substrate molecules in an antiparallel orientation. With the enzyme of M.jannaschii, 13C NMR spectroscopy with 13C-labeled 6,7-dimethyl-8-ribityllumazine samples as substrates showed that the regiochemistry of the dismutation reaction is the same as observed in eubacteria and eukaryotes, however, in a non-pseudo-c2 symmetric environment. Whereas the riboflavin synthases of M.jannaschii and M.thermoautotrophicus are devoid of similarity with those of eubacteria and eukaryotes, they have significant sequence similarity with 6,7-dimethyl-8-ribityllumazine synthases catalyzing the penultimate step of riboflavin biosynthesis. 6,7-Dimethyl-8-ribityllumazine synthase and the archaeal riboflavin synthase appear to have diverged early in the evolution of Archaea from a common ancestor. Some Archaea have eubacterial type riboflavin synthases which may have been acquired by lateral gene transfer.
Asunto(s)
Archaea/enzimología , Archaea/genética , Riboflavina/biosíntesis , Secuencia de Aminoácidos , Secuencia de Bases , Codón , Secuencia Conservada , Evolución Molecular , Cinética , Espectroscopía de Resonancia Magnética , Methanococcus/enzimología , Methanococcus/genética , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Riboflavina/química , Riboflavina/genética , Riboflavina/aislamiento & purificación , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Ionización de Electrospray , Especificidad por Sustrato , Temperatura , UltracentrifugaciónRESUMEN
NusB is a prokaryotic transcription factor involved in antitermination processes, during which it interacts with the boxA portion of the mRNA nut site. Previous studies have shown that NusB exhibits an all-helical fold, and that the protein from Escherichia coli forms monomers, while Mycobacterium tuberculosis NusB is a dimer. The functional significance of NusB dimerization is unknown. We have determined five crystal structures of NusB from Thermotoga maritima. In three crystal forms the protein appeared monomeric, whereas the two other crystal forms contained assemblies, which resembled the M. tuberculosis dimers. In solution, T. maritima NusB could be cross-linked as dimers, but it migrated as a monomer in gel-filtration analyses, suggesting a monomer/dimer equilibrium with a preference for the monomer. Binding to boxA-like RNA sequences could be detected by gel-shift analyses and UV-induced cross-linking. An N-terminal arginine-rich sequence is a probable RNA binding site of the protein, exhibiting aromatic residues as potential stacking partners for the RNA bases. Anions located in various structures support the assignment of this RNA binding site. The proposed RNA binding region is hidden in the subunit interface of dimeric NusB proteins, such as NusB from M. tuberculosis, suggesting that such dimers have to undergo a considerable conformational change or dissociate for engagement with RNA. Therefore, in certain organisms, dimerization may be employed to package NusB in an inactive form until recruitment into antitermination complexes.
Asunto(s)
Thermotoga maritima/química , Factores de Transcripción/química , Secuencia de Aminoácidos/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión/genética , Clonación Molecular/métodos , Cristalización/métodos , Bases de Datos de Proteínas , Dimerización , Proteínas de Escherichia coli/química , Regulación de la Expresión Génica/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Mycobacterium tuberculosis/química , Péptidos/química , Filogenia , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína/genética , ARN Bacteriano/metabolismo , Regiones Terminadoras Genéticas/genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Transcripción Genética/genética , Difracción de Rayos X/métodosRESUMEN
An open reading frame (Acc. no. P50740) on the Bacillus subtilis chromosome extending from bp 184,997-186,043 with similarity to the idi-2 gene of Streptomyces sp. CL190 specifying type II isopentenyl diphosphate isomerase was expressed in a recombinant Escherichia coli strain. The recombinant protein with a subunit mass of 39 kDa was purified to apparent homogeneity by column chromatography. The protein was shown to catalyse the conversion of dimethylallyl diphosphate into isopentenyl diphosphate and vice versa at rates of 0.23 and 0.63 micromol.mg(-1).min(-1), respectively, as diagnosed by 1H spectroscopy. FMN and divalent cations are required for catalytic activity; the highest rates were found with Ca2+. NADPH is required under aerobic but not under anaerobic assay conditions. The enzyme is related to a widespread family of (S)-alpha-hydroxyacid oxidizing enzymes including flavocytochrome b2 and L-lactate dehydrogenase and was shown to catalyse the formation of [2,3-13C2]lactate from [2,3-13C2]pyruvate, albeit at a low rate of 1 nmol.mg(-1).min(-1). Putative genes specifying type II isopentenyl diphosphate isomerases were found in the genomes of Archaea and of certain eubacteria but not in the genomes of fungi, animals and plants. The analysis of the occurrence of idi-1 and idi-2 genes in conjunction with the mevalonate and nonmevalonate pathway in 283 completed and unfinished prokaryotic genomes revealed 10 different classes. Type II isomerase is essential in some important human pathogens including Staphylococcus aureus and Enterococcus faecalis where it may represent a novel target for anti-infective therapy.
Asunto(s)
Bacillus subtilis/enzimología , Isomerasas/metabolismo , Terpenos/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Catálisis , Cartilla de ADN , Isomerasas/química , Isomerasas/genética , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Filogenia , Homología de Secuencia de Aminoácido , Difracción de Rayos XRESUMEN
The Arabidopsis thaliana open reading frame At4g20960 predicts a protein whose N-terminal part is similar to the eubacterial 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate deaminase domain. A synthetic open reading frame specifying a pseudomature form of the plant enzyme directed the synthesis of a recombinant protein which was purified to apparent homogeneity and was shown by NMR spectroscopy to convert 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate into 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate at a rate of 0.9 micromol mg(-1) min(-1). The substrate and product of the enzyme are both subject to spontaneous anomerization of the ribosyl side chain as shown by (13)C NMR spectroscopy. The protein contains 1 eq of Zn(2+)/subunit. The deaminase activity could be assigned to the N-terminal section of the plant protein. The deaminase domains of plants and eubacteria share a high degree of similarity, in contrast to deaminases from fungi. These data show that the riboflavin biosynthesis in plants proceeds by the same reaction steps as in eubacteria, whereas fungi use a different pathway.
Asunto(s)
Nucleótido Desaminasas/química , Nucleótido Desaminasas/metabolismo , Riboflavina/biosíntesis , Secuencia de Aminoácidos , Arabidopsis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Secuencia de Bases , Fenómenos Bioquímicos , Bioquímica , Proteínas Portadoras/química , Clonación Molecular , ADN/metabolismo , Enzimas de Restricción del ADN/farmacología , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Evolución Molecular , GTP Ciclohidrolasa/química , Prueba de Complementación Genética , Guanosina Trifosfato/química , Cinética , Espectroscopía de Resonancia Magnética , Proteínas de Unión a Maltosa , Modelos Químicos , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Oligonucleótidos/química , Sistemas de Lectura Abierta , Filogenia , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Ionización de Electrospray , Espectrofotometría Atómica , Deshidrogenasas del Alcohol de Azúcar/química , Factores de Tiempo , Zinc/químicaRESUMEN
BACKGROUND: Riboflavin synthase catalyzes the transformation of 6,7-dimethyl-8-ribityllumazine into riboflavin in the last step of the riboflavin biosynthetic pathway. Gram-negative bacteria and certain yeasts are unable to incorporate riboflavin from the environment and are therefore absolutely dependent on endogenous synthesis of the vitamin. Riboflavin synthase is therefore a potential target for the development of antiinfective drugs. RESULTS: A cDNA sequence from Schizosaccharomyces pombe comprising a hypothetical open reading frame with similarity to riboflavin synthase of Escherichia coli was expressed in a recombinant E. coli strain. The recombinant protein is a homotrimer of 23 kDa subunits as shown by sedimentation equilibrium centrifugation. The protein sediments at an apparent velocity of 4.1 S at 20 degrees C. The amino acid sequence is characterized by internal sequence similarity indicating two similar folding domains per subunit. The enzyme catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a rate of 158 nmol mg(-1) min(-1) with an apparent KM of 5.7 microM. 19F NMR protein perturbation experiments using fluorine-substituted intermediate analogs show multiple signals indicating that a given ligand can be bound in at least 4 different states. 19F NMR signals of enzyme-bound intermediate analogs were assigned to ligands bound by the N-terminal respectively C-terminal folding domain on basis of NMR studies with mutant proteins. CONCLUSION: Riboflavin synthase of Schizosaccharomyces pombe is a trimer of identical 23-kDa subunits. The primary structure is characterized by considerable similarity of the C-terminal and N-terminal parts. Riboflavin synthase catalyzes a mechanistically complex dismutation of 6,7-dimethyl-8-ribityllumazine affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. The 19F NMR data suggest large scale dynamic mobility in the trimeric protein which may play an important role in the reaction mechanism.
Asunto(s)
Riboflavina Sintasa/química , Schizosaccharomyces/enzimología , Secuencia de Aminoácidos , Flúor , Datos de Secuencia Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Pteridinas/metabolismo , Riboflavina/biosíntesis , Riboflavina Sintasa/genética , Riboflavina Sintasa/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Skeletal rearrangements of carbohydrates are crucial for many biosynthetic pathways. In riboflavin biosynthesis ribulose 5-phosphate is converted into 3,4-dihydroxy-2-butanone 4-phosphate while its C4 atom is released as formate in a sequence of metal-dependent reactions. Here, we present the crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate ribulose 5-phosphate at a dimetal center presumably consisting of non-catalytic zinc and calcium ions at 1.7-A resolution. The carbonyl group (O2) and two out of three free hydroxyl groups (OH3 and OH4) of the substrate are metal-coordinated. We correlate previous mutational studies on this enzyme with the present structural results. Residues of the first coordination sphere involved in metal binding are indispensable for catalytic activity. Only Glu-185 of the second coordination sphere cannot be replaced without complete loss of activity. It contacts the C3 hydrogen atom directly and probably initiates enediol formation in concert with both metal ions to start the reaction sequence. Mechanistic similarities to Rubisco acting on the similar substrate ribulose 1,5-diphosphate in carbon dioxide fixation as well as other carbohydrate (reducto-) isomerases are discussed.
Asunto(s)
Transferasas Intramoleculares/química , Metales Pesados/química , Methanococcus/enzimología , Ribulosafosfatos/química , Sitios de Unión , Catálisis , Cationes Bivalentes , Cristalografía por Rayos X , Manganeso/química , Modelos Moleculares , Estructura Molecular , Unión Proteica , Riboflavina/biosíntesisRESUMEN
The regulation of ribosomal RNA biosynthesis in Escherichia coli by antitermination requires binding of NusB protein to a dodecamer sequence designated boxA on the nascent RNA. The affinity of NusB protein for boxA RNA exceeds that for the homologous DNA segment by more than three orders of magnitude as shown by surface plasmon resonance measurements. DNA RNA discrimination by NusB protein was shown to involve methyl groups (i.e. discrimination of uracil versus thymine) and 2' hydroxyl groups (i.e. discrimination of ribose versus deoxyribose side-chains) in the RNA motif. Ligand perturbation experiments monitored by 1H15N correlation NMR experiments identified amide NH groups whose chemical shifts are affected selectively by ribose/deoxyribose exchange in the 5' and the central part of the dodecameric boxA motif respectively. The impact of structural modification of the boxA motif on the affinity for NusB protein as observed by 1H15N heterocorrelation was analysed by a generic algorithm.
Asunto(s)
Proteínas Bacterianas/fisiología , ADN/metabolismo , Proteínas de Escherichia coli , Proteínas de Unión al ARN/fisiología , ARN/metabolismo , Factores de Transcripción/fisiología , Secuencia de Bases , ADN/química , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Regiones Promotoras Genéticas , ARN/química , Homología de Secuencia de Ácido Nucleico , Resonancia por Plasmón de SuperficieRESUMEN
The LOV2 domain of Avena sativa phototropin and its C450A mutant were expressed as recombinant fusion proteins and were examined by optical spectroscopy, electron paramagnetic resonance, and electron-nuclear double resonance. Upon irradiation (420-480 nm), the LOV2 C450A mutant protein gave an optical absorption spectrum characteristic of a flavin radical even in the absence of exogenous electron donors, thus demonstrating that the flavin mononucleotide (FMN) cofactor in its photogenerated triplet state is a potent oxidant for redox-active amino acid residues within the LOV2 domain. The FMN radical in the LOV2 C450A mutant is N(5)-protonated, suggesting that the local pH close to the FMN is acidic enough so that the cysteine residue in the wild-type protein is likely to be also protonated. An electron paramagnetic resonance analysis of the photogenerated FMN radical gave information on the geometrical and electronic structure and the environment of the FMN cofactor. The experimentally determined hyperfine couplings of the FMN radical point to a highly restricted delocalization of the unpaired electron spin in the isoalloxazine moiety. In the light of these results a possible radical-pair mechanism for the formation of the FMN-C(4a)-cysteinyl adduct in LOV domains is discussed.
Asunto(s)
Avena/fisiología , Proteínas de Drosophila , Proteínas del Ojo , Flavinas/biosíntesis , Flavoproteínas/química , Luz , Células Fotorreceptoras de Invertebrados , Criptocromos , Espectroscopía de Resonancia por Spin del Electrón , Flavinas/química , Flavoproteínas/genética , Datos de Secuencia Molecular , Plásmidos , Receptores Acoplados a Proteínas G , Espectrofotometría UltravioletaRESUMEN
Flavocoenzymes labeled with stable isotopes are important reagents for the study of flavoproteins using isotope-sensitive methods such as NMR, ENDOR, infrared, and Raman spectroscopy. We describe highly versatile one-pot methods for the preparation of riboflavin isotopomers labeled with (13)C in every desired position of the xylene moiety. The starting materials are commercially available (13)C-labeled glucose samples, which are converted into riboflavin using enzymes of the oxidative pentose phosphate pathway in combination with recombinant enzymes of the riboflavin biosynthetic pathway. The overall reaction comprises six enzyme-catalyzed reaction steps for the synthesis of the vitamin and two auxiliary enzymes for in situ recycling of cofactors. The overall yields of riboflavin based on isotope-labeled glucose are 35-50%.
Asunto(s)
Mononucleótido de Flavina/síntesis química , Riboflavina/análisis , Riboflavina/síntesis química , Bacillus subtilis/enzimología , Isótopos de Carbono , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/enzimología , Indicadores y Reactivos , Transferasas Intramoleculares/metabolismo , Isomerismo , Marcaje Isotópico/métodos , Espectroscopía de Resonancia Magnética , Estructura Molecular , Complejos Multienzimáticos/metabolismo , Riboflavina Sintasa/metabolismo , Espectrofotometría Infrarroja , Espectrometría RamanRESUMEN
The hypothetical protein predicted by the open reading frame MJ0055 of Methanococcus jannaschii was expressed in a recombinant Escherichia coli strain under the control of a synthetic gene optimized for translation in an eubacterial host. The recombinant protein catalyzes the formation of the riboflavin precursor 3,4-dihydroxy-2-butanone 4-phosphate from ribulose 5-phosphate at a rate of 174 nmol mg(-1) min(-1) at 37 degrees C. The homodimeric 51.6-kDa protein requires divalent metal ions, preferentially magnesium, for activity. The reaction involves an intramolecular skeletal rearrangement as shown by (13)C NMR spectroscopy using [U-(13)C(5)]ribulose 5-phosphate as substrate. A cluster of charged amino acid residues comprising arginine 25, glutamates 26 and 28, and aspartates 21 and 30 is essential for catalytic activity. Histidine 164 and glutamate 185 were also shown to be essential for catalytic activity.