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Background & Aims: Induction of potent, HBV-specific immune responses is crucial to control and finally cure HBV. The therapeutic hepatitis B vaccine TherVacB combines protein priming with a Modified Vaccinia virus Ankara (MVA)-vector boost to break immune tolerance in chronic HBV infection. Particulate protein and vector vaccine components, however, require a constant cooling chain for storage and transport, posing logistic and financial challenges to vaccine applications. We aimed to identify an optimal formulation to maintain stability and immunogenicity of the protein and vector components of the vaccine using a systematic approach. Methods: We used stabilizing amino acid (SAA)-based formulations to stabilize HBsAg and HBV core particles (HBcAg), and the MVA-vector. We then investigated the effect of lyophilization and short- and long-term high-temperature storage on their integrity. Immunogenicity and safety of the formulated vaccine was validated in HBV-naïve and adeno-associated virus (AAV)-HBV-infected mice. Results: In vitro analysis proved the vaccine's stability against thermal stress during lyophilization and the long-term stability of SAA-formulated HBsAg, HBcAg and MVA during thermal stress at 40 °C for 3 months and at 25 °C for 12 months. Vaccination of HBV-naïve and AAV-HBV-infected mice demonstrated that the stabilized vaccine was well tolerated and able to brake immune tolerance established in AAV-HBV mice as efficiently as vaccine components constantly stored at 4 °C/-80 °C. Even after long-term exposure to elevated temperatures, stabilized TherVacB induced high titre HBV-specific antibodies and strong CD8+ T-cell responses, resulting in anti-HBs seroconversion and strong suppression of the virus in HBV-replicating mice. Conclusion: SAA-formulation resulted in highly functional and thermostable HBsAg, HBcAg and MVA vaccine components. This will facilitate global vaccine application without the need for cooling chains and is important for the development of prophylactic as well as therapeutic vaccines supporting vaccination campaigns worldwide. Impact and implications: Therapeutic vaccination is a promising therapeutic option for chronic hepatitis B that may enable its cure. However, its application requires functional cooling chains during transport and storage that can hardly be guaranteed in many countries with high demand. In this study, the authors developed thermostable vaccine components that are well tolerated and that induce immune responses and control the virus in preclinical mouse models, even after long-term exposure to high surrounding temperatures. This will lower costs and ease application of a therapeutic vaccine and thus be beneficial for the many people affected by hepatitis B around the world.
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Specifically tailored amino acid-based formulations were previously shown to have a high potential to avoid stress-mediated degradation of complex molecules such as monoclonal antibodies and viral vectors. By using adenovirus 5 (Ad5) as a model, we studied whether such formulations may also efficiently protect viral vectors in thermal stress experiments and during long-term liquid storage. Algorithm-based amino acid preselection using an excipient database and subsequent application of design of experiments (DoE) in combination with a 37°C challenging model enabled the prediction of long-term storage stability of Ad5. By statistical analysis of the Ad5 infectivity, amino acids with significant influence on Ad5 stability were detected after 2 and 3 weeks of liquid storage at 37°C. Ad5 formulations comprising positively selected amino acids did not reveal any loss of infectivity after 24 months in liquid storage at 5°C. By contrast, a 2 log reduction after 3 months and complete loss of infectivity after 18 months was observed with a standard viral vector formulation. By an optimization round, we designed a simple and well-balanced formulation avoiding MgCl2, previously considered essential in Ad5 formulations. This work demonstrates the efficacy of an algorithm-based development approach in the formulation development for viral vectors.
Assuntos
Adenovírus Humanos/genética , Algoritmos , Aminoácidos/química , DNA Viral/química , Excipientes/química , Técnicas de Transferência de Genes , Vetores Genéticos , DNA Viral/metabolismo , Células HEK293 , Humanos , Desnaturação de Ácido Nucleico , Temperatura , Fatores de TempoRESUMO
To develop highly concentrated therapeutic antibodies enabling convenient subcutaneous application, well stabilizing pharmaceutical formulations with low viscosities are considered to be key. The purpose of this study is to select specific amino acid combinations that reduce and balance aggregation, fragmentation and chemical degradation, and also lower viscosity of highly concentrated liquid antibodies. As a model, the therapeutically well-established antibody trastuzumab (25->200 mg mL-1 ) in liquid formulation is used. Pre-testing of formulations based on a stabilizing and protecting solutions (SPS®) platform is conducted in a thermal unfolding model using differential scanning fluorimetry (DSF) and accelerated aging at 37 and 45 °C. Pre-selected amino acid combinations are further iteratively adjusted to obtain stable highly concentrated antibody formulations with low viscosity. Size exclusion chromatography (SE-HPLC) reveals significantly lower aggregation and fragmentation at specific amino acid:sugar and protein:excipient ratios. Dynamic viscosities <20 mPa * s of highly concentrated trastuzumab (≥200 mg mL-1 ) are measured by falling ball viscosimetry. Moreover, less chemical degradation is found by cationic exchange chromatography (CEX-HPLC) even after 6 months liquid storage at 25 °C. In conclusion, specifically tailored and advanced amino acid-based liquid formulations avoid aggregation and enable the development of stable and low viscous highly concentrated biopharmaceuticals.
Assuntos
Aminoácidos/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Animais , Anticorpos Monoclonais/análise , Células CHO , Cromatografia em Gel , Cricetinae , Cricetulus , Fluorometria , Humanos , Estabilidade Proteica , Desdobramento de Proteína , Temperatura , Trastuzumab , ViscosidadeRESUMO
The implementation of expanded newborn screening programs reduced mortality and morbidity in medium-chain acyl-CoA dehydrogenase deficiency (MCADD) caused by mutations in the ACADM gene. However, the disease is still potentially fatal. Missense induced MCADD is a protein misfolding disease with a molecular loss-of-function phenotype. Here we established a comprehensive experimental setup to analyze the structural consequences of eight ACADM missense mutations (p.Ala52Val, p.Tyr67His, p.Tyr158His, p.Arg206Cys, p.Asp266Gly, p.Lys329Glu, p.Arg334Lys, p.Arg413Ser) identified after newborn screening and linked the corresponding protein misfolding phenotype to the site of side-chain replacement with respect to the domain. With fever being the crucial risk factor for metabolic decompensation of patients with MCADD, special emphasis was put on the analysis of structural and functional derangements related to thermal stress. Based on protein conformation, thermal stability and kinetic stability, the molecular phenotype in MCADD depends on the structural region that is affected by missense-induced conformational changes with the central ß-domain being particularly prone to structural derangement and destabilization. Since systematic classification of conformational derangements induced by ACADM mutations may be a helpful tool in assessing the clinical risk of patients, we scored the misfolding phenotype of the variants in comparison to p.Lys329Glu (K304E), the classical severe mutation, and p.Tyr67His (Y42H), discussed to be mild. Experiments assessing the impact of thermal stress revealed that mutations in the ACADM gene lower the temperature threshold at which MCAD loss-of-function occurs. Consequently, increased temperature as it occurs during intercurrent infections, significantly increases the risk of further conformational derangement and loss of function of the MCAD enzyme explaining the life-threatening clinical courses observed during fever episodes. Early and aggressive antipyretic treatment thus may be life-saving in patients suffering from MCADD.
Assuntos
Acil-CoA Desidrogenase/química , Acil-CoA Desidrogenase/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Dobramento de Proteína , Temperatura , Animais , Células COS , Chlorocebus aethiops , Dicroísmo Circular , Ativação Enzimática , Flavina-Adenina Dinucleotídeo/metabolismo , Fluorescência , Temperatura Alta , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Mutação de Sentido Incorreto/genética , Fenótipo , Agregados Proteicos , Desnaturação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Currently, the need for cooled storage and the impossibility of terminal sterilisation are major drawbacks in vaccine manufacturing and distribution. To overcome current restrictions a preclinical safety and efficacy study was conducted to evaluate new influenza A vaccine formulations regarding thermal resistance, resistance against irradiation-mediated damage and storage stability. We evaluated the efficacy of novel antigen stabilizing and protecting solutions (SPS) to protect influenza A(H1N1)pdm09 split virus antigen under experimental conditions in vitro and in vivo. Original or SPS re-buffered vaccine (Pandemrix) was spray-dried and terminally sterilised by irradiation with 25 kGy (e-beam). Antigen integrity was monitored by SDS-PAGE, dynamic light scattering, size exclusion chromatography and functional haemagglutination assays. In vitro screening experiments revealed a number of highly stable compositions containing glycyrrhizinic acid (GA) and/or chitosan. The most stable composition was selected for storage tests and in vivo assessment of seroconversion in non-human primates (Macaca fascicularis) using a prime-boost strategy. Redispersed formulations with original adjuvant were administered intramuscularly. Storage data revealed high stability of protected vaccines at 4°C and 25°C, 60% relative humidity, for at least three months. Animals receiving original Pandemrix exhibited expected levels of seroconversion after 21 days (prime) and 48 days (boost) as assessed by haemagglutination inhibition and microneutralisation assays. Animals vaccinated with spray-dried and irradiated Pandemrix failed to exhibit seroconversion after 21 days whereas spray-dried and irradiated, SPS-protected vaccines elicited similar seroconversion levels to those vaccinated with original Pandemrix. Boost immunisation with SPS-protected vaccine resulted in a strong increase in seroconversion but had only minor effects in animals treated with non SPS-protected vaccine. In conclusion, utilising the SPS formulation technology, spray-drying and terminal sterilisation of influenza A(H1N1)pdm09 split virus vaccine is feasible. Findings indicate the potential utility of such formulated vaccines e.g. for needle-free vaccination routes and delivery to countries with uncertain cold chain facilities.
Assuntos
Vacinas contra Influenza/química , Vacinas contra Influenza/uso terapêutico , Infecções por Orthomyxoviridae/prevenção & controle , Potência de Vacina , Animais , Dessecação , Armazenamento de Medicamentos , Excipientes/química , Feminino , Ácido Glicirrízico/química , Testes de Inibição da Hemaglutinação , Vírus da Influenza A Subtipo H1N1 , Macaca fascicularis , Masculino , Manitol/química , Testes de Neutralização , Pós , Esterilização , Trealose/químicaRESUMO
Phenylketonuria (PKU) is caused by inherited phenylalanine-hydroxylase (PAH) deficiency and, in many genotypes, it is associated with protein misfolding. The natural cofactor of PAH, tetrahydrobiopterin (BH(4)), can act as a pharmacological chaperone (PC) that rescues enzyme function. However, BH(4) shows limited efficacy in some PKU genotypes and its chemical synthesis is very costly. Taking an integrated drug discovery approach which has not been applied to this target before, we identified alternative PCs for the treatment of PKU. Shape-focused virtual screening of the National Cancer Institute's chemical library identified 84 candidate molecules with potential to bind to the active site of PAH. An in vitro evaluation of these yielded six compounds that restored the enzymatic activity of the unstable PAHV106A variant and increased its stability in cell-based assays against proteolytic degradation. During a 3-day treatment study, two compounds (benzylhydantoin and 6-amino-5-(benzylamino)-uracil) substantially improved the in vivo Phe oxidation and blood Phe concentrations of PKU mice (Pah(enu1)). Notably, benzylhydantoin was twice as effective as tetrahydrobiopterin. In conclusion, we identified two PCs with high in vivo efficacy that may be further developed into a more effective drug treatment of PKU.
Assuntos
Hidantoínas/metabolismo , Fenilalanina Hidroxilase/metabolismo , Fenilcetonúrias/tratamento farmacológico , Uracila/análogos & derivados , Animais , Sítios de Ligação , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Domínio Catalítico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Estabilidade Enzimática , Humanos , Hidantoínas/química , Hidantoínas/farmacologia , Hidantoínas/toxicidade , Camundongos , Oxirredução , Fenilalanina/metabolismo , Fenilalanina Hidroxilase/química , Fenilalanina Hidroxilase/deficiência , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/metabolismo , Dobramento de Proteína , Bibliotecas de Moléculas Pequenas , Uracila/química , Uracila/metabolismo , Uracila/farmacologia , Uracila/toxicidadeRESUMO
The discovery of a pharmacological treatment for phenylketonuria (PKU) raised new questions about function and dysfunction of phenylalanine hydroxylase (PAH), the enzyme deficient in this disease. To investigate the interdependence of the genotype, the metabolic state (phenylalanine substrate) and treatment (BH(4) cofactor) in the context of enzyme function in vitro and in vivo, we (i) used a fluorescence-based method for fast enzyme kinetic analyses at an expanded range of phenylalanine and BH(4) concentrations, (ii) depicted PAH function as activity landscapes, (iii) retraced the analyses in eukaryotic cells, and (iv) translated this into the human system by analyzing the outcome of oral BH(4) loading tests. PAH activity landscapes uncovered the optimal working range of recombinant wild-type PAH and provided new insights into PAH kinetics. They demonstrated how mutations might alter enzyme function in the space of varying substrate and cofactor concentrations. Experiments in eukaryotic cells revealed that the availability of the active PAH enzyme depends on the phenylalanine-to-BH(4) ratio. Finally, evaluation of data from BH(4) loading tests indicated that the patient's genotype influences the impact of the metabolic state on drug response. The results allowed for visualization and a better understanding of PAH function in the physiological and pathological state as well as in the therapeutic context of cofactor treatment. Moreover, our data underscore the need for more personalized procedures to safely identify and treat patients with BH(4)-responsive PAH deficiency.
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Biopterinas/análogos & derivados , Coenzimas/uso terapêutico , Genótipo , Fenilalanina Hidroxilase/genética , Fenilalanina Hidroxilase/metabolismo , Fenilalanina/metabolismo , Fenilcetonúrias , Biopterinas/farmacologia , Biopterinas/uso terapêutico , Coenzimas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Cinética , Chaperonas Moleculares/metabolismo , Mutação/genética , Fenilalanina Hidroxilase/deficiência , Fenilcetonúrias/tratamento farmacológico , Fenilcetonúrias/enzimologia , Fenilcetonúrias/genéticaRESUMO
Protein misfolding with loss-of-function of the enzyme phenylalanine hydroxylase (PAH) is the molecular basis of phenylketonuria in many individuals carrying missense mutations in the PAH gene. PAH is complexly regulated by its substrate L-Phenylalanine and its natural cofactor 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)). Sapropterin dihydrochloride, the synthetic form of BH(4), was recently approved as the first pharmacological chaperone to correct the loss-of-function phenotype. However, current knowledge about enzyme function and regulation in the therapeutic setting is scarce. This illustrates the need for comprehensive analyses of steady state kinetics and allostery beyond single residual enzyme activity determinations to retrace the structural impact of missense mutations on the phenylalanine hydroxylating system. Current standard PAH activity assays are either indirect (NADH) or discontinuous due to substrate and product separation before detection. We developed an automated fluorescence-based continuous real-time PAH activity assay that proved to be faster and more efficient but as precise and accurate as standard methods. Wild-type PAH kinetic analyses using the new assay revealed cooperativity of activated PAH toward BH(4), a previously unknown finding. Analyses of structurally preactivated variants substantiated BH(4)-dependent cooperativity of the activated enzyme that does not rely on the presence of l-Phenylalanine but is determined by activating conformational rearrangements. These findings may have implications for an individualized therapy, as they support the hypothesis that the patient's metabolic state has a more significant effect on the interplay of the drug and the conformation and function of the target protein than currently appreciated.
Assuntos
Biopterinas/análogos & derivados , Coenzimas/química , Fenilalanina Hidroxilase/química , Fenilalanina/química , Regulação Alostérica/genética , Biopterinas/química , Biopterinas/metabolismo , Biopterinas/uso terapêutico , Coenzimas/metabolismo , Coenzimas/uso terapêutico , Ativação Enzimática/genética , Fluorescência , Humanos , Cinética , Mutação de Sentido Incorreto , Fenilalanina/genética , Fenilalanina/metabolismo , Fenilalanina Hidroxilase/genética , Fenilalanina Hidroxilase/metabolismo , Fenilcetonúrias/tratamento farmacológico , Fenilcetonúrias/enzimologia , Fenilcetonúrias/genéticaRESUMO
The recent approval of sapropterin dihydrochloride, the synthetic form of 6[R]-l-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)), for the treatment of phenylketonuria (PKU) as the first pharmacological chaperone drug initiated a paradigm change in the treatment of monogenetic diseases. Symptomatic treatment is now replaced by a causal pharmacological therapy correcting misfolding of the defective phenylalanine hydroxylase (PAH) in numerous patients. Here, we disclose BH(4) responsiveness in Pah(enu1), a mouse model for PAH deficiency. Loss of function resulted from loss of PAH, a consequence of misfolding, aggregation, and accelerated degradation of the enzyme. BH(4) attenuated this triad by conformational stabilization augmenting the effective PAH concentration. This led to the rescue of the biochemical phenotype and enzyme function in vivo. Combined in vitro and in vivo analyses revealed a selective pharmaceutical action of BH(4) confined to the pathological metabolic state. Our data provide new molecular-level insights into the mechanisms underlying protein misfolding with loss of function and support a general model of pharmacological chaperone-induced stabilization of protein conformation to correct this intracellular phenotype. Pah(enu1) will be essential for pharmaceutical drug optimization and to design individually tailored therapies.
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Biopterinas/análogos & derivados , Modelos Animais de Doenças , Chaperonas Moleculares/metabolismo , Fenilalanina Hidroxilase/deficiência , Substituição de Aminoácidos/genética , Animais , Biopterinas/farmacologia , Células COS , Chlorocebus aethiops , Humanos , Hidroxilação/efeitos dos fármacos , Cinética , Camundongos , Mutação/genética , Fenilalanina/metabolismo , Fenilalanina Hidroxilase/química , Fenilalanina Hidroxilase/metabolismo , Dobramento de Proteína/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estrutura Quaternária de ProteínaRESUMO
Newborn screening (NBS) for medium-chain acyl-CoA dehydrogenase deficiency (MCADD) revealed a higher birth prevalence and genotypic variability than previously estimated, including numerous novel missense mutations in the ACADM gene. On average, these mutations are associated with milder biochemical phenotypes raising the question about their pathogenic relevance. In this study, we analyzed the impact of 10 ACADM mutations identified in NBS (A27V, Y42H, Y133H, R181C, R223G, D241G, K304E, R309K, I331T and R388S) on conformation, stability and enzyme kinetics of the corresponding proteins. Partial to total rescue of aggregation by co-overexpression of GroESL indicated protein misfolding. This was confirmed by accelerated thermal unfolding in all variants, as well as decreased proteolytic stability and accelerated thermal inactivation in most variants. Catalytic function varied from high residual activity to markedly decreased activity or substrate affinity. Mutations mapping to the beta-domain of the protein predisposed to severe destabilization. In silico structural analyses of the affected amino acid residues revealed involvement in functionally relevant networks. Taken together, our results substantiate the hypothesis of protein misfolding with loss-of-function being the common molecular basis in MCADD. Moreover, considerable structural alterations in all analyzed variants do not support the view that novel mutations found in NBS bear a lower risk of metabolic decompensation than that associated with mutations detected in clinically ascertained patients. Finally, the detailed insight into how ACADM missense mutations induce loss of MCAD function may provide guidance for risk assessment and counseling of patients, and in future may assist delineation of novel pharmacological strategies.
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Acil-CoA Desidrogenase/química , Acil-CoA Desidrogenase/deficiência , Erros Inatos do Metabolismo Lipídico/enzimologia , Triagem Neonatal , Dobramento de Proteína , Acil-CoA Desidrogenase/genética , Substituição de Aminoácidos , Estabilidade Enzimática , Feminino , Humanos , Recém-Nascido , Cinética , Erros Inatos do Metabolismo Lipídico/genética , Masculino , Conformação Molecular , Dados de Sequência Molecular , Mutação de Sentido IncorretoRESUMO
A significant share of patients with phenylalanine hydroxylase (PAH) deficiency benefits from pharmacological doses of tetrahydrobiopterin (BH(4)), the natural PAH cofactor. Phenylketonuria (PKU) is hypothesized to be a conformational disease, with loss of function due to protein destabilization, and the restoration of enzyme function that is observed in BH(4) treatment might be transmitted by correction of protein misfolding. To elucidate the molecular basis of functional impairment in PAH deficiency, we investigated the impact of ten PAH gene mutations identified in patients with BH(4)-responsiveness on enzyme kinetics, stability, and conformation of the protein (F55L, I65S, H170Q, P275L, A300S, S310Y, P314S, R408W, Y414C, Y417H). Residual enzyme activity was generally high, but allostery was disturbed in almost all cases and pointed to altered protein conformation. This was confirmed by reduced proteolytic stability, impaired tetramer assembly or aggregation, increased hydrophobicity, and accelerated thermal unfolding--with particular impact on the regulatory domain--observed in most variants. Three-dimensional modeling revealed the involvement of functionally relevant amino acid networks that may communicate misfolding throughout the protein. Our results substantiate the view that PAH deficiency is a protein-misfolding disease in which global conformational changes hinder molecular motions essential for physiological enzyme function. Thus, PKU has evolved from a model of a genetic disease that leads to severe neurological impairment to a model of a treatable protein-folding disease with loss of function.
Assuntos
Movimento (Física) , Fenilalanina Hidroxilase/deficiência , Fenilalanina Hidroxilase/metabolismo , Fenilcetonúrias/enzimologia , Fenilcetonúrias/genética , Administração Oral , Regulação Alostérica , Erros Inatos do Metabolismo dos Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Biopterinas/administração & dosagem , Biopterinas/análogos & derivados , Biopterinas/uso terapêutico , Domínio Catalítico , Simulação por Computador , Dimerização , Endopeptidase K/farmacologia , Estabilidade Enzimática , Feminino , Temperatura Alta , Humanos , Ligação de Hidrogênio , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Recém-Nascido , Cinética , Luminescência , Masculino , Modelos Moleculares , Mutação de Sentido Incorreto , Fenilalanina/sangue , Fenilalanina/metabolismo , Fenilalanina Hidroxilase/análise , Fenilalanina Hidroxilase/química , Fenilalanina Hidroxilase/genética , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína/genética , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Eletricidade EstáticaRESUMO
Riboflavin synthase from Escherichia coli is a homotrimer of 23.4 kDa subunits and catalyzes the formation of one molecule each of riboflavin and 5-amino-6-ribitylamino- 2,4(1H,3H)-pyrimidinedione by the transfer of a 4-carbon moiety between two molecules of the substrate, 6,7- dimethyl-8-ribityllumazine. Each subunit comprises two closely similar folding domains. Recombinant expression of the N-terminal domain is known to provide a c(2)-symmetric homodimer. In this study, the binding properties of wild type as well as two mutated proteins of N-terminal domain of riboflavin synthase with various ligands were tested. The replacement of the amino acid residue A43, located in the second shell of riboflavin synthase active center, in the recombinant N-terminal domain dimer reduces the affinity for 6,7-dimethyl-8-ribityllumazine. The mutation of the amino acid residue C48 forming part of activity cavity of the enzyme causes significant (19)F NMR chemical shift modulation of trifluoromethyl derivatives of 6,7-dimethyl-8-ribityllumazine in complex with the protein, while substitution of A43 results in smaller chemical shift changes.
Assuntos
Escherichia coli/enzimologia , Riboflavina Sintase/química , Riboflavina Sintase/metabolismo , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Estereoisomerismo , Especificidade por Substrato , TitulometriaRESUMO
Thioredoxin reductase (TrxR) from Escherichia coli, the mutant proteins E159Y and C138S, and the mutant protein C138S treated with phenylmercuric acetate were reconstituted with [U-(13)C(17),U-(15)N(4)]FAD and analysed, in their oxidized and reduced states, by (13)C-, (15)N- and (31)P-NMR spectroscopy. The enzymes studied showed very similar (31)P-NMR spectra in the oxidized state, consisting of two peaks at -9.8 and -11.5 p.p.m. In the reduced state, the two peaks merge into one apparent peak (at -9.8 p.p.m.). The data are compared with published (31)P-NMR data of enzymes closely related to TrxR. (13)C and (15)N-NMR chemical shifts of TrxR and the mutant proteins in the oxidized state provided information about the electronic structure of the protein-bound cofactor and its interactions with the apoproteins. Strong hydrogen bonds exist between protein-bound flavin and the apoproteins at C(2)O, C(4)O, N(1) and N(5). The N(10) atoms in the enzymes are slightly out of the molecular plane of the flavin. Of the ribityl carbon atoms C(10alpha,gamma,delta) are the most affected upon binding to the apoprotein and the large downfield shift of the C(10gamma) atom indicates strong hydrogen bonding with the apoprotein. The hydrogen bonding pattern observed is in excellent agreement with X-ray data, except for the N(1) and the N(3) atoms where a reversed situation was observed. Some chemical shifts observed in C138S deviate considerably from those of the other enzymes. From this it is concluded that C138S is in the FO conformation and the others are in the FR conformation, supporting published data. In the reduced state, strong hydrogen bonding interactions are observed between C(2)O and C(4)O and the apoprotein. As revealed by the (15)N chemical shifts and the N(5)H coupling constant the N(5) and the N(10) atom are highly sp(3) hybridized. The calculation of the endocyclic angles for the N(5) and the N(10) atoms shows the angles to be approximately 109 degrees, in perfect agreement with X-ray data showing that the flavin assumes a bent conformation along the N(10)/N(5) axis of the flavin. In contrast, the N(1) is highly sp(2) hybridized and is protonated, i.e. in the neutral state. Upon reduction of the enzymes, the (13)C chemical shifts of some atoms of the ribityl side chain undergo considerable changes also indicating conformational rearrangements of the side-chain interactions with the apoproteins. The chemical shifts between native TrxR and C138S are now rather similar and differ from those of the two other mutant proteins. This strongly indicates that the former enzymes are in the FO conformation and the other two are in the FR conformation. The data are discussed briefly in the context of published NMR data obtained with a variety of flavoproteins.
Assuntos
Tiorredoxina Dissulfeto Redutase/química , Substituição de Aminoácidos , Isótopos de Carbono , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Flavinas/química , Flavinas/metabolismo , Peso Molecular , Mutação , NADH NADPH Oxirredutases/química , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular/métodos , Oxirredução , Isótopos de Fósforo , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrofotometria Ultravioleta , Tiorredoxina Dissulfeto Redutase/genéticaRESUMO
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.
Assuntos
Riboflavina Sintase/química , Schizosaccharomyces/enzimologia , Sequência de Aminoácidos , Flúor , Dados de Sequência Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Pteridinas/metabolismo , Riboflavina/biossíntese , Riboflavina Sintase/genética , Riboflavina Sintase/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The essential redox cofactors riboflavin monophosphate (FMN) and flavin adenine dinucleotide (FAD) are synthesised from their precursor, riboflavin, in sequential reactions by the metal-dependent riboflavin kinase and FAD synthetase. Here, we describe the 1.6A crystal structure of the Schizosaccharomyces pombe riboflavin kinase. The enzyme represents a novel family of phosphoryl transferring enzymes. It is a monomer comprising a central beta-barrel clasped on one side by two C-terminal helices that display an L-like shape. The opposite side of the beta-barrel serves as a platform for substrate binding as demonstrated by complexes with ADP and FMN. Formation of the ATP-binding site requires significant rearrangements in a short alpha-helix as compared to the substrate free form. The diphosphate moiety of ADP is covered by the glycine-rich flap I formed from parts of this alpha-helix. In contrast, no significant changes are observed upon binding of riboflavin. The ribityl side-chain might be covered by a rather flexible flap II. The unusual metal-binding site involves, in addition to the ADP phosphates, only the strictly conserved Thr45. This may explain the preference for zinc observed in vitro.
Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Mononucleotídeo de Flavina/metabolismo , Complexos Multienzimáticos/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Riboflavina/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/enzimologia , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Mononucleotídeo de Flavina/química , Técnicas In Vitro , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Riboflavina/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
The two epimeric covalent hydrates A and B of 6,7-bis(trifluoromethyl)-8-D-ribityllumazine are metabolically stable analogues of hypothetical intermediates proposed in the reactions catalyzed by riboflavin synthase and lumazine synthase. To confirm the stereochemical assignments previously based solely on results for epimer B, a (15)N[(19)F] REDOR NMR study was performed on the complex formed from epimer A and a recombinant, uniformly (15)N-labeled F22W mutant of Bacillus subtilis lumazine synthase. The results indicate that the fluorines of the ligands are closer to the side chain nitrogens of Arg127 and farther away from the side chain nitrogens of Lys135 in epimer B than in epimer A. These results are consistent with the assignment of the earlier 7R configuration of epimer A and the 7S configuration of epimer B.