RESUMEN
The health benefits of green tea are associated with its high catechin content. In scientific studies, green tea is often prepared with deionized water. However, casual consumers will simply use their local tap water, which differs in alkalinity and mineral content depending on the region. To assess the effect of water hardness on catechin and caffeine content, green tea infusions were prepared with synthetic freshwater in five different hardness levels, a sodium bicarbonate solution, a mineral salt solution, and deionized water. HPLC analysis was performed with a superficially porous pentafluorophenyl column. As water hardness increased, total catechin yield decreased. This was mostly due to the autoxidation of epigallocatechin (EGC) and epigallocatechin gallate (EGCG). Epicatechin (EC), epicatechin gallate (ECG), and caffeine showed greater chemical stability. Autoxidation was promoted by alkaline conditions and resulted in the browning of the green tea infusions. High levels of alkaline sodium bicarbonate found in hard water can render some tap waters unsuitable for green tea preparation.
Asunto(s)
Cafeína/química , Catequina/química , Té/química , Agua/química , Camellia sinensis/química , Catequina/análogos & derivados , Cromatografía Líquida de Alta Presión/métodos , Dureza , Minerales/química , Extractos Vegetales/químicaRESUMEN
Poplar hybrids can be used for selenium (Se) and boron (B) phytoremediation under saline conditions. The phenolic antioxidant stress response of two salt and B tolerant poplar hybrids of parentage Populus trichocarpa × nigra × deltoides was studied using high-performance liquid chromatography (HPLC) and absorption-based assays to determine the antioxidant capacity, total phenolic content, hydroxycinnamic acid levels, and the enzyme activity of l-phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), phenol peroxidase (POD), and laccase. Most remarkable was the contrasting response of the two poplar clones for PPO activity and phenolic levels to irrigation with high salt/B water. To cope with stressful growing conditions, only one clone increased its phenolic antioxidant level, and each clone displayed different PPO isoform patterns. Our study shows that poplar hybrids of the same parentage can differ in their salt/B stress coping mechanism.
Asunto(s)
Antioxidantes/análisis , Boro/análisis , Catecol Oxidasa/metabolismo , Fenoles/análisis , Proteínas de Plantas/metabolismo , Populus/enzimología , Populus/genética , Cloruro de Sodio/metabolismo , Antioxidantes/metabolismo , Boro/metabolismo , Fenoles/metabolismo , Populus/química , Populus/crecimiento & desarrollo , Cloruro de Sodio/análisisRESUMEN
An enzyme with catechol oxidase activity was identified in Thermomicrobium roseum extracts via solution assays and activity-stained SDS-PAGE. Yet, the genome of T. roseum does not harbor a catecholase gene. The enzyme was purified with two anion exchange chromatography steps and ultimately identified to be a manganese catalase with additional peroxidase and catecholase activity. Catalase activity (6280 ± 430 IU/mg) clearly dominated over pyrogallol peroxidase (231 ± 53 IU/mg) and catecholase (3.07 ± 0.56 IU/mg) activity as determined at 70 °C. Most enzyme kinetic properties were comparable to previously characterized manganese catalase enzymes. Catalase activity was highest at alkaline pH values and showed inhibition by excess substrate and chloride. The apparent K m and k cat values were 20 mM and 2.02 × 104 s-1 subunit-1 at 25 °C and pH 7.0.
Asunto(s)
Proteínas Bacterianas/metabolismo , Catalasa/metabolismo , Catecoles/metabolismo , Chloroflexi/enzimología , Peroxidasa/metabolismo , Proteínas Bacterianas/química , Catalasa/química , Estabilidad de Enzimas , Calor , Concentración de Iones de Hidrógeno , Peroxidasa/química , Pirogalol/metabolismo , Especificidad por SustratoRESUMEN
Tyramine ß-monooxygenase (TßM) belongs to a family of physiologically important dinuclear copper monooxygenases that function with a solvent-exposed active site. To accomplish each enzymatic turnover, an electron transfer (ET) must occur between two solvent-separated copper centers. In wild-type TßM, this event is too fast to be rate limiting. However, we have recently shown [Osborne, R. L.; et al. Biochemistry 2013, 52, 1179] that the Tyr216Ala variant of TßM leads to rate-limiting ET. In this study, we present a pH-rate profile study of Tyr216Ala, together with deuterium oxide solvent kinetic isotope effects (KIEs). A solvent KIE of 2 on kcat is found in a region where kcat is pH/pD independent. As a control, the variant Tyr216Trp, for which ET is not rate determining, displays a solvent KIE of unity. We conclude, therefore, that the observed solvent KIE arises from the rate-limiting ET step in the Tyr216Ala variant, and show how small solvent KIEs (ca. 2) can be fully accommodated from equilibrium effects within the Marcus equation. To gain insight into the role of the enzyme in the long-range ET step, a temperature dependence study was also pursued. The small enthalpic barrier of ET (Ea = 3.6 kcal/mol) implicates a significant entropic barrier, which is attributed to the requirement for extensive rearrangement of the inter-copper environment during PCET catalyzed by the Tyr216Ala variant. The data lead to the proposal of a distinct inter-domain pathway for PCET in the dinuclear copper monooxygenases.
Asunto(s)
Proteínas de Drosophila/metabolismo , Oxigenasas de Función Mixta/metabolismo , Protones , Temperatura , Animales , Cromatografía Líquida de Alta Presión , Drosophila/citología , Drosophila/metabolismo , Proteínas de Drosophila/aislamiento & purificación , Transporte de Electrón , Concentración de Iones de Hidrógeno , Cinética , Oxigenasas de Función Mixta/aislamiento & purificación , Solventes/químicaRESUMEN
Crude extracts of Ataulfo exhibited polyphenol oxidase (PPO) activity with pyrogallol, 3-methylcatechol, catechol, gallic acid, and protocatechuic acid. The substrate dependent pH optima ranged from pH 5.4 to 6.4 with Michaelis-Menten constants between 0.84 ± 0.09 and 4.6 ± 0.7 mM measured in MES or phosphate buffers. The use of acetate buffers resulted in larger Michaelis-Menten constants, up to 14.62 ± 2.03 mM. Sodium ascorbate, glutathione, and kojic acid are promising inhibitors to prevent enzymatic browning in Ataulfo. PPO activity increased with ripeness and was always higher in the skin compared to the pulp. Sodium dodecyl sulphate (SDS) enhanced PPO activity, with pulp showing a stronger increase than skin. SDS-PAGE gels stained for catecholase activity showed multiple bands, with the most prominent bands at apparent molecular weights of 53, 112, and 144 kDa.
Asunto(s)
Catecol Oxidasa/metabolismo , Mangifera/enzimología , Ácido Ascórbico/química , Ácido Ascórbico/metabolismo , Catecol Oxidasa/antagonistas & inhibidores , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Glutatión/química , Glutatión/metabolismo , Cinética , Peso Molecular , Extractos Vegetales/metabolismo , Unión Proteica , Pironas/química , Pironas/metabolismo , Dodecil Sulfato de Sodio/química , Dodecil Sulfato de Sodio/metabolismoRESUMEN
The hyperthermophilic bacterium Thermotoga maritima has a noncanonical nucleoside triphosphatase that catalyzes the conversion of inosine triphosphate (ITP), deoxyinosine triphosphate (dITP) and xanthosine triphosphate (XTP) into inosine monophosphate (IMP), deoxyinosine monophosphate (IMP) and xanthosine monophosphate (XMP), respectively. The k(cat)/K(m) values determined at 323 and 353 K fall between 1.31 × 10(4) and 7.80 × 10(4) M(-1) s(-1). ITP and dITP are slightly preferred over XTP. Activity towards canonical nucleoside triphosphates (ATP and GTP) was not detected. The enzyme has an absolute requirement for Mg(2+) as a cofactor and has a preference for alkaline conditions. A protein X-ray structure of the enzyme with bound IMP was obtained at 2.15 Å resolution. The active site houses a well conserved network of residues that are critical for substrate recognition and catalysis. The crystal structure shows a tetramer with two possible dimer interfaces. One of these interfaces strongly resembles the dimer interface that is found in the structures of other noncanonical nucleoside pyrophosphatases from human (human ITPase) and archaea (Mj0226 and PhNTPase).
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Pirofosfatasas/química , Pirofosfatasas/fisiología , Thermotoga maritima/enzimología , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Humanos , Biosíntesis de Proteínas , Conformación Proteica , Pirofosfatasas/genética , Distribución Aleatoria , Thermotoga maritima/genéticaRESUMEN
Design and chemical synthesis of de novo heme proteins with enzymatic activity on cellulose membranes is described. 352 antiparallel four-helix bundle proteins with a single histidine for heme ligation were assembled from three different sets of short amphipathic helices on membrane-bound peptide templates. The templates were coupled by linkers to cellulose membranes of microplate format, which could be cleaved for control of intermediate and final products. The incorporation of heme and the heme oxygenase activity of the 352 proteins were monitored by measuring UV-visible spectra directly on the cellulose. The kinetics of the heme oxygenase reaction was studied by monitoring the decrease of the Soret band and the transient absorbance of verdoheme being an intermediate product in the formation of biliverdin. Four of the proteins covering a broad range of the enzymatic rate constants were selected and synthesized in solution for further characterization. Detailed studies by redox potentiometry, analytical ultracentrifugation, and electron paramagnetic resonance spectroscopy yielded information about the aggregation state of the proteins, the spin state and the putative coordination environment of the iron. The amount of five-coordinated high-spin iron and a positive reduction potential were found to promote the oxygenase activity of the proteins.
Asunto(s)
Hemo Oxigenasa (Desciclizante)/metabolismo , Hemoproteínas/química , Hemoproteínas/síntesis química , Biblioteca de Péptidos , Secuencia de Aminoácidos , Catálisis , Celulosa/metabolismo , Centrifugación , Electroquímica , Espectroscopía de Resonancia por Spin del Electrón , Hemo/química , Hemoproteínas/metabolismo , Cinética , Datos de Secuencia Molecular , Oxidación-Reducción , Péptidos/química , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Protoporfirinas/química , Soluciones , Espectrometría de Masa por Ionización de Electrospray , Espectrofotometría Ultravioleta , Factores de Tiempo , VolumetríaRESUMEN
The heterodimeric hemoprotein SoxXA, essential for lithotrophic sulfur oxidation of the aerobic bacterium Paracoccus pantotrophus, was examined by a combination of spectroelectrochemistry and EPR spectroscopy. The EPR spectra for SoxXA showed contributions from three paramagnetic heme iron centers. One highly anisotropic low-spin (HALS) species (gmax = 3.45) and two "standard" cytochrome-like low-spin heme species with closely spaced g-tensor values were identified, LS1 (gz = 2.54, gy = 2.30, and gx = 1.87) and LS2 (gz = 2.43, gy = 2.26, and gx = 1.90). The crystal structure of SoxXA from P. pantotrophus confirmed the presence of three heme groups, one of which (heme 3) has a His/Met axial coordination and is located on the SoxX subunit [Dambe et al. (2005) J. Struct. Biol. 152, 229-234]. This heme was assigned to the HALS species in the EPR spectra of the isolated SoxX subunit. The LS1 and LS2 species were associated with heme 1 and heme 2 located on the SoxA subunit, both of which have EPR parameters characteristic for an axial His/thiolate coordination. Using thin-layer spectroelectrochemistry the midpoint potentials of heme 3 and heme 2 were determined: Em3 = +189 +/- 15 mV and Em2 = -432 +/- 15 mV (vs NHE, pH 7.0). Heme 1 was not reducible even with 20 mM titanium(III) citrate. The Em2 midpoint potential turned out to be pH dependent. It is proposed that heme 2 participates in the catalysis and that the cysteine persulfide ligation leads to the unusually low redox potential (-436 mV). The pH dependence of its redox potential may be due to (de)protonation of the Arg247 residue located in the active site.
Asunto(s)
Proteínas Bacterianas/fisiología , Grupo Citocromo c/fisiología , Hemo/química , Paracoccus pantotrophus/enzimología , Proteínas Bacterianas/química , Grupo Citocromo c/química , Electroquímica , Espectroscopía de Resonancia por Spin del Electrón , Modelos Químicos , Espectrofotometría Ultravioleta , Tiosulfatos/metabolismoRESUMEN
COMMD1 is the prototype of a new protein family that plays a role in several important cellular processes, including NF-kappaB signaling, sodium transport, and copper metabolism. The COMMD proteins interact with one another via a conserved C-terminal domain, whereas distinct functions are predicted to result from a variable N-terminal domain. The COMMD proteins have not been characterized biochemically or structurally. Here, we present the solution structure of the N-terminal domain of COMMD1 (N-COMMD1, residues 1-108). This domain adopts an alpha-helical structure that bears little resemblance to any other helical protein. The compact nature of N-COMMD1 suggests that full-length COMMD proteins are modular, consistent with specific functional properties for each domain. Interactions between N-COMMD1 and partner proteins may occur via complementary electrostatic surfaces. These data provide a new foundation for biochemical characterization of COMMD proteins and for probing COMMD1 protein-protein interactions at the molecular level.
Asunto(s)
Proteínas/química , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Proteínas Portadoras , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Soluciones , Electricidad EstáticaRESUMEN
A new crystal form of wild-type ribonucleotide reductase R2 from Escherichia coli was obtained. Crystals grow in space group P6(1)22 with one R2 monomer in the asymmetric unit. A twofold crystallographic symmetry axis generates the physiological dimeric form of R2. Co-crystallization with CoCl(2) or MnCl(2) results in full occupancy of the dinuclear metal site. The structure of the Mn(II)-loaded form was determined to 2.6 Angstroms resolution by molecular replacement. The crystallization conditions, backbone conformation, crystal-packing interactions and metal centers are compared with those of previously determined crystal forms.
Asunto(s)
Ribonucleótido Reductasas/química , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Escherichia coli/enzimología , Modelos Moleculares , Conformación ProteicaRESUMEN
Metalloenzyme crystal structures have a major impact on our understanding of biological metal centers. They are often the starting point for mechanistic and computational studies and inspire synthetic modeling chemistry. The strengths and limitations of X-ray crystallography in determining properties of biological metal centers and their corresponding ligand spheres are explored through examples, including ribonucleotide reductase R2 and particulate methane monooxygenase. Protein crystal structures locate metal ions within a protein fold and reveal the identities and coordination geometries of amino acid ligands. Data collection strategies that exploit the anomalous scattering effect of metal ions can establish metal ion identity. The quality of crystallographic data, particularly the resolution, determines the level of detail that can be extracted from a protein crystal structure. Complementary spectroscopic techniques can provide crucial information regarding the redox state of the metal center as well as the presence, type, and protonation state of exogenous ligands. The final result of the crystallographic characterization of a metalloenzyme is a model based on crystallographic data, supported by information from biophysical and modeling studies, influenced by sample handling, and interpreted carefully by the crystallographer.
Asunto(s)
Metaloproteínas/química , Metales/química , Modelos Moleculares , Oxigenasas/química , Ribonucleótido Reductasas/química , Aminoácidos/química , Aminoácidos/metabolismo , Productos Biológicos , Cationes , Cristalografía por Rayos X , Ligandos , Metaloproteínas/metabolismo , Metales/metabolismo , Oxidación-Reducción , Oxigenasas/metabolismo , Conformación Proteica , Pliegue de Proteína , Ribonucleótido Reductasas/metabolismoRESUMEN
Class I ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides. Eukaryotic RNRs comprise two subunits, the R1 subunit, which contains substrate and allosteric effector binding sites, and the R2 subunit, which houses a catalytically essential diiron-tyrosyl radical cofactor. In Saccharomyces cerevisiae, there are two variants of the R2 subunit, called Rnr2 and Rnr4. Rnr4 is unique in that it lacks three iron-binding residues conserved in all other R2s. Nevertheless, Rnr4 is required to activate Rnr2, and the functional species in vivo is believed to be a heterodimeric complex between the two proteins. The crystal structures of the Rnr2 and Rnr4 homodimers have been determined and are compared to that of the heterodimer. The homodimers are very similar to the heterodimer and to mouse R2 in overall fold, but there are several key differences. In the Rnr2 homodimer, one of the iron-binding helices, helix alphaB, is not well-ordered. In the heterodimer, interactions with a loop region connecting Rnr4 helices alphaA and alpha3 stabilize this Rnr2 helix, which donates iron ligand Asp 145. Sequence differences between Rnr2 and Rnr4 prevent the same interactions from occurring in the Rnr2 homodimer. These findings provide a structural rationale for why the heterodimer is the preferred complex in vivo. The active-site region in the Rnr4 homodimer reveals interactions not apparent in the heterodimer, supporting previous conclusions that this subunit does not bind iron. When taken together, these results support a model in which Rnr4 stabilizes Rnr2 for cofactor assembly and activity.
Asunto(s)
Ribonucleótido Reductasas/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Dimerización , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
The R2 subunit of Escherichia coli ribonucleotide reductase contains a dinuclear iron center that generates a catalytically essential stable tyrosyl radical by one electron oxidation of a nearby tyrosine residue. After acquisition of Fe(II) ions by the apo protein, the resulting diiron(II) center reacts with O(2) to initiate formation of the radical. Knowledge of the structure of the reactant diiron(II) form of R2 is a prerequisite for a detailed understanding of the O(2) activation mechanism. Whereas kinetic and spectroscopic studies of the reaction have generally been conducted at pH 7.6 with reactant produced by the addition of Fe(II) ions to the apo protein, the available crystal structures of diferrous R2 have been obtained by chemical or photoreduction of the oxidized diiron(III) protein at pH 5-6. To address this discrepancy, we have generated the diiron(II) states of wildtype R2 (R2-wt), R2-D84E, and R2-D84E/W48F by infusion of Fe(II) ions into crystals of the apo proteins at neutral pH. The structures of diferrous R2-wt and R2-D48E determined from these crystals reveal diiron(II) centers with active site geometries that differ significantly from those observed in either chemically or photoreduced crystals. Structures of R2-wt and R2-D48E/W48F determined at both neutral and low pH are very similar, suggesting that the differences are not due solely to pH effects. The structures of these "ferrous soaked" forms are more consistent with circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopic data and provide alternate starting points for consideration of possible O(2) activation mechanisms.