RESUMEN
A gene encoding a galactose oxidase (GalOx) was isolated from Fusarium sambucinum cultures and overexpressed in Escherichia coli yielding 4.4mg enzyme per L of growth culture with a specific activity of 159Umg(-1). By adding a C-terminal His-tag the enzyme could be easily purified with a single affinity chromatography step with high recovery rate (90%). The enzyme showed a single band on SDS-PAGE with an apparent molecular mass of 68.5kDa. The pH optimum for the oxidation of galactose was in the range of pH 6-7.5. Optimum temperature for the enzyme activity was 35°C, with a half-life of 11.2min, 5.3min, and 2.7min for incubation at 40°C, 50°C, and 60°C, respectively. From all tested substrates, the highest relative activity was found for 1-methyl-ß-galactopyranoside (226Umg(-1)) and the highest catalytic efficiency (kcat/Km) for melibiose (2700mM(-1)s(-1)). The enzyme was highly specific for molecular oxygen as an electron acceptor, and showed no appreciable activity with a range of alternative acceptors investigated. Different chemicals were tested for their effect on GalOx activity. The activity was significantly reduced by EDTA, NaN3, and KCN.
Asunto(s)
Escherichia coli/metabolismo , Proteínas Fúngicas , Fusarium/enzimología , Galactosa Oxidasa , Expresión Génica , Escherichia coli/genética , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Fusarium/genética , Galactosa Oxidasa/biosíntesis , Galactosa Oxidasa/química , Galactosa Oxidasa/genética , Galactosa Oxidasa/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
MicroRNAs are short non-coding RNAs that play an important role in the regulation of gene expression. Hence, microRNAs are considered as potential targets for engineering of Chinese hamster ovary (CHO) cells to improve recombinant protein production. Here, we analyzed and compared the microRNA expression patterns of high, low, and non-producing recombinant CHO cell lines expressing two structurally different model proteins in order to identify microRNAs that are involved in heterologous protein synthesis and secretion and thus might be promising targets for cell engineering to increase productivity. To generate reproducible and comparable data, the cells were cultivated in a bioreactor under steady-state conditions. Global microRNA expression analysis showed that mature microRNAs were predominantly upregulated in the producing cell lines compared to the non-producer. Several microRNAs were significantly differentially expressed between high and low producers, but none of them commonly for both model proteins. The identification of target messenger RNAs (mRNAs) is essential to understand the biological function of microRNAs. Therefore, we negatively correlated microRNA and global mRNA expression data and combined them with computationally predicted and experimentally validated targets. However, statistical analysis of the identified microRNA-mRNA interactions indicated a considerable false positive rate. Our results and the comparison to published data suggest that the reaction of CHO cells to the heterologous protein expression is strongly product- and/or clone-specific. In addition, this study highlights the urgent need for reliable CHO-specific microRNA target prediction tools and experimentally validated target databases in order to facilitate functional analysis of high-throughput microRNA expression data in CHO cells.
Asunto(s)
Células CHO/fisiología , MicroARNs/metabolismo , Animales , Reactores Biológicos , Técnicas de Cultivo de Célula/métodos , Cricetulus , Perfilación de la Expresión Génica , Regulación de la Expresión GénicaRESUMEN
Investigations of excited-state redox processes have an exceptional impact on the field of organic photochemistry and its application to the synthesis of complex target molecules. In such a photochemical process a single-electron transfer takes place to produce ion-radical intermediates, if the reduction and oxidation potentials, as well as excited-state energies of electron donors and acceptors are chosen appropriately. The Witkop cyclization constitutes an intramolecular variant of such a process, typically with an indole heterocycle as an electron donor. The specific synthetic value of this reaction lies in a CC bond formation without requiring any prefunctionalization of the indole system. Although this photoreaction has been applied to the total synthesis of natural products, it has still not been used to its full capacity. The following review details synthetic efforts using the Witkop cyclization, and aims to incite further applications of reaction in the synthesis of complex molecular architectures.
RESUMEN
Aim: The metabolite α-hydroxybutyrate (α-HB) is an important marker of insulin resistance and impaired glucose tolerance allowing to identify patients at risk of developing diabetes and related metabolic disorders before any symptoms become apparent. At present, its exact quantification requires mass spectrometry (LC-MS), which is not compatible with routine laboratory use. Accordingly, a simple enzymatic-based method was assessed and its applicability and measuring accuracy compared with LC-MS was investigated. Methods: Standards, serum, and plasma samples containing α-HB were prepared with routine procedures and their α-HB contents measured with the XpressGT® enzymatic test kit photometrically or with LC-MS and multiple reaction monitoring. Results: α-HB detection with XpressGT® yielded highly linear calibration curves and 102 % recovery of stocks added to commercial samples. Stability of the analyte in serum and plasma samples prepared with various anti-coagulants was >90 % after 46 h for several widely used preparations and recovery after 3 freeze-thaw cycles was ≥95 % with these anti-coagulants. A direct comparison of 75 samples indicated very good agreement of α-HB levels determined by both methods, 86 % of XpressGT® samples being within ±20 % of LC-MS values and even 93 % within ±20 % considering only samples above 30 µM concentration. Conclusion: XpressGT®-based detection of α-HB is an easily applicable method which can be used for accurate and reliable quantification of the metabolite in clinical practice. Routine α-HB determination in patients at risk of developing diabetes would allow early establishment of preventive measures or pharmacological intervention reducing the risk for the onset of serious diabetes-related health problems.
RESUMEN
There is an alarming scarcity of novel chemical matter with bioactivity against multidrug-resistant Gram-negative bacterial pathogens. Cystobactamids, recently discovered natural products from myxobacteria, are an exception to this trend. Their unusual chemical structure, composed of oligomeric para-aminobenzoic acid moieties, is associated with a high antibiotic activity through the inhibition of gyrase. In this study, structural determinants of cystobactamid's antibacterial potency were defined at five positions, which were varied using three different synthetic routes to the cystobactamid scaffold. The potency against Acinetobacter baumannii could be increased ten-fold to an MIC (minimum inhibitory concentration) of 0.06 µg mL-1, and the previously identified spectrum gap of Klebsiella pneumoniae could be closed compared to the natural products (MIC of 0.5 µg mL-1). Proteolytic degradation of cystobactamids by the resistance factor AlbD was prevented by an amide-triazole replacement. Conjugation of cystobactamid's N-terminal tetrapeptide to a Bodipy moiety induced the selective localization of the fluorophore for bacterial imaging purposes. Finally, a first in vivo proof of concept was obtained in an E. coli infection mouse model, where derivative 22 led to the reduction of bacterial loads (cfu, colony-forming units) in muscle, lung and kidneys by five orders of magnitude compared to vehicle-treated mice. These findings qualify cystobactamids as highly promising lead structures against infections caused by Gram-positive and Gram-negative bacterial pathogens.
RESUMEN
BACKGROUND: Lytic polysaccharide monooxygenases (LPMO) release a spectrum of cleavage products from their polymeric substrates cellulose, hemicellulose, or chitin. The correct identification and quantitation of these released products is the basis of MS/HPLC-based detection methods for LPMO activity. The duration, effort, and intricate analysis allow only specialized laboratories to measure LPMO activity in day-to-day work. A spectrophotometric assay will simplify the screening for LPMO in culture supernatants, help monitor recombinant LPMO expression and purification, and support enzyme characterization. RESULTS: Based on a newly discovered peroxidase activity of LPMO, we propose a fast, robust, and sensitive spectrophotometric activity assay using 2,6-dimethoxyphenol (2,6-DMP) and H2O2. The fast enzymatic assay (300 s) consists of 1 mM 2,6-DMP as chromogenic substrate, 100 µM H2O2 as cosubstrate, and an adequate activity of LPMO in a suitable buffer. The high molar absorption coefficient of the formed product coerulignone (ε469 = 53,200 M-1 cm-1) makes the assay sensitive and allows reliable activity measurements of LPMO in concentrations of approx. 0.5-50 mg L-1. CONCLUSIONS: The activity assay based on 2,6-DMP detects a novel peroxidase activity of LPMO. This activity can be accurately measured and used for enzyme screening, production, and purification, and can also be applied to study binding constants or thermal stability. However, the assay has to be used with care in crude extracts, because other enzymes such as laccase or peroxidase will interfere with the assay. We also want to stress that the peroxidase activity is a homogeneous reaction with soluble substrates and should not be correlated to heterogeneous LPMO activity on polymeric substrates.
RESUMEN
We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose-methanol-choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi-covalent flavinylation in PDH, 9-S-cysteinyl, 8-alpha-N3-histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are D-glucose, D-galactose, L-arabinose, and D-xylose. As shown by in situ NMR analysis, D-glucose and D-galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino-bis-(3-ethylbenzthiazolin-6-sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (k(cat)/K(m)). The enzyme may play a role in lignocellulose degradation.
Asunto(s)
Agaricus/enzimología , Deshidrogenasas de Carbohidratos/metabolismo , Proteínas Fúngicas/metabolismo , Monosacáridos/metabolismo , Agaricus/genética , Secuencia de Aminoácidos , Deshidrogenasas de Carbohidratos/genética , Deshidrogenasas de Carbohidratos/aislamiento & purificación , Celulosa/metabolismo , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Galactosa/química , Galactosa/metabolismo , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica , Cinética , Lignina/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Monosacáridos/química , Oxidación-Reducción , Estructura Secundaria de Proteína , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrofotometría/métodos , Especificidad por Sustrato , TemperaturaRESUMEN
A total synthesis to the pseudoaspidospermidine family via a C-H activation/transannular cyclization strategy has been accomplished. The applicability of this approach is showcased in the concise synthesis (ten steps) of (±)-20S-hydroxy-1,2-dehydro-pseudoaspidospermidine (4) starting from literature known compound 11. Via a joint synthetic sequence we were also able to address the related iboga alkaloid (±)-isovelbanamine (7) in nine steps. Key features of this synthesis are a transannular cyclization to generate the pseudoaspidospermidine skeleton (C-H activation) and a Witkop photocyclization reaction providing a 9-membered lactam. It is also worth mentioning that the joint synthetic sequence can be carried out on a multigram scale.
RESUMEN
The flavin-dependent enzyme pyranose oxidase catalyses the oxidation of several pyranose sugars at position C-2. In a second reaction step, oxygen is reduced to hydrogen peroxide. POx is of interest for biocatalytic carbohydrate oxidations, yet it was found that the enzyme is rapidly inactivated under turnover conditions. We studied pyranose oxidase from Trametes multicolor (TmPOx) inactivated either during glucose oxidation or by exogenous hydrogen peroxide using mass spectrometry. MALDI-MS experiments of proteolytic fragments of inactivated TmPOx showed several peptides with a mass increase of 16 or 32 Da indicating oxidation of certain amino acids. Most of these fragments contain at least one methionine residue, which most likely is oxidised by hydrogen peroxide. One peptide fragment that did not contain any amino acid residue that is likely to be oxidised by hydrogen peroxide (DAFSYGAVQQSIDSR) was studied in detail by LC-ESI-MS/MS, which showed a +16 Da mass increase for Phe454. We propose that oxidation of Phe454, which is located at the flexible active-site loop of TmPOx, is the first and main step in the inactivation of TmPOx by hydrogen peroxide. Oxidation of methionine residues might then further contribute to the complete inactivation of the enzyme.
Asunto(s)
Deshidrogenasas de Carbohidratos/química , Deshidrogenasas de Carbohidratos/metabolismo , Fenilalanina/metabolismo , Trametes/enzimología , Secuencia de Aminoácidos , Dominio Catalítico , Activación Enzimática , Estabilidad de Enzimas , Peróxido de Hidrógeno/metabolismo , Cinética , Espectrometría de Masas , Metionina/metabolismo , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Oxidación-Reducción , Péptidos/química , Especificidad por SustratoRESUMEN
Pyranose 2-oxidase (P2Ox) is a 270 kDa homotetramer localized preferentially in the hyphal periplasmic space of lignocellulolytic fungi and has a proposed role in lignocellulose degradation to produce the essential co-substrate, hydrogen peroxide, for lignin peroxidases. P2Ox oxidizes D-glucose and other aldopyranoses regioselectively at C2 to the corresponding 2-keto sugars; however, for some substrates, the enzyme also displays specificity for oxidation at C3. The crystal structure of P2Ox from Trametes multicolor has been determined using single anomalous dispersion with mercury as anomalous scatterer. The model was refined at 1.8A resolution to R and Rfree values of 0.134 and 0.171, respectively. The overall fold of the P2Ox subunit resembles that of members of the glucose-methanol-choline family of long-chain oxidoreductases, featuring a flavin-binding Rossmann domain of class alpha/beta and a substrate-binding subdomain with a six-stranded central beta sheet and three alpha helices. The homotetramer buries a large internal cavity of roughly 15,000 A3, from which the four active sites are accessible. Four solvent channels lead from the surface into the cavity through which substrate must enter before accessing the active site. The present structure shows an acetate molecule bound in the active site with the carboxylate group positioned immediately below the flavin N5 atom, and with one carboxylate oxygen atom interacting with the catalytic residues His548 and Asn593. The entrance to the active site is blocked by a loop (residues 452 to 461) with excellent electron density but elevated temperature factors. We predict that this loop is dynamic and opens to allow substrate entry and exit. In silico docking of D-glucose in the P2Ox active site shows that with the active-site loop in the closed conformation, monosaccharides cannot be accommodated; however, after removing the loop from the model, a tentative set of protein-substrate interactions for beta-D-glucose have been outlined.
Asunto(s)
Deshidrogenasas de Carbohidratos/química , Proteínas Fúngicas/química , Lignina/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Bases de Datos como Asunto , Electrones , Flavinas/química , Hongos/enzimología , Glucosa/química , Cinética , Modelos Químicos , Modelos Moleculares , Monosacáridos/química , Oxígeno/metabolismo , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Dispersión de RadiaciónRESUMEN
The white-rot fungus Trametes multicolor MB 49 has been identified as an excellent producer of the industrially important enzyme laccase. The formation of extracellular laccase could be considerably stimulated by the addition of Cu(II) to a simple, glycerol-based culture medium. In this study, optimal concentrations of copper were found to be 0.5-1 mM, which were added during the growth phase of the fungus. Other medium components important for laccase production are the carbon and nitrogen sources employed. When using an optimized medium containing glycerol (40 g/L), peptone from meat (15 g/L), and MgSO4 x 7H2O and stimulating enzyme formation by the addition of 1.0 mM Cu, maximal laccase activities obtained in shake-flask cultures were approx 85 U/mL. These results, however, could not be scaled up to a laboratory fermentor cultivation. Laccase production by T. multicolor decreased considerably when the fungus was grown in a stirred-tank reactor, presumably because of damage of the mycelia caused by shear stress and/or changes in the morphology of the fungus.
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Ascomicetos/enzimología , Oxidorreductasas/metabolismo , Ascomicetos/efectos de los fármacos , Ascomicetos/crecimiento & desarrollo , Cobre/farmacología , Medios de Cultivo , Cinética , LacasaRESUMEN
The wood-degrading fungus Trametes multicolor secretes several laccase isoforms when grown on a simple medium containing copper in the millimolar range for stimulating laccase synthesis. The main isoenzyme laccase II was purified to apparent homogeneity from the culture supernatant by using anion-exchange chromatography and gel filtration. Laccase II is a monomeric glycoprotein with a molecular mass of 63 kDa as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis, contains 18% glycosylation, and has a pI of 3.0. It oxidizes a variety of phenolic substrates as well as ferrocyanide and iodide. The pH optimum depends on the substrate employed and shows a bell-shaped pH activity profile with an optimum of 4.0 to 5.0 for the phenolic substrates, while the nonphenolic substrates ferrocyanide and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) show a monotonic pH profile with a rate decreasing with increasing pH.
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Basidiomycota/enzimología , Oxidorreductasas/aislamiento & purificación , Oxidorreductasas/metabolismo , Basidiomycota/crecimiento & desarrollo , Concentración de Iones de Hidrógeno , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Cinética , Lacasa , Peso Molecular , Especificidad por Sustrato , TermodinámicaRESUMEN
A short stereoselective synthesis of the Elisabethin A skeleton 4 is described, which opens a formal access to the diterpenes Elisapterosin B and Colombiasin A as well. Key reactions were an intermolecular endo-selective Diels-Alder reaction to generate the decalin part of the molecule, a chemo- and diastereoselective allylation of an aldehyde with allylzinc, a palladium ene annulation of the cyclopentane ring, and a novel sulfonium ylide induced fragmentation of a polycyclic ketone. Additional insights have been gained for the crucial epimerization at C-2.
Asunto(s)
Hidrocarburos Aromáticos con Puentes/síntesis química , Diterpenos/síntesis química , Hidrocarburos Aromáticos con Puentes/química , Diterpenos/química , Estructura Molecular , EstereoisomerismoRESUMEN
A gene coding for galactose 6-oxidase from Fusarium oxysporum G12 was cloned together with its native preprosequence and a C-terminal His-tag, and successfully expressed both in Escherichia coli and Pichia pastoris. The enzyme was subsequently purified and characterized. Among all tested substrates, the highest catalytic efficiency (kcat/Km) was found with 1-methyl-ß-D-galactopyranoside (2.2 mM(-1) s(-1)). The Michaelis constant (Km) for D-galactose was determined to be 47 mM. Optimal pH and temperature for the enzyme activity were 7.0 and 40°C, respectively, and the enzyme was thermoinactivated at temperatures above 50°C. GalOx contains a unique metalloradical complex consisting of a copper atom and a tyrosine residue covalently attached to the sulphur of a cysteine. The correct formation of this thioether bond during the heterologous expression in E. coli and P. pastoris could be unequivocally confirmed by MALDI mass spectrometry, which offers a convenient alternative to prove this Tyr-Cys crosslink, which is essential for the catalytic activity of GalOx.
Asunto(s)
Escherichia coli/genética , Fusarium/enzimología , Galactosa Oxidasa/genética , Galactosa Oxidasa/metabolismo , Pichia/genética , Secuencia de Aminoácidos , Clonación Molecular , Éteres/química , Fusarium/genética , Galactosa Oxidasa/química , Galactosa Oxidasa/aislamiento & purificación , Expresión Génica , Concentración de Iones de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
Chinese hamster ovary (CHO) cells are currently the workhorse of the biopharmaceutical industry. However, yeasts such as Pichia pastoris are about to enter this field. To compare their capability for recombinant protein secretion, P. pastoris strains and CHO cell lines producing human serum albumin (HSA) and the 3D6 single chain Fv-Fc anti-HIV-1 antibody (3D6scFv-Fc) were cultivated in comparable fed batch processes. In P. pastoris, the mean biomass-specific secretion rate (qp ) was 40-fold lower for 3D6scFv-Fc compared to HSA. On the contrary, qp was similar for both proteins in CHO cells. When comparing both organisms, the mean qp of the CHO cell lines was 1011-fold higher for 3D6scFv-Fc and 26-fold higher for HSA. Due to the low qp of the 3D6scFv-Fc producing strain, the space-time yield (STY) was 9.6-fold lower for P. pastoris. In contrast, the STY of the HSA producer was 9.2-fold higher compared to CHO cells because of the shorter process time and higher biomass density. The results indicate that the protein secretion machinery of P. pastoris is much less efficient and the secretion rate strongly depends on the complexity of the recombinant protein. However, process efficiency of the yeast system allows higher STYs for less complex proteins.
Asunto(s)
Biotecnología/métodos , Pichia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Proteínas Recombinantes/análisis , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/metabolismoRESUMEN
A primary objective of cell line development and process optimisation in animal cell culture is the improvement of culture performance as indicated by desirable properties such as high cell concentration, viability, productivity and product quality. The inefficient energy metabolism of mammalian cells in culture is still a major limiting factor for improvements in process performance. It results in high uptake rates of glucose and glutamine and the concomitant accumulation of waste products which in turn limits final cell concentrations and growth. To avoid these negative side effects, a CHO host cell line was established recently which is able to grow in completely glutamine free medium (Hernandez Bort et al., 2010). To determine the influence of this adaptation on productivity and product quality, the same procedure was repeated with a recombinant CHO cell line producing an erythropoietin-Fc fusion protein (CHO-EpoFc) for this publication. After adaptation to higher cell densities and glutamine free medium, culture performance was monitored in batch bioprocesses and revealed comparable growth properties and EpoFc product formation in both cell lines. The level of reactive oxygen species was elevated in the adapted cells, reflecting a higher level of oxidative stress, however, at the same time the level of the oxido-protective glutathione was also higher, so that cells seem adequately protected against cellular damage. Analysis of nucleotides and nucleotide sugars revealed elevated UDP-sugars in cells grown in the absence of glutamine. Furthermore, the antennarity of N-glycans was moderately higher on the Epo part of the protein produced by the adapted cell line compared to the parental cell line. Except for this, the glycosylation, with respect to site occupancy, degree of sialylation and glycoform structure, was highly comparable, both for the Epo and the Fc part of the protein.
Asunto(s)
Células CHO , Proteínas Recombinantes de Fusión/biosíntesis , Animales , Reactores Biológicos , Células CHO/citología , Células CHO/metabolismo , Supervivencia Celular , Cricetinae , Medios de Cultivo , Eritropoyetina/genética , Fermentación , Glutamina/química , Glutamina/metabolismo , Glicosilación , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Metaboloma , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The gene gaoA encoding the copper-dependent enzyme galactose oxidase (GAO) from Fusarium graminearum PH-1 was cloned and successfully overexpressed in E. coli. Culture conditions for cultivations in shaken flasks were optimized, and optimal conditions were found to be double-strength LB medium, 0.5% lactose as inducer, and induction at the reduced temperature of 25°C. When using these cultivation conditions ~24 mg of active GAO could be produced in shaken flasks per litre medium. Addition of copper to the fermentation medium decreased the enzyme production significantly. The His-tagged recombinant enzyme could be purified conveniently with a single affinity chromatography step. The purified enzyme showed a single band on SDS-PAGE with an apparent molecular mass of 66 kDa and had kinetic properties similar to those of the fungal wild-type enzyme.
RESUMEN
The fungal homotetrameric flavoprotein pyranose 2-oxidase (P2Ox; EC 1.1.3.10) catalyses the oxidation of various sugars at position C2, while, concomitantly, electrons are transferred to oxygen as well as to alternative electron acceptors (e.g. oxidized ferrocenes). These properties make P2Ox an interesting enzyme for various biotechnological applications. Random mutagenesis has previously been used to identify variant E542K, which shows increased thermostability. In the present study, we selected position Leu537 for saturation mutagenesis, and identified variants L537G and L537W, which are characterized by a higher stability and improved catalytic properties. We report detailed studies on both thermodynamic and kinetic stability, as well as the kinetic properties of the mutational variants E542K, E542R, L537G and L537W, and the respective double mutants (L537G/E542K, L537G/E542R, L537W/E542K and L537W/E542R). The selected substitutions at positions Leu537 and Glu542 increase the melting temperature by approximately 10 and 14 degrees C, respectively, relative to the wild-type enzyme. Although both wild-type and single mutants showed first-order inactivation kinetics, thermal unfolding and inactivation was more complex for the double mutants, showing two distinct phases, as revealed by microcalorimetry and CD spectroscopy. Structural information on the variants does not provide a definitive answer with respect to the stabilizing effects or the alteration of the unfolding process. Distinct differences, however, are observed for the P2Ox Leu537 variants at the interfaces between the subunits, which results in tighter association.
Asunto(s)
Biocatálisis , Deshidrogenasas de Carbohidratos/metabolismo , Evolución Molecular Dirigida , Temperatura , Trametes/enzimología , Deshidrogenasas de Carbohidratos/química , Deshidrogenasas de Carbohidratos/genética , Deshidrogenasas de Carbohidratos/aislamiento & purificación , Dicroismo Circular , Cristalografía por Rayos X , Diseño de Fármacos , Estabilidad de Enzimas , Expresión Génica , Cinética , Modelos Moleculares , Mutación/genética , Desnaturalización Proteica , Estructura Cuaternaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Trametes/genéticaRESUMEN
Pyranose 2-oxidase (P2Ox) participates in fungal lignin degradation by producing the H2O2 needed for lignin-degrading peroxidases. The enzyme oxidizes cellulose- and hemicellulose-derived aldopyranoses at C2 preferentially, but also on C3, to the corresponding ketoaldoses. To investigate the structural determinants of catalysis, covalent flavinylation, substrate binding, and regioselectivity, wild-type and mutant P2Ox enzymes were produced and characterized biochemically and structurally. Removal of the histidyl-FAD linkage resulted in a catalytically competent enzyme containing tightly, but noncovalently bound FAD. This mutant (H167A) is characterized by a 5-fold lower kcat, and a 35-mV lower redox potential, although no significant structural changes were seen in its crystal structure. In previous structures of P2Ox, the substrate loop (residues 452-457) covering the active site has been either disordered or in a conformation incompatible with carbohydrate binding. We present here the crystal structure of H167A in complex with a slow substrate, 2-fluoro-2-deoxy-D-glucose. Based on the details of 2-fluoro-2-deoxy-D-glucose binding in position for oxidation at C3, we also outline a probable binding mode for D-glucose positioned for regioselective oxidation at C2. The tentative determinant for discriminating between the two binding modes is the position of the O6 hydroxyl group, which in the C2-oxidation mode can make favorable interactions with Asp452 in the substrate loop and, possibly, a nearby arginine residue (Arg472). We also substantiate our hypothesis with steady-state kinetics data for the alanine replacements of Asp452 and Arg472 as well as the double alanine 452/472 mutant.
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Deshidrogenasas de Carbohidratos/metabolismo , Monosacáridos/química , Monosacáridos/metabolismo , Basidiomycota/enzimología , Conformación de Carbohidratos , Oxidación-Reducción , Unión Proteica , Especificidad por SustratoRESUMEN
Pyranose 2-oxidase (P2Ox) is a 270 kDa homotetrameric flavoenzyme that catalyzes the oxidation of D-glucose to 2-keto-D-glucose. P2Ox participates in lignin degradation by white-rot fungi and a tentative role of the enzyme is the production of H(2)O(2) for lignin peroxidases. Crystals of Trametes multicolor P2Ox were grown from monomethylether PEG 2000, sodium acetate, MgCl(2) and Ta(6)Br(12). They belong to space group P2(1), with unit-cell parameters a = 99.9, b = 101.7, c = 135.6 A, beta = 90.85 degrees. X-ray diffraction data to 2.0 A resolution were collected using synchrotron radiation. Self-rotation function calculations suggest that the asymmetric unit contains one homotetramer with 222 point-group symmetry.