RESUMO
Unspecific Peroxygenases (UPOs) are increasingly significant enzymes for selective oxygenations as they are stable, highly active and catalyze their reactions at the expense of only hydrogen peroxide as the oxidant. Their structural similarity to chloroperoxidase (CPO) means that UPOs can also catalyze halogenation reactions based upon the generation of hypohalous acids from halide and H2O2. Here we show that the halogenation and oxygenation modes of a UPO can be stimulated at different pH values. Using simple aromatic compounds such as thymol, we show that, at a pH of 3.0 and 6.0, either brominated or oxygenated products respectively are produced. Preparative 100â mg scale transformations of substrates were performed with 60-72 % isolated yields of brominated products obtained. A one-pot bromination-oxygenation cascade reaction on 4-ethylanisole, in which the pH was adjusted from 3.0 to 6.0 at the halfway stage, yielded sequentially brominated and oxygenated products 1-(3-bromo-4-methoxyphenyl)ethyl alcohol and 3-bromo-4-methoxy acetophenone with 82 % combined conversion. These results identify UPOs as an unusual example of a biocatalyst that is tunable for entirely different chemical reactions, dependent upon the reaction conditions.
RESUMO
The unspecific peroxygenase (UPO) from Agrocybe aegerita (rAaeUPO-PaDa-I-H) is an effective and practical biocatalyst for the oxidative expansion of furfuryl alcohols/amines on a preparative scale, using the Achmatowicz and aza-Achmatowicz reaction. The high activity and stability of the enzyme, which can be produced on a large scale as an air-stable lyophilised powder, renders it a versatile and scalable biocatalyst for the preparation of synthetically valuable 6-hydroxypyranones and dihydropiperidinones. In several cases, the biotransformation out-performed the analogous chemo-catalysed process, and operates under milder and greener reaction conditions.
Assuntos
Agrocybe , Oxigenases de Função Mista , Agrocybe/enzimologia , Aminas/química , Aminas/metabolismo , Biocatálise , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/química , Estrutura MolecularRESUMO
Unspecific peroxygenases (UPOs) have emerged as valuable tools for the oxygenation of non-activated carbon atoms, as they exhibit high turnovers, good stability and depend only on hydrogen peroxide as the external oxidant for activity. However, the isolation of UPOs from their natural fungal sources remains a barrier to wider application. We have cloned the gene encoding an 'artificial' peroxygenase (artUPO), close in sequence to the 'short' UPO from Marasmius rotula (MroUPO), and expressed it in both the yeast Pichia pastoris and E.â coli to compare the catalytic and structural characteristics of the enzymes produced in each system. Catalytic efficiency for the UPO substrate 5-nitro-1,3-benzodioxole (NBD) was largely the same for both enzymes, and the structures also revealed few differences apart from the expected glycosylation of the yeast enzyme. However, the glycosylated enzyme displayed greater stability, as determined by nano differential scanning fluorimetry (nano-DSF) measurements. Interestingly, while artUPO hydroxylated ethylbenzene derivatives to give the (R)-alcohols, also given by a variant of the 'long' UPO from Agrocybe aegerita (AaeUPO), it gave the opposite (S)-series of sulfoxide products from a range of sulfide substrates, broadening the scope for application of the enzymes. The structures of artUPO reveal substantial differences to that of AaeUPO, and provide a platform for investigating the distinctive activity of this and related'short' UPOs.
Assuntos
Escherichia coli , Saccharomyces cerevisiae , Escherichia coli/genética , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/química , Pichia/genéticaRESUMO
A lyophilized preparation of an unspecific peroxygenase variant from Agrocybe aegerita (rAaeUPO-PaDa-I-H) is a highly effective catalyst for the oxygenation of a diverse range of N-heterocyclic compounds. Scalable biocatalytic oxygenations (27 preparative examples, ca. 100â mg scale) have been developed across a wide range of substrates, including alkyl pyridines, bicyclic N-heterocycles and indoles. H2 O2 is the only stoichiometric oxidant needed, without auxiliary electron transport proteins, which is key to the practicality of the method. Reaction outcomes can be altered depending on whether hydrogen peroxide was delivered by syringe pump or through in situ generation using an alcohol oxidase from Pichia pastoris (PpAOX) and methanol as a co-substrate. Good synthetic yields (up to 84 %), regioselectivity and enantioselectivity (up to 99 % ee) were observed in some cases, highlighting the promise of UPOs as practical, versatile and scalable oxygenation biocatalysts.
Assuntos
Oxigenases de Função Mista , Oxigenases de Função Mista/metabolismo , Catálise , BiocatáliseRESUMO
H2 O2 -driven enzymes are of great interest for industrial biotransformations. Herein, we show for the first time that oxalate oxidase (OXO) is an efficient in situ source of H2 O2 for one of these biocatalysts, which is known as unspecific peroxygenase (UPO). OXO is reasonably robust, produces only CO2 as a by-product and uses oxalate as a cheap sacrificial electron donor. UPO has significant potential as an industrial catalyst for selective C-H oxyfunctionalisations, as we confirm herein by testing a diverse drug panel using miniaturised high-throughput assays and mass spectrometry. 33 out of 64 drugs were converted in 5â µL-scale reactions by the UPO with OXO (conversion >70 % for 11 drugs). Furthermore, oxidation of the drug tolmetin was achieved on a 50â mg scale (TONUPO 25 664) with 84 % yield, which was further improved via enzyme immobilization. This one-pot approach ensures adequate H2 O2 levels, enabling rapid access to industrially relevant molecules that are difficult to obtain by other routes.
Assuntos
Tolmetino , Dióxido de Carbono , Oxigenases de Função Mista , Oxalatos , OxirredutasesRESUMO
Controlling the selectivity of a chemical reaction with external stimuli is common in thermal processes, but rare in visible-light photocatalysis. Here we show that the redox potential of a carbon nitride photocatalyst (CN-OA-m) can be tuned by changing the irradiation wavelength to generate electron holes with different oxidation potentials. This tuning was the key to realizing photo-chemo-enzymatic cascades that give either the (S)- or the (R)-enantiomer of phenylethanol. In combination with an unspecific peroxygenase from Agrocybe aegerita, green light irradiation of CN-OA-m led to the enantioselective hydroxylation of ethylbenzene to (R)-1-phenylethanol (99 % ee). In contrast, blue light irradiation triggered the photocatalytic oxidation of ethylbenzene to acetophenone, which in turn was enantioselectively reduced with an alcohol dehydrogenase from Rhodococcus ruber to form (S)-1-phenylethanol (93 % ee).
Assuntos
Acetofenonas/química , Álcool Desidrogenase/química , Derivados de Benzeno/química , Oxigenases de Função Mista/química , Nitrilas/química , Álcool Feniletílico/química , Acetofenonas/metabolismo , Agrocybe/enzimologia , Álcool Desidrogenase/metabolismo , Derivados de Benzeno/metabolismo , Catálise , Luz , Oxigenases de Função Mista/metabolismo , Estrutura Molecular , Nitrilas/metabolismo , Oxirredução , Álcool Feniletílico/metabolismo , Processos Fotoquímicos , Rhodococcus/enzimologia , EstereoisomerismoRESUMO
Cytochromes c are ubiquitous proteins, essential for life in most organisms. Their distinctive characteristic is the covalent attachment of heme to their polypeptide chain. This post-translational modification is performed by a dedicated protein system, which in many Gram-negative bacteria and plant mitochondria is a nine-protein apparatus (CcmA-I) called System I. Despite decades of study, mechanistic understanding of the protein-protein interactions in this highly complex maturation machinery is still lacking. Here, we focused on the interaction of CcmC, the protein that sources the heme cofactor, with CcmE, the pivotal component of System I responsible for the transfer of the heme to the apocytochrome. Using in silico analyses, we identified a putative interaction site between these two proteins (residues Asp47, Gln50, and Arg55 on CcmC; Arg73, Asp101, and Glu105 on CcmE), and we validated our findings by in vivo experiments in Escherichia coli Moreover, employing NMR spectroscopy, we examined whether a heme-binding site on CcmE contributes to this interaction and found that CcmC and CcmE associate via protein-protein rather than protein-heme contacts. The combination of in vivo site-directed mutagenesis studies and high-resolution structural techniques enabled us to determine at the residue level the mechanism for the formation of one of the key protein complexes for cytochrome c maturation by System I.
Assuntos
Apoproteínas/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Citocromos c/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Heme/metabolismo , Hemeproteínas/metabolismo , Proteínas de Membrana/metabolismo , Substituição de Aminoácidos , Apoproteínas/química , Apoproteínas/genética , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Sítios de Ligação , Cristalografia por Raios X , Citocromos c/química , Citocromos c/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Heme/química , Heme/genética , Hemeproteínas/química , Hemeproteínas/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Conformação Proteica , Domínios e Motivos de Interação entre ProteínasRESUMO
Flow cytometry is a powerful tool for the quantitation of fluorescence and is proven to be able to correlate the fluorescence intensity to the number of protein on cells surface. Mass spectroscopy can also be used to determine the number of proteins per cell. Here we have developed two methods, using flow cytometry and mass spectroscopy to quantify number of transporters in human cells. These two approaches were then used to analyse the same samples so that a direct comparison could be made. Transporters have a major impact on the behaviour of a diverse number of drugs in human systems. While active uptake studies by transmembrane protein transporters using model substrates are routinely undertaken in human cell lines and hepatocytes as part of drug discovery and development, the interpretation of these results is currently limited by the inability to quantify the number of transporters present in the test samples. Here we provide a flow cytometric method for accurate quantification of transporter levels both on the cell surface and within the cell, and compare this to a quantitative mass spectrometric approach. Two transporters were selected for the study: OATP1B1 (also known as SLCO1B1, LST-1, OATP-C, OATP2) due to its important role in hepatic drug uptake and elimination; P-gp (also known as P-glycoprotein, MDR1, ABCB1) as a well characterised system and due to its potential impact on oral bioavailability, biliary and renal clearance, and brain penetration of drugs that are substrates for this transporter. In all cases the mass spectrometric method gave higher levels than the flow cytometry method. However, the two methods showed very similar trends in the relative ratios of both transporters in the hepatocyte samples investigated. The P-gp antibody allowed quantitative discrimination between externally facing transporters located in the cytoplasmic membrane and the total number of transporters on and in the cell. The proportion of externally facing transporter varied considerably in the four hepatocyte samples analysed, ranging from only 6% to 35% of intact and viable cells. The sample with only 6% externally facing transporter was further analysed by confocal microscopy which qualitatively confirmed the low level of transporter in the membrane and the large internal population. Here we prove that flow cytometry is an important tool for future protein analysis as it can not only quantify the number of proteins that a cell express but also identify the number of proteins on the surface and it is easy to apply for routine assays.
Assuntos
Citometria de Fluxo , Hepatócitos/metabolismo , Espectrometria de Massas , Transportadores de Ânions Orgânicos/análise , Subfamília B de Transportador de Cassetes de Ligação de ATP/análise , Linhagem Celular , Hepatócitos/química , Humanos , Transportador 1 de Ânion Orgânico Específico do FígadoRESUMO
The Baeyer-Villiger monooxygenase (BVMO) 'MO14' from Rhodococcus jostii RHA1, is an enantioselective BVMO that catalyses the resolution of the model ketone substrate bicyclo[3.2.0]hept-2-en-6-one to the (1S,5R)-2-oxa lactone and the residual (1S,5R)-substrate enantiomer. This regio-plus enantioselective behaviour is highly unusual for BVMOs, which often perform enantiodivergent biotransformations of this substrate. The scaleability of the transformation was investigated using fermentor-based experiments, in which variables including gene codon optimisation, temperature and substrate concentration were investigated. E. coli cells expressing MO14 catalysed the resolution of bicyclo[3.2.0]hept-2-en-6-one to yield (1S,5R)-2-oxa lactone of >99% ee and (1S,5R)-ketone of 96% ee after 14 h at a temperature of 16 °C and a substrate concentration of 0.5 g L(-1) (4.5 mM). MO14 is thus a promising biocatalyst for the production of enantio-enriched ketones and lactones derived from the [3.2.0] platform.
Assuntos
Reatores Biológicos , Escherichia coli/genética , Cetonas/metabolismo , Oxigenases/genética , Rhodococcus/enzimologia , Biocatálise , Escherichia coli/citologia , Escherichia coli/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Cetonas/química , Conformação Molecular , Oxigenases/química , Oxigenases/metabolismo , Rhodococcus/metabolismo , Especificidade por SubstratoRESUMO
ARF GTPases are central regulators of membrane trafficking that control local membrane identity and remodeling facilitating vesicle formation. Unraveling their function is complicated by the overlapping association of ARFs with guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and numerous interactors. Through a functional genomic screen of three-dimensional (3D) prostate cancer cell behavior, we explore the contribution of ARF GTPases, GEFs, GAPs, and interactors to collective invasion. This revealed that ARF3 GTPase regulates the modality of invasion, acting as a switch between leader cell-led chains of invasion or collective sheet movement. Functionally, the ability of ARF3 to control invasion modality is dependent on association and subsequent control of turnover of N-cadherin. In vivo, ARF3 levels acted as a rheostat for metastasis from intraprostatic tumor transplants and ARF3/N-cadherin expression can be used to identify prostate cancer patients with metastatic, poor-outcome disease. Our analysis defines a unique function for the ARF3 GTPase in controlling how cells collectively organize during invasion and metastasis.
Assuntos
Fatores de Ribosilação do ADP , Proteínas Ativadoras de GTPase , Proteínas Monoméricas de Ligação ao GTP , Neoplasias da Próstata , Humanos , Masculino , Fatores de Ribosilação do ADP/genética , Caderinas/genética , Endocitose , Proteínas Ativadoras de GTPase/genética , Neoplasias da Próstata/genéticaRESUMO
A gene from the marine bacterium Stenotrophomonas maltophilia encodes a 38.6 kDa FAD-containing flavoprotein (Uniprot B2FLR2) named S. maltophilia flavin-containing monooxygenase (SMFMO), which catalyses the oxidation of thioethers and also the regioselective Baeyer-Villiger oxidation of the model substrate bicyclo[3.2.0]hept-2-en-6-one. The enzyme was unusual in its ability to employ either NADH or NADPH as nicotinamide cofactor. The K(M) and k(cat) values for NADH were 23.7±9.1 µM and 0.029 s(-1) and 27.3±5.3 µM and 0.022 s(-1) for NADPH. However, k(cat) /K(M) value for the ketone substrate in the presence of 100 µM cofactor was 17 times greater for NADH than for NADPH. SMFMO catalysed the quantitative conversion of 5 mM ketone in the presence of substoichiometric concentrations of NADH with the formate dehydrogenase cofactor recycling system, to give the 2-oxa and 3-oxa lactone products of Baeyer-Villiger reaction in a ratio of 5:1, albeit with poor enantioselectivity. The conversion with NADPH was 15 %. SMFMO also catalysed the NADH-dependent transformation of prochiral aromatic thioethers, giving in the best case, 80 % ee for the transformation of p-chlorophenyl methyl sulfide to its R enantiomer. The structure of SMFMO reveals that the relaxation in cofactor specificity appears to be accomplished by the substitution of an arginine residue, responsible for recognition of the 2'-phosphate on the NADPH ribose in related NADPH-dependent FMOs, with a glutamine residue in SMFMO. SMFMO is thus representative of a separate class of single-component, flavoprotein monooxygenases that catalyse NADH-dependent oxidations from which possible sequences and strategies for developing NADH-dependent biocatalysts for asymmetric oxygenation reactions might be identified.
Assuntos
Flavoproteínas/química , Niacinamida/química , Oxigenases/química , Sulfetos/química , Sequência de Aminoácidos , Animais , Catálise , Flavoproteínas/genética , Flavoproteínas/metabolismo , Dados de Sequência Molecular , NAD/química , NAD/genética , NAD/metabolismo , NADP/genética , NADP/metabolismo , Niacinamida/genética , Niacinamida/metabolismo , Oxirredução , Oxigenases/genética , Oxigenases/metabolismo , Stenotrophomonas maltophilia/enzimologia , Stenotrophomonas maltophilia/genética , Especificidade por Substrato , Sulfetos/metabolismoRESUMO
Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.
Assuntos
Clonagem Molecular/métodos , Escherichia coli/genética , Vetores Genéticos/normas , Complexos Multiproteicos/biossíntese , Proteínas Recombinantes/biossíntese , Academias e Institutos , Fator de Ligação a CCAAT/biossíntese , Fator de Ligação a CCAAT/genética , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Europa (Continente) , Geminina , Cooperação Internacional , Israel , Complexos Multiproteicos/química , Complexos Multiproteicos/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Fatores de Transcrição TFII/biossíntese , Fatores de Transcrição TFII/genéticaRESUMO
Controlling the selectivity of a chemical reaction with external stimuli is common in thermal processes, but rare in visible-light photocatalysis. Here we show that the redox potential of a carbon nitride photocatalyst (CN-OA-m) can be tuned by changing the irradiation wavelength to generate electron holes with different oxidation potentials. This tuning was the key to realizing photo-chemo-enzymatic cascades that give either the (S)- or the (R)-enantiomer of phenylethanol. In combination with an unspecific peroxygenase from Agrocybe aegerita, green light irradiation of CN-OA-m led to the enantioselective hydroxylation of ethylbenzene to (R)-1-phenylethanol (99 % ee). In contrast, blue light irradiation triggered the photocatalytic oxidation of ethylbenzene to acetophenone, which in turn was enantioselectively reduced with an alcohol dehydrogenase from Rhodococcus ruber to form (S)-1-phenylethanol (93 % ee).
RESUMO
BACKGROUND: The development of a schistosome vaccine has proved challenging but we have suggested that characterisation of the self-cure mechanism in rhesus macaques might provide a route to an effective product. The schistosome esophagus is a complex structure where blood processing is initiated by secretions from anterior and posterior glands, achieved by a mixture of ~40 unique proteins. The mechanism of self-cure in macaques involves cessation of feeding, after which worms slowly starve to death. Antibody coats the esophagus lumen and disrupts the secretory processes from the glands, potentially making their secretions ideal vaccine targets. METHODOLOGY/PRINCIPAL FINDINGS: We have designed three peptide arrays comprising overlapping 15-mer peptides encompassing 32 esophageal gland proteins, and screened them for reactivity against 22-week infection serum from macaques versus permissive rabbit and mouse hosts. There was considerable intra- and inter-species variation in response and no obvious unique target was associated with self-cure status, which suggests that self-cure is achieved by antibodies reacting with multiple targets. Some immuno-dominant sequences/regions were evident across species, notably including: MEGs 4.1C, 4.2, and 11 (Array 1); MEG-12 and Aspartyl protease (Array 2); a Tetraspanin 1 loop and MEG-n2 (Array 3). Responses to MEGs 8.1C and 8.2C were largely confined to macaques. As proof of principle, three synthetic genes were designed, comprising several key targets from each array. One of these was expressed as a recombinant protein and used to vaccinate rabbits. Higher antibody titres were obtained to the majority of reactive regions than those elicited after prolonged infection. CONCLUSIONS/SIGNIFICANCE: It is feasible to test simultaneously the additive potential of multiple esophageal proteins to induce protection by combining their most reactive regions in artificial constructs that can be used to vaccinate suitable hosts. The efficacy of the approach to disrupt esophageal function now needs to be tested by a parasite challenge.
Assuntos
Antígenos de Helmintos , Schistosoma japonicum/imunologia , Vacinas Sintéticas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/biossíntese , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/genética , Modelos Animais de Doenças , Mapeamento de Epitopos , Epitopos/genética , Esôfago/imunologia , Genes de Helmintos , Genes Sintéticos , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Macaca mulatta , Camundongos , Análise Serial de Proteínas , Coelhos , Ratos , Schistosoma japonicum/genética , Esquistossomose Japônica/imunologia , Esquistossomose Japônica/prevenção & controle , Vacinas Sintéticas/genéticaRESUMO
CotE is a coat protein that is present in the spores of Clostridium difficile, an obligate anaerobic bacterium and a pathogen that is a leading cause of antibiotic-associated diarrhoea in hospital patients. Spores serve as the agents of disease transmission, and CotE has been implicated in their attachment to the gut epithelium and subsequent colonization of the host. CotE consists of an N-terminal peroxiredoxin domain and a C-terminal chitinase domain. Here, a C-terminal fragment of CotE comprising residues 349-712 has been crystallized and its structure has been determined to reveal a core eight-stranded ß-barrel fold with a neighbouring subdomain containing a five-stranded ß-sheet. A prominent groove running across the top of the barrel is lined by residues that are conserved in family 18 glycosyl hydrolases and which participate in catalysis. Electron density identified in the groove defines the pentapeptide Gly-Pro-Ala-Met-Lys derived from the N-terminus of the protein following proteolytic cleavage to remove an affinity-purification tag. These observations suggest the possibility of designing peptidomimetics to block C. difficile transmission.
Assuntos
Proteínas de Bactérias/química , Quitinases/química , Clostridioides difficile/metabolismo , Peroxirredoxinas/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Quitinases/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Peroxirredoxinas/metabolismo , Proteínas de Plantas/metabolismo , Conformação ProteicaRESUMO
Morphinan alkaloids from the opium poppy are used for pain relief. The direction of metabolites to morphinan biosynthesis requires isomerization of (S)- to (R)-reticuline. Characterization of high-reticuline poppy mutants revealed a genetic locus, designated STORR [(S)- to (R)-reticuline] that encodes both cytochrome P450 and oxidoreductase modules, the latter belonging to the aldo-keto reductase family. Metabolite analysis of mutant alleles and heterologous expression demonstrate that the P450 module is responsible for the conversion of (S)-reticuline to 1,2-dehydroreticuline, whereas the oxidoreductase module converts 1,2-dehydroreticuline to (R)-reticuline rather than functioning as a P450 redox partner. Proteomic analysis confirmed that these two modules are contained on a single polypeptide in vivo. This modular assembly implies a selection pressure favoring substrate channeling. The fusion protein STORR may enable microbial-based morphinan production.
Assuntos
Benzilisoquinolinas/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Isoquinolinas/metabolismo , Morfinanos/metabolismo , Papaver/enzimologia , Proteínas de Plantas/metabolismo , Compostos de Amônio Quaternário/metabolismo , Sequência de Bases , Benzilisoquinolinas/química , Sistema Enzimático do Citocromo P-450/genética , Loci Gênicos , Isoquinolinas/química , Dados de Sequência Molecular , Morfinanos/química , Mutação , Oxirredução , Papaver/genética , Proteínas de Plantas/genética , Compostos de Amônio Quaternário/químicaRESUMO
BACKGROUND: Nudix hydrolases form a protein family whose function is to hydrolyse intracellular nucleotides and so regulate their levels and eliminate potentially toxic derivatives. The genome of the radioresistant bacterium Deinococcus radiodurans encodes 25 nudix hydrolases, an unexpectedly large number. These may contribute to radioresistance by removing mutagenic oxidised and otherwise damaged nucleotides. Characterisation of these hydrolases is necessary to understand the reason for their presence. Here, we report the cloning and characterisation of the DR0975 gene product, a nudix hydrolase that appears to be unique to this organism. RESULTS: The DR0975 gene was cloned and expressed as a 20 kDa histidine-tagged recombinant product in Escherichia coli. Substrate analysis of the purified enzyme showed it to act primarily as a phosphatase with a marked preference for (deoxy)nucleoside 5'-diphosphates (dGDP > ADP > dADP > GDP > dTDP > UDP > dCDP > CDP). Km for dGDP was 110 microM and kcat was 0.18 s-1 under optimal assay conditions (pH 9.4, 7.5 mM Mg2+). 8-Hydroxy-2'-deoxyguanosine 5'-diphosphate (8-OH-dGDP) was also a substrate with a Km of 170 microM and kcat of 0.13 s-1. Thus, DR0975 showed no preference for 8-OH-dGDP over dGDP. Limited pyrophosphatase activity was also observed with NADH and some (di)adenosine polyphosphates but no other substrates. Expression of the DR0975 gene was undetectable in logarithmic phase cells but was induced at least 30-fold in stationary phase. Superoxide, but not peroxide, stress and slow, but not rapid, dehydration both caused a slight induction of the DR0975 gene. CONCLUSION: Nucleotide substrates for nudix hydrolases conform to the structure NDP-X, where X can be one of several moieties. Thus, a preference for (d)NDPs themselves is most unusual. The lack of preference for 8-OH-dGDP over dGDP as a substrate combined with the induction in stationary phase, but not by peroxide or superoxide, suggests that the function of DR09075 may be to assist in the recycling of nucleotides under the very different metabolic requirements of stationary phase. Thus, if DR0975 does contribute to radiation resistance, this contribution may be indirect.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Deinococcus/enzimologia , Nucleotídeos/metabolismo , Pirofosfatases/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Deinococcus/efeitos dos fármacos , Deinococcus/genética , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Difosfatos/metabolismo , Indução Enzimática/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Cinética , Estrutura Molecular , Nucleotídeos/química , Pirofosfatases/genética , Pirofosfatases/metabolismo , Especificidade por Substrato , Superóxidos/farmacologia , Nudix HidrolasesAssuntos
Amidas/metabolismo , Artemia/enzimologia , Fosfatos de Dinucleosídeos/metabolismo , Nucleotidiltransferases/metabolismo , Ácidos Fosfóricos/metabolismo , Amidas/química , Aminoácidos/química , Aminoácidos/metabolismo , Animais , Artemia/fisiologia , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Estabilidade Enzimática , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Estrutura Molecular , Nucleotidiltransferases/química , Ácidos Fosfóricos/química , Radioisótopos de Fósforo/química , Radioisótopos de Fósforo/metabolismoRESUMO
A transcriptional product of a gene encoding cathepsin B-like peptidase in the bird schistosome Trichobilharzia regenti was identified and cloned. The enzyme was named TrCB2 due to its 77% sequence similarity to cathepsin B2 from the important human parasite Schistosoma mansoni. The zymogen was expressed in the methylotropic yeast Pichia pastoris; procathepsin B2 underwent self-processing in yeast media. The peptidolytic activity of the recombinant enzyme was characterised using synthetic fluorogenic peptide substrates at optimal pH 6.0. Functional studies using different specific inhibitors proved the typical cathepsin B-like nature of the enzyme. The S(2) subsite specificity profile of recombinant TrCB2 was obtained. Using monospecific antibodies against the recombinant enzyme, the presence of cathepsin B2 was confirmed in extracts from cercariae (infective stage) and schistosomula (early post-cercarial stage) of T. regenti on Western blots. Also, cross-reactivity was observed between T. regenti and S. mansoni cathepsins B2 in extracts of cercariae, schistosomula or adults. In T. regenti, the antisera localised the enzyme to post-acetabular penetration glands of cercariae implying an important role in the penetration of host skin. The ability of recombinant TrCB2 to degrade skin, serum and nervous tissue proteins was evident. Elastinolytic activity suggests that the enzyme might functionally substitute the histolytic role of the serine class elastase known from S. mansoni and Schistosoma haematobium but not found in Schistosoma japonicum or in bird schistosomes.
Assuntos
Cisteína Proteases/biossíntese , Cisteína Proteases/fisiologia , Cisteína/biossíntese , Schistosoma/enzimologia , Sequência de Aminoácidos , Animais , Western Blotting , Catepsina B/biossíntese , Bovinos , Cisteína Proteases/genética , Patos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Pichia , Coelhos , Schistosoma mansoni/enzimologia , Caramujos , Especificidade por Substrato , PerusRESUMO
Proteasomes are molecular machines found in virtually all cells that provide one of the mechanisms for protein turnover. We have analysed the 20S proteasome of Schistosoma mansoni, the first multimeric complex isolated from this helminth parasite. Three chromatographic steps were employed to yield a highly homogeneous preparation. 2-DE of the purified complex revealed 58 spots, of which 46 could be assigned either an alpha or a beta proteasome signature by MS. Most of the 14 transcripts (7alpha and 7beta) encoded by the parasite genome were represented by multiple spots and we suggest that this diversity is due to PTMs of subunits. For most of the isoforms, variations in pI predominated although alterations in mass were also observed. 2-DE separations of extracts from infective cercariae and blood-dwelling adult worms probed by Western blotting, using a human anti-alpha subunit antibody, revealed different patterns of reactivity, most probably in alpha3 and alpha6 subunits, on the basis of sequence conservation. This difference was rapidly lost following transformation of the cercaria to the skin schistosomulum stage, suggesting that changes in the proteasome structure, likely caused by the introduction of a new set of PTMs, precede remodelling of the parasite body prior to intravascular migration.