RESUMEN
UNLABELLED: Flavohemoglobins (flavoHbs) serve various microorganisms as the major protective enzymes against NOË-mediated toxicity. FlavoHbs dominantly function as an NOË dioxygenase (O2+ NOâ NO3 -), the required electron being shuttled from NAD(P)H via FAD to the heme iron. The X-ray structures of the flavoHb from Saccharomyces cerevisae presented in complex with an unknown small ligand (Yhb) and with econazole (Yhb(E) ) at 2.1 and 3.0 Å resolutions, respectively, reveal a high architectural accordance between prokaryotic and eukaryotic family members. The active site is characterized by a proximal heme side with a strictly conserved histidine, glutamate and tyrosine triad and a highly variable distal heme side with helix shifts up to 10 Å mainly dependent on the presence/absence and size of the bound ligand. In yeast flavoHb, the small heme iron ligand adjusts a catalytically productive active site geometry that reliably suggests the NO and O(2) binding site. O(2) is activated by its ligation to an electron-rich heme iron and a hydrogen bond to Tyr29 and Gln53. High active site similarities between eukaryotic Yhb and bacterial single-domain globins argue for identical biochemical reactions. Binding of the bulky econazole implies a large-scale induced-fit process concerning, in particular, an outwards shift of helices B and E to increase the active site pocket. Yeast Yhb and Ralstonia eutropha flavoHb both structurally studied in complex with econazole indicate conformational differences between the inhibitors and the polypeptide primarily caused by stable binding of a phospholipid to the latter and by distinct loop D structures. DATABASE: Structural data and final coordinates of Yhb and Yhb-econazole are available in the Protein Data Bank under the accession numbers 4G1V and 4G1B.
Asunto(s)
Dioxigenasas/metabolismo , Econazol/farmacología , Hemoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sitios de Unión , Catálisis , Dominio Catalítico , Dioxigenasas/química , Dioxigenasas/efectos de los fármacos , Hemoproteínas/química , Hemoproteínas/efectos de los fármacos , Ligandos , Modelos Moleculares , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/efectos de los fármacosRESUMEN
Flavohemoglobins (flavoHbs) are enzymes that operate primarily as nitric oxide dioxygenases and shuttle thereby electrons among NAD(P)H, FAD, heme, and a ligated redox-active substrate such as O(2). They function in the bacterial defense against nitrosative stress and are therefore considered as targets for new antibiotic drugs. Recently, azole derivatives were proven to be attractive nitric oxide dioxygenase inhibitors, and to explore their binding characteristics, we determined the X-ray structure of the flavoHb from Ralstonia eutropha in a complex with miconazole (FHP(M)), econazole (FHP(E)), and ketoconazole (FHP(K)). In agreement with UV-vis spectroscopic data, one azole compound binds inside the distal heme pocket and ligates to the heme iron by its imidazole substituent. The two additional substituents, mostly chlorinated phenyl groups, form a series of van der Waals contacts with the protein matrix. Both interactions explain their high affinity for flavoHbs, the binding constants being 2.6, 1.2, and 11.6 µM for miconazole, econazole, and ketoconazole, respectively. The FHP(M) and FHP(Lip) (flavoHbs originally loaded with a phospholipid) structures share an "open" state and the FHP(E) and FHP(K) structures a "closed" state. Although the azole compounds were able to push the lipid out of its binding site, a fatty acid fragment is still bound inside the heme pocket of FHP(E) and FHP(K) and dictates the state of the protein. The ligand-induced open-to-closed transition involves a reorientation of the NADH domain accompanied by conformational changes in the C-terminal arm, helix E, and the CE loop resulting in an encapsulation of the heme-binding pocket. Implications of the observed open-to-closed process on the catalytic cycle are discussed.
Asunto(s)
Azoles/química , Azoles/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cupriavidus necator/metabolismo , Hemoproteínas/química , Hemoproteínas/metabolismo , Sustancias Macromoleculares/química , Antibacterianos/química , Antibacterianos/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Cupriavidus necator/química , Modelos Biológicos , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , EspectrofotometríaRESUMEN
Polyphenol oxidase (PPO) catalyzes the oxidation of o-diphenols to their respective quinones which undergo autopolymerization and form dark pigments. The interaction of PPO with various substrates and effectors remains the focus of intensive investigations due to the enzyme's key role in pigments biosynthesis including animal melanogenesis and fruit/fungi enzymatic browning. In this study, the effect of a range of commonly used pesticides on the enzyme activity has been evaluated using the purified quince (Cydonia oblonga Miller) PPO. The biochemical analysis showed that, in the presence of high pesticide concentrations, the enzyme was competitively inhibited, particularly with benomyl, carbaryl, deltamethrine and parathion methyl for which inhibition constants (K(i)) were 8.3, 5.7, 12 and 4 microM, respectively. At lower pesticide concentrations (2-10 microM), however, the catecholase activity was significantly activated (p<0.01), suggesting a homotropic behavior of these chemical compounds. Furthermore, the use of in silico structure-based analyses, known as computational docking, highlighted the nature of the PPO-pesticides interactions and confirmed the in vitro observations. Catechol substrate and parathion methyl inhibitor showed lower total energy scores of -120.06 and -117.4 3 kcal mol(-1), indicating that these ligands had higher PPO-binding affinities. The obtained data bring to light new pesticide functional features of great interest in the medicinal, agro-chemical and environmental circles.
Asunto(s)
Catecol Oxidasa/antagonistas & inhibidores , Catecol Oxidasa/metabolismo , Plaguicidas/toxicidad , Rosaceae/enzimología , Secuencia de Aminoácidos , Catecol Oxidasa/genética , Biología Computacional , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Frutas/química , Ipomoea batatas/enzimología , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Técnicas de Amplificación de Ácido Nucleico , Conformación Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The Pol6 mutant of Penicillium occitanis fungus is of great biotechnological interest since it possesses a high capacity of cellulases and beta-glucosidase production with high cellulose degradation efficiency (Jain et al., Enzyme Microb Technol, 12:691-696, 1990; Hadj-Taieb et al., Appl Microbiol Biotechnol, 37:197-201, 1992; Ellouz Chaabouni et al., Enzyme Microb Technol, 16:538-542, 1994; Ellouz Chaabouni et al., Appl Microbiol Biotechnol, 43:267-269, 1995). In this work, two forms of beta-glucosidase (beta-glu 1 and beta-glu 2) were purified from the culture supernatant of the Pol6 strain by gel filtration, ion exchange chromatography, and preparative anionic native electrophoresis. These enzymes were eluted as two distinct species from the diethylamino ethanol Sepharose CL6B and anionic native electrophoresis. However, both behaved identically on sodium dodecyl sulfate polyacrylamide gel electrophoresis (MW, 98 kDa), shared the same amino acid composition, carbohydrate content (8%), and kinetic properties. Moreover, they strongly cross-reacted immunologically. They were active on cellobiose and pNPG with Km values of 1.43 and 0.37 mM, respectively. beta-glu 1 and beta-glu 2 were competitively inhibited by 1 mM of glucose and 0.03 mM of delta-gluconolactone. They were also significantly inhibited by Hg(2+) and Cu(2) at 2 mM. The addition of purified enzymes to the poor beta-glucosidase crude extract of Trichoderma reesei increased its hydrolytic efficiency on H(3)P0(4) swollen cellulose but had no effect with P. occitanis crude extract. Besides their hydrolytic activities, beta-glu 1 and beta-glu 2 were endowed with trans-glycosidase activity at high concentration of glucose.
Asunto(s)
Mutación , Penicillium/enzimología , beta-Glucosidasa/aislamiento & purificación , Celulasa/metabolismo , Celulosa/metabolismo , Glucosa/metabolismo , beta-Glucosidasa/metabolismoRESUMEN
Quince (Cydonia oblonga Miller) fruit aqueous acetone extracts were evaluated. High-performance liquid chromatography-diode array detection and electrospray ionization-mass spectrometry were used for the identification and quantification of the phenolic compounds. The total phenolic content of the pulp and peel parts ranged from 37 to 47 and 105 to 157 mg/100 g of fresh weight, respectively. Chlorogenic acid (5-O-caffeoylquinic acid) was the most abundant phenolic compound in the pulp (37%), whereas rutin (quercetin 3-O-rutinoside) was the main one in the peel (36%). The radical scavenging potential of the extracts was determined and compared with that of synthetic antioxidants. The stronger properties corresponded to those obtained from peel material with a 70-80% inhibitory effect on DPPH radicals. The antimicrobial activity of the extracts against different microorganism strains was also investigated. Quince peel extract was the most active for inhibiting bacteria growth with minimum inhibitory and bactericide concentrations in the range of 102-5 x 103 microg polyphenol/mL. It seems that chlorogenic acid acts in synergism with other components of the extracts to exhibit their total antimicrobial activities.
Asunto(s)
Antiinfecciosos/farmacología , Flavonoides/análisis , Flavonoides/farmacología , Frutas/química , Fenoles/análisis , Fenoles/farmacología , Rosaceae/química , Antioxidantes/farmacología , Ácido Clorogénico/análisis , Extractos Vegetales/química , Extractos Vegetales/farmacología , Polifenoles , Rutina/análisisRESUMEN
A new approach to galacto-oligosaccharides and galacto-conjugates synthesis performed by the beta-galactosidase from Kluyveromyces lactis is reported. The enzymatic galactosylation of eight kinds of adsorbed aromatic primary alcohols, in particular the two drugs guaifenesin and chlorphenesin, gave the corresponding beta-D-galacto-pyranosides in yields ranging between approximately 10% and 96%. For the first time, we have showed that the adsorption of acceptor substrates onto solid supports such as silica gel influences the yield and the selectivity of galacto-conjugates synthesis. In particular, we observed that adsorption of acceptor favored the synthesis of digalactosylated compounds.
Asunto(s)
Hidrocarburos Aromáticos/metabolismo , Kluyveromyces/enzimología , beta-Galactosidasa/metabolismo , Adsorción , Catálisis , Clorfenesina/química , Clorfenesina/metabolismo , Glicosilación , Guaifenesina/química , Guaifenesina/metabolismo , Hidrocarburos Aromáticos/química , Estructura MolecularRESUMEN
Two endoglucanases (EGs), EG A and EG B, were purified to homogeneity from Penicillium occitanis mutant Pol 6 culture medium. The molecular weights of EG A and EG B were 31,000 and 28,000 kDa, respectively. The pI was about 3 for EG A and 7.5 for EG B. Optimal activity was obtained at pH 3.5 for both endoglucanases. Optimal temperature for enzyme activity was 60 degrees C for EG A and 50 degrees C for EG B. EG A was thermostable at 60 degrees C and remained active after 1 h at 70 degrees C. EGs hydrolyzed carboxymethylcellulose, phosphoric acid swollen cellulose, and beta-glucan efficiently, whereas microcrystalline cellulose (Avicel) and laminarin were poorly hydrolyzed. Only EG B showed xylanase activity. Furthermore, these EGs were insensitive to the action of glucose and cellobiose but were inhibited by the divalent cations Hg2+, Co2+, and Mn2+.
Asunto(s)
Celulasa/aislamiento & purificación , Celulasa/metabolismo , Penicillium/enzimología , Carboximetilcelulosa de Sodio/metabolismo , Cationes Bivalentes/química , Celulasa/química , Celulosa/metabolismo , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Glucanos , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Cinética , Peso Molecular , Mutación , Penicillium/genética , Polisacáridos/metabolismo , Especificidad por Sustrato , Temperatura , Xilanos/metabolismo , beta-Glucanos/metabolismoRESUMEN
A domain of epiglucan was synthesized by beta-glucosidases. Two beta-glucosidases, an extracellular beta-glucosidase derived from Sclerotinia sclerotiorum grown on xylose, and a commercial lyophilized preparation of beta-glucosidase from Aspergillus niger, were used to synthesize gluco-oligosaccharides from cellobiose and, specially, beta-(1-6) branched beta-(1-3) gluco-oligosaccharides, corresponding to the structure of epiglucan. Gentiobiose, cellotriose, cellotetraose, beta-Glc-(1-3)-beta-Glc-(1-4)-Glc, beta-Glc-(1-6)-beta-Glc-(1-4)-Glc and beta-Glc-(1-6)-beta-Glc-(1-3)-Glc were synthesized from cellobiose by both enzymes. The latter compound was preferentially synthesized by the beta-glycosidase from Sclerotinia sclerotiorum. Under the best conditions, only 7 g l(-1) of beta-Glc-(1-6)-beta-Glc-(1-3)-Glc was synthesized by the beta-glycosidase from Aspergillus niger compared to 20 g l(-1) synthesized with beta-glycosidase from Sclerotinia sclerotiorum.