RESUMO
Photosynthesis must maintain stability and robustness throughout fluctuating natural environments. In cyanobacteria, dark-to-light transition leads to drastic metabolic changes from dark respiratory metabolism to CO2 fixation through the Calvin-Benson-Bassham (CBB) cycle using energy and redox equivalents provided by photosynthetic electron transfer. Previous studies have shown that catabolic metabolism supports the smooth transition into CBB cycle metabolism. However, metabolic mechanisms for robust initiation of photosynthesis are poorly understood due to lack of dynamic metabolic characterizations of dark-to-light transitions. Here, we show rapid dynamic changes (on a time scale of seconds) in absolute metabolite concentrations and 13C tracer incorporation after strong or weak light irradiation in the cyanobacterium Synechocystis sp. PCC 6803. Integration of this data enabled estimation of time-resolved nonstationary metabolic flux underlying CBB cycle activation. This dynamic metabolic analysis indicated that downstream glycolytic intermediates, including phosphoglycerate and phosphoenolpyruvate, accumulate under dark conditions as major substrates for initial CO2 fixation. Compared with wild-type Synechocystis, significant decreases in the initial oxygen evolution rate were observed in 12 h dark preincubated mutants deficient in glycogen degradation or oxidative pentose phosphate pathways. Accordingly, the degree of decrease in the initial oxygen evolution rate was proportional to the accumulated pool size of glycolytic intermediates. These observations indicate that the accumulation of glycolytic intermediates is essential for efficient metabolism switching under fluctuating light environments.
Assuntos
Dióxido de Carbono , Synechocystis , Dióxido de Carbono/metabolismo , Fotossíntese/fisiologia , Transporte de Elétrons , Synechocystis/metabolismo , Oxigênio/metabolismoRESUMO
BACKGROUND: Computational mining of useful enzymes and biosynthesis pathways is a powerful strategy for metabolic engineering. Through systematic exploration of all conceivable combinations of enzyme reactions, including both known compounds and those inferred from the chemical structures of established reactions, we can uncover previously undiscovered enzymatic processes. The application of the novel alternative pathways enables us to improve microbial bioproduction by bypassing or reinforcing metabolic bottlenecks. Benzylisoquinoline alkaloids (BIAs) are a diverse group of plant-derived compounds with important pharmaceutical properties. BIA biosynthesis has developed into a prime example of metabolic engineering and microbial bioproduction. The early bottleneck of BIA production in Escherichia coli consists of 3,4-dihydroxyphenylacetaldehyde (DHPAA) production and conversion to tetrahydropapaveroline (THP). Previous studies have selected monoamine oxidase (MAO) and DHPAA synthase (DHPAAS) to produce DHPAA from dopamine and oxygen; however, both of these enzymes produce toxic hydrogen peroxide as a byproduct. RESULTS: In the current study, in silico pathway design is applied to relieve the bottleneck of DHPAA production in the synthetic BIA pathway. Specifically, the cytochrome P450 enzyme, tyrosine N-monooxygenase (CYP79), is identified to bypass the established MAO- and DHPAAS-mediated pathways in an alternative arylacetaldoxime route to DHPAA with a peroxide-independent mechanism. The application of this pathway is proposed to result in less formation of toxic byproducts, leading to improved production of reticuline (up to 60 mg/L at the flask scale) when compared with that from the conventional MAO pathway. CONCLUSIONS: This study showed improved reticuline production using the bypass pathway predicted by the M-path computational platform. Reticuline production in E. coli exceeded that of the conventional MAO-mediated pathway. The study provides a clear example of the integration of pathway mining and enzyme design in creating artificial metabolic pathways and suggests further potential applications of this strategy in metabolic engineering.
Assuntos
Benzilisoquinolinas , Escherichia coli , Engenharia Metabólica , Engenharia Metabólica/métodos , Benzilisoquinolinas/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Vias Biossintéticas , Simulação por Computador , Tetra-Hidropapaverolina/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/análogos & derivadosRESUMO
Synthesis of the A/D/E-ring core compounds of maoecrystal V was achieved. The key Diels-Alder reactions between tricyclic α-methylene lactones and Kitahara-Danishefsky dienes afforded the spirocyclic core compounds in a regioselective and stereoselective manner.
Assuntos
Lactonas , Estereoisomerismo , Lactonas/química , Lactonas/síntese química , Reação de Cicloadição , Técnicas de Química Sintética , Diterpenos/síntese química , Diterpenos/química , Compostos de Espiro/química , Compostos de Espiro/síntese química , Estrutura MolecularRESUMO
Unannotated gene sequences in databases are increasing due to sequencing advances. Therefore, computational methods to predict functions of unannotated genes are needed. Moreover, novel enzyme discovery for metabolic engineering applications further encourages annotation of sequences. Here, enzyme functions are predicted using two general approaches, each including several machine learning algorithms. First, Enzyme-models (E-models) predict Enzyme Commission (EC) numbers from amino acid sequence information. Second, Substrate-Enzyme models (SE-models) are built to predict substrates of enzymatic reactions together with EC numbers, and Substrate-Enzyme-Product models (SEP-models) are built to predict substrates, products, and EC numbers. While accuracy of E-models is not optimal, SE-models and SEP-models predict EC numbers and reactions with high accuracy using all tested machine learning-based methods. For example, a single Random Forests-based SEP-model predicts EC first digits with an Average AUC score of over 0.94. Various metrics indicate that the current strategy of combining sequence and chemical structure information is effective at improving enzyme reaction prediction.
Assuntos
Biologia Computacional , Aprendizado de Máquina , Algoritmos , Sequência de Aminoácidos , Bases de Dados FactuaisRESUMO
Influenza virus neuraminidase (NA) drug resistance is one of the challenges to preparedness against epidemic and pandemic influenza virus infections. NA N1- and N2-containing influenza viruses are the primary cause of seasonal epidemics and past pandemics. The structural and functional basis underlying drug resistance of the influenza virus N1 NA is well characterized. Yet drug resistance of the N2 strain is not well understood. Here, we confirm that replacement of N2 E119 or I222 results in multidrug resistance, and when the replacements occur together, the sensitivity to NA inhibitors (NAI) is reduced severely. Using crystallographic studies, we showed that E119 replacement results in a loss of hydrogen bonding to oseltamivir and zanamivir, whereas I222 replacement results in a change in the hydrophobic environment that is critical for oseltamivir binding. Moreover, we found that MS-257, a zanamivir-oseltamivir hybrid inhibitor, is less susceptible to drug resistance. The binding mode of MS-257 shows that increased hydrogen bonding interactions between the inhibitor and NA active site anchor the inhibitor within the active site and allow adjustments in response to active-site modifications. Such stability is likely responsible for the observed reduced susceptibility to drug resistance. MS-257 serves as a next-generation anti-influenza virus drug candidate and serves also as a scaffold for further design of NAIs. IMPORTANCE: Oseltamivir and zanamivir are the two major antiviral drugs available for the treatment of influenza virus infections. However, multidrug-resistant viruses have emerged in clinical cases, which pose a challenge for the development of new drugs. N1 and N2 subtypes exist in the viruses which cause seasonal epidemics and past pandemics. Although N1 drug resistance is well characterized, the molecular mechanisms underlying N2 drug resistance are unknown. A previous report showed that an N2 E119V/I222L dual mutant conferred drug resistance to seasonal influenza virus. Here, we confirm that these substitutions result in multidrug resistance and dramatically reduced sensitivity to NAI. We further elucidate the molecular mechanism underlying N2 drug resistance by solving crystal structures of the N2 E119V and I222L mutants and the dual mutant. Most importantly, we found that a novel oseltamivir-zanamivir hybrid inhibitor, MS-257, remains more effective against drug-resistant N2 and is a promising candidate as a next-generation anti-influenza virus drug.
Assuntos
Antivirais/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/enzimologia , Mutação , Neuraminidase/antagonistas & inibidores , Neuraminidase/genética , Oseltamivir/farmacologia , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética , Zanamivir/farmacologia , Substituição de Aminoácidos , Farmacorresistência Viral Múltipla/genética , Inibidores Enzimáticos/farmacologia , Humanos , Vírus da Influenza A/genética , Modelos Moleculares , Neuraminidase/química , Proteínas Virais/químicaRESUMO
In our previous study, we found unique free N-glycans (FNGs), which carry a single GlcNAc residue (GN1) at the reducing-end side and the Lewis-a epitope at the non-reducing-end side, in the culture broth of rice cells. Based on the FNG structural features and the substrate specificity of plant ENGase, we hypothesized that there might be a novel biosynthetic mechanism responsible for the production of these unique GN1-FNGs, in which high-mannose type (HMT)-GN1-FNGs produced in the cytosol from misfolded glycoproteins by ENGase are transported back into the endoplasmic reticulum and processed to plant complex type (PCT)-GN1-FNGs in the Golgi apparatus. Until now, however, PCT-GN1-FNGs had only been found in the culture broth of rice cultured cells and never in plants, suggesting that the formation of PCT-GN1-FNGs might be generated under special or artificial conditions. In this study, we confirm the presence of PCT-GN1-FNGs, HMT-GN1-FNGs and PCT-GN2-FNGs in the fresh-water plant Egeria densa. These results suggest that a mechanism responsible for the production of PCT-GN1-FNG is present in native plant tissues.
Assuntos
Acetilglucosamina/metabolismo , Magnoliopsida/metabolismo , Polissacarídeos/metabolismo , Acetilglucosamina/química , Configuração de Carboidratos , Magnoliopsida/química , Polissacarídeos/químicaRESUMO
Two tetranortriterpenoids with new skeletons, xylomexicanins I and J (1 and 2), were isolated during the investigation of chemical constituents from seeds of the Chinese mangrove, Xylocarpus granatum. Xylomexicanin I (1) is an unprecedented limonoid with bridged B- and C-rings. A biosynthesis pathway for 1 from xylomexicanin F is proposed.
Assuntos
Limoninas/isolamento & purificação , Meliaceae/química , Sementes/química , Limoninas/química , Estrutura MolecularRESUMO
UNLABELLED: There are nine subtypes of influenza A virus neuraminidase (NA), N1 to N9. In addition, influenza B virus also contains NA, and there are two influenza virus NA-like molecules, N10 and N11, which were recently identified from bats. Crystal structures for all of these proteins have been solved, with the exception of N7, and there is no published report of N6, although a structure has been deposited in the Protein Data Bank. Here, we present the N7 and N6 structures at 2.1 Å and 1.8 Å, respectively. Structural comparison of all NA subtypes shows that both N7 and N6 highly resemble typical group 2 NA structures with some special characteristics, including an additional cavity adjacent to their active sites formed by novel 340-loop conformations. Comparative analysis also revealed new structural insights into the N-glycosylation, calcium binding, and second sialic acid binding site of influenza virus NA. This comprehensive study is critical for understanding the complexity of the most successful influenza drug target and for the structure-based design of novel influenza inhibitors. IMPORTANCE: Influenza viruses impose a great burden on society, by the human-adapted seasonal types as well as by variants that occasionally jump from the avian reservoir to infect humans. The surface glycoprotein neuraminidase (NA) is essential for the propagation of the virus and currently the most successfully drug-targeted molecule. Therefore, the structural and functional analysis of NA is critical for the prevention and control of influenza infections. There are nine subtypes of influenza A virus NA (N1 to N9). In addition, influenza B virus also contains NA, and there are two influenza NA-like molecules, N10 and N11, which were recently identified in bats. Crystal structures for all of these proteins have been solved and reported with the exception of N7 and N6. The structural analysis of influenza virus N7 and N6 presented in this study therefore completes the puzzle and adds to a comprehensive understanding of influenza virus NA.
Assuntos
Neuraminidase/química , Orthomyxoviridae/química , Proteínas Virais/química , Cálcio/metabolismo , Domínio Catalítico , Glicosilação , Ácido N-Acetilneuramínico/metabolismo , Orthomyxoviridae/metabolismo , Conformação ProteicaRESUMO
The recent discovery of the unique genome of influenza virus H17N10 in bats raises considerable doubt about the origin and evolution of influenza A viruses. It also identifies a neuraminidase (NA)-like protein, N10, that is highly divergent from the nine other well-established serotypes of influenza A NA (N1-N9). The structural elucidation and functional characterization of influenza NAs have illustrated the complexity of NA structures, thus raising a key question as to whether N10 has a special structure and function. Here the crystal structure of N10, derived from influenza virus A/little yellow-shouldered bat/Guatemala/153/2009 (H17N10), was solved at a resolution of 2.20 Å. Overall, the structure of N10 was found to be similar to that of the other known influenza NA structures. In vitro enzymatic assays demonstrated that N10 lacks canonical NA activity. A detailed structural analysis revealed dramatic alterations of the conserved active site residues that are unfavorable for the binding and cleavage of terminally linked sialic acid receptors. Furthermore, an unusual 150-loop (residues 147-152) was observed to participate in the intermolecular polar interactions between adjacent N10 molecules of the N10 tetramer. Our study of influenza N10 provides insight into the structure and function of the sialidase superfamily and sheds light on the molecular mechanism of bat influenza virus infection.
Assuntos
Quirópteros/virologia , Vírus da Influenza A/enzimologia , Neuraminidase/química , Proteínas Virais/química , Animais , Cristalografia por Raios X , Evolução Molecular , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Neuraminidase/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Virais/genéticaRESUMO
The influenza virus neuraminidase H274Y substitution is a highly prevalent amino acid substitution associated with resistance to the most heavily used influenza drug, oseltamivir. Previous structural studies suggest that the group specific 252 residue (Y252 in group 1 and T252 in group 2) might be a key factor underlying H274Y resistance. However, H274Y has only been reported in N1 subtypes, which indicates that there must be additional key residues that determine H274Y resistance. Furthermore, we found that members of NA serotype N3 also possess Y252, raising the key question as to whether or not H274Y resistance may also be possible for some group 2 NAs. Here, we demonstrate that the H274Y substitution results in mild oseltamivir resistance for N3. Comparative structural analysis of N3, N1, and their 274Y variants indicates that the interaction of residue 296 (H in N1 and nonaromatic for other serotypes) with conserved W295 is another important determinant of oseltamivir resistance.
Assuntos
Antivirais/farmacologia , Farmacorresistência Viral , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/enzimologia , Neuraminidase/química , Neuraminidase/metabolismo , Oseltamivir/farmacologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Substituição de Aminoácidos , Cristalografia por Raios X , Humanos , Mutação de Sentido Incorreto , Neuraminidase/genética , Conformação Proteica , Proteínas Virais/genéticaRESUMO
The high mutation rate of RNA viruses has resulted in limitation of vaccine effectiveness and increased emergence of drug-resistant viruses. New effective antivirals are therefore needed to control of the highly mutative RNA viruses. The n-butanol fraction of the stem bark of Mangifera indica exhibited inhibitory activity against influenza neuraminidase (NA) and coxsackie virus 3C protease. Bioassay guided phytochemical study of M. indica stem bark afforded two new compounds including one benzophenone C-glycoside (4) and one xanthone dimer (7), together with eleven known compounds. The structures of these isolated compounds were elucidated on the basis of spectroscopic evidences and correlated with known compounds. Anti-influenza and anti-coxsackie virus activities were evaluated by determining the inhibition of anti-influenza neuraminidase (NA) from pandemic A/RI/5+/1957 H2N2 influenza A virus and inhibition of coxsackie B3 virus 3C protease, respectively. The highest anti-influenza activity was observed for compounds 8 and 9 with IC50 values of 11.9 and 9.2µM, respectively. Compounds 8 and 9 were even more potent against coxsackie B3 virus 3C protease, with IC50 values of 1.1 and 2.0µM, respectively. Compounds 8 and 9 showed weak cytotoxic effect against human hepatocellular carcinoma and human epithelial carcinoma cell lines through MTT assay.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Antivirais/farmacologia , Benzofenonas/farmacologia , Taninos Hidrolisáveis/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Mangifera/química , Inibidores de Proteases/farmacologia , Proteases Virais 3C , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Antivirais/química , Antivirais/isolamento & purificação , Benzofenonas/química , Benzofenonas/isolamento & purificação , Proliferação de Células/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Enterovirus Humano B/enzimologia , Células HeLa , Células Hep G2 , Humanos , Taninos Hidrolisáveis/química , Taninos Hidrolisáveis/isolamento & purificação , Estrutura Molecular , Casca de Planta/química , Caules de Planta/química , Inibidores de Proteases/química , Inibidores de Proteases/isolamento & purificação , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismoRESUMO
The 2009 H1N1 influenza pandemic (pH1N1) led to record sales of neuraminidase (NA) inhibitors, which has contributed significantly to the recent increase in oseltamivir-resistant viruses. Therefore, development and careful evaluation of novel NA inhibitors is of great interest. Recently, a highly potent NA inhibitor, laninamivir, has been approved for use in Japan. Laninamivir is effective using a single inhaled dose via its octanoate prodrug (CS-8958) and has been demonstrated to be effective against oseltamivir-resistant NA in vitro. However, effectiveness of laninamivir octanoate prodrug against oseltamivir-resistant influenza infection in adults has not been demonstrated. NA is classified into 2 groups based upon phylogenetic analysis and it is becoming clear that each group has some distinct structural features. Recently, we found that pH1N1 N1 NA (p09N1) is an atypical group 1 NA with some group 2-like features in its active site (lack of a 150-cavity). Furthermore, it has been reported that certain oseltamivir-resistant substitutions in the NA active site are group 1 specific. In order to comprehensively evaluate the effectiveness of laninamivir, we utilized recombinant N5 (typical group 1), p09N1 (atypical group 1) and N2 from the 1957 pandemic H2N2 (p57N2) (typical group 2) to carry out in vitro inhibition assays. We found that laninamivir and its octanoate prodrug display group specific preferences to different influenza NAs and provide the structural basis of their specific action based upon their novel complex crystal structures. Our results indicate that laninamivir and zanamivir are more effective against group 1 NA with a 150-cavity than group 2 NA with no 150-cavity. Furthermore, we have found that the laninamivir octanoate prodrug has a unique binding mode in p09N1 that is different from that of group 2 p57N2, but with some similarities to NA-oseltamivir binding, which provides additional insight into group specific differences of oseltamivir binding and resistance.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Neuraminidase/antagonistas & inibidores , Pró-Fármacos/farmacologia , Zanamivir/análogos & derivados , Antivirais/química , Caprilatos/química , Caprilatos/farmacologia , Domínio Catalítico/efeitos dos fármacos , Domínio Catalítico/fisiologia , Farmacorresistência Viral/efeitos dos fármacos , Inibidores Enzimáticos/química , Guanidinas , Humanos , Vírus da Influenza A Subtipo H1N1/enzimologia , Concentração Inibidora 50 , Oseltamivir/química , Oseltamivir/farmacologia , Pró-Fármacos/química , Piranos , Ácidos Siálicos , Zanamivir/química , Zanamivir/farmacologiaRESUMO
Recombination of biosynthetic gene clusters including those of non-ribosomal peptide synthetases (NRPSs) is essential for understanding the mechanisms of biosynthesis. Due to relatively huge gene cluster sizes ranging from 10 to 150 kb, the prevalence of sequence repeats, and inability to clearly define optimal points for manipulation, functional characterization of recombinant NRPSs with maintained activity has been hindered. In this study, we introduce a simple yet rapid approach named "Seamed Express Assembly Method (SEAM)" coupled with Ordered Gene Assembly in Bacillus subtilis (OGAB) to reconstruct fully functional plipastatin NRPS. This approach is enabled by the introduction of restriction enzyme sites as seams at module borders. SEAM-OGAB is then first demonstrated by constructing the ppsABCDE NRPS (38.4 kb) to produce plipastatin, a cyclic decapeptide in B. subtilis. The introduced amino acid level seams do not hinder the NRPS function and enable successful production of plipastatin at a commensurable titer. It is challenging to modify the plipastatin NRPS gene cluster due to the presence of three long direct-repeat sequences; therefore, this study demonstrates that SEAM-OGAB can be readily applied towards the recombination of various NRPSs. Compared to previous NRPS gene assembly methods, the advantage of SEAM-OGAB is that it readily enables the shuffling of NRPS gene modules, and therefore, chimeric NRPSs can be rapidly constructed for the production of novel peptides. This chimeric assembly application of SEAM-OGAB is demonstrated by swapping plipastatin NRPS and surfactin NRPS modules to produce two novel lipopeptides in B. subtilis.
Assuntos
Bacillus subtilis , Peptídeo Sintases , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Peptídeo Sintases/metabolismo , Sequência de Bases , Lipopeptídeos/genéticaRESUMO
Conventional analysis of microbial bioproducers requires the extraction of metabolites from liquid cultures, where the culturing steps are time consuming and greatly limit throughput. To break through this barrier, the current study aims to directly evaluate microbial bioproduction colonies by way of supercritical fluid extraction-supercritical fluid chromatography-triple quadrupole mass spectrometry (SFE-SFC-MS/MS). The online SFE-SFC-MS/MS system offers great potential for high-throughput analysis due to automated metabolite extraction without any need for pretreatment. This is the first report of SFE-SFC-MS/MS as a method for direct colony screening, as demonstrated in the high-throughput screening of (-)-limonene bioproducers. Compared with conventional analysis, the SFE-SFC-MS/MS system enables faster and more convenient screening of highly productive strains.
Assuntos
Cromatografia com Fluido Supercrítico , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Limoneno , Cromatografia com Fluido Supercrítico/métodos , Cromatografia Líquida , Ensaios de Triagem em Larga Escala/métodosRESUMO
There are 9 serotypes of neuraminidase (NA) from influenza A virus (N1 to N9), which are classified into two groups based on primary sequences (groups 1 and 2). The structural hallmark of the two groups is the presence or absence of an extra 150-cavity (formed by the 150-loop) in the active site. Thus far, structures of NAs from 6 out of the 9 serotypes have been solved. Here, we solved the N5 structure, the last unknown structure group 1 serotype with a unique Asn147 residue in its 150-loop, demonstrating that it has an extended 150-cavity that closes upon inhibitor binding.
Assuntos
Vírus da Influenza A/química , Neuraminidase/química , Sequência de Aminoácidos , Antivirais/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Virus da Influenza A Subtipo H5N1/química , Virus da Influenza A Subtipo H5N1/genética , Vírus da Influenza A/genética , Modelos Moleculares , Neuraminidase/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Zanamivir/metabolismoRESUMO
Methods to synthesize influenza virus inhibitors with fluoro, phosphono, and/or sulfo functional groups are described. The resulting sialic acid analogues are produced from the natural substrate N-acetylneuraminic acid as starting material. Fluorescent assay methods for inhibition of influenza neuraminidase and virus proliferation are also provided.
Assuntos
Influenza Humana , Ácido N-Acetilneuramínico , Corantes , Humanos , Influenza Humana/tratamento farmacológico , Ácido N-Acetilneuramínico/farmacologia , NeuraminidaseRESUMO
Depending on the strain, influenza A virus causes animal, zoonotic, pandemic, or seasonal influenza with varying degrees of severity. Two surface glycoprotein spikes, hemagglutinin (HA) and neuraminidase (NA), are the most important influenza A virus antigens. NA plays an important role in the propagation of influenza virus by removing terminal sialic acid from sialyl decoy receptors and thereby facilitating the release of viruses from traps such as in mucus and on infected cells. Some NA inhibitors have become widely used drugs for treatment of influenza. However, attempts to develop effective and safe NA inhibitors that can be used for treatment of anti-NA drugs-resistant influenza viruses have continued. In this chapter, we describe the following updates on influenza A NA inhibitor development: (i) N-acetylneuraminic acid (Neu5Ac)-based derivatives, (ii) covalent NA inhibitors, (iii) sulfo-sialic acid analogs, (iv) N-acetyl-6-sulfo-ß-D-glucosaminide-based inhibitors, (v) inhibitors targeting the 150-loop of group 1 NAs, (vi) conjugation inhibitors, (vii) acylhydrazone derivatives, (viii) monoclonal antibodies, (ix) PVP-I, and (x) natural products. Finally, we provide future perspectives on the next-generation anti-NA drugs.
Assuntos
Produtos Biológicos , Vírus da Influenza A , Influenza Humana , Animais , Anticorpos Monoclonais , Antivirais/farmacologia , Hemaglutininas , Humanos , Ácido N-Acetilneuramínico , Neuraminidase , Povidona-IodoRESUMO
New enzyme functions exist within the increasing number of unannotated protein sequences. Novel enzyme discovery is necessary to expand the pathways that can be accessed by metabolic engineering for the biosynthesis of functional compounds. Accordingly, various machine learning models have been developed to predict enzymatic reactions. However, the ability to predict unknown reactions that are not included in the training data has not been clarified. In order to cover uncertain and unknown reactions, a wider range of reaction types must be demonstrated by the models. Here, we establish 16 expanded enzymatic reaction prediction models developed using various machine learning algorithms, including deep neural network. Improvements in prediction performances over that of our previous study indicate that the updated methods are more effective for the prediction of enzymatic reactions. Overall, the deep neural network model trained with combined substrate-enzyme-product information exhibits the highest prediction accuracy with Macro F1 scores up to 0.966 and with robust prediction of unknown enzymatic reactions that are not included in the training data. This model can predict more extensive enzymatic reactions in comparison to previously reported models. This study will facilitate the discovery of new enzymes for the production of useful substances.
Assuntos
Aprendizado de Máquina , Redes Neurais de Computação , AlgoritmosRESUMO
Thelepamide, an unique ketide-amino acid isolated from a marine annelid worm Thelepus crispus, has a unique oxazolidinone ring derived from cysteine, glycine and valine. Rareness in nature as well as promising bioactive possibility make the oxazolidinone ring an attractive synthetic target. The hydroxy oxazolidinone fragment of thelepamide was prepared by acid-catalysed N,O-acetal formation between a ketoamide and formaldehyde. Lactone-carbonyl selective isopropyl addition to an oxazilidine-dione under Grignard conditions also forms the target compound.
Assuntos
Oxazolidinonas , Policetídeos , Oxazolidinonas/químicaRESUMO
Engineering the microbial production of secondary metabolites is limited by the known reactions of correctly annotated enzymes. Therefore, the machine learning discovery of specialized enzymes offers great potential to expand the range of biosynthesis pathways. Benzylisoquinoline alkaloid production is a model example of metabolic engineering with potential to revolutionize the paradigm of sustainable biomanufacturing. Existing bacterial studies utilize a norlaudanosoline pathway, whereas plants contain a more stable norcoclaurine pathway, which is exploited in yeast. However, committed aromatic precursors are still produced using microbial enzymes that remain elusive in plants, and additional downstream missing links remain hidden within highly duplicated plant gene families. In the current study, machine learning is applied to predict and select plant missing link enzymes from homologous candidate sequences. Metabolomics-based characterization of the selected sequences reveals potential aromatic acetaldehyde synthases and phenylpyruvate decarboxylases in reconstructed plant gene-only benzylisoquinoline alkaloid pathways from tyrosine. Synergistic application of the aryl acetaldehyde producing enzymes results in enhanced benzylisoquinoline alkaloid production through hybrid norcoclaurine and norlaudanosoline pathways.