Papaverine 7-O-demethylase, a novel 2-oxoglutarate/Fe(2+)-dependent dioxygenase from opium poppy.

Opium poppy (Papaver somniferum) produces several pharmacologically important benzylisoquinoline alkaloids including the vasodilator papaverine. Pacodine and palaudine are tri-O-methylated analogs of papaverine, which contains four O-linked methyl groups. However, the biosynthetic origin of pacodine and palaudine has not been established. Three members of the 2-oxoglutarate/Fe(2+)-dependent dioxygenases (2ODDs) family in opium poppy display widespread O-dealkylation activity on several benzylisoquinoline alkaloids with diverse structural scaffolds, and two are responsible for the antepenultimate and ultimate steps in morphine biosynthesis. We report a novel 2ODD from opium poppy catalyzing the efficient substrate- and regio-specific 7-O-demethylation of papaverine yielding pacodine. The occurrence of papaverine 7-O-demethylase (P7ODM) expands the enzymatic scope of the 2ODD family in opium poppy and suggests an unexpected biosynthetic route to pacodine.

Electrochemical biosensor for DNA demethylase detection based on demethylation triggered endonuclease BstUI and Exonuclease III digestion.

Herein, an electrochemical biosensor was fabricated for DNA demethylase detection based on DNA demethylation triggered endonuclease BstUI and Exonuclease III digestion. After the double-strand DNA was demethylated, it can be further digested by BstUI and formed a blunt end at the electrode surface. Then, the remained fragment of DNA-DNA duplex was further cleaved by exonuclease III and led to increased electrochemical signal. Based on this detection strategy, the biosensor showed high sensitivity with low detection limit of 0.15ng/mL. Moreover, the developed method also presented high selectivity and acceptable reproducibility. This work provides a novel detection platform for DNA demethylase detection.

Three-Component O-Demethylase System Essential for Catabolism of a Lignin-Derived Biphenyl Compound in Sphingobium sp. Strain SYK-6.

Sphingobium sp. strain SYK-6 is able to assimilate lignin-derived biaryls, including a biphenyl compound, 5,5'-dehydrodivanillate (DDVA). Previously, ligXa (SLG_07770), which is similar to the gene encoding oxygenase components of Rieske-type nonheme iron aromatic-ring-hydroxylating oxygenases, was identified to be essential for the conversion of DDVA; however, the genes encoding electron transfer components remained unknown. Disruption of putative electron transfer component genes scattered through the SYK-6 genome indicated that SLG_08500 and SLG_21200, which showed approximately 60% amino acid sequence identities with ferredoxin and ferredoxin reductase of dicamba O-demethylase, were essential for the normal growth of SYK-6 on DDVA. LigXa and the gene products of SLG_08500 (LigXc) and SLG_21200 (LigXd) were purified and were estimated to be a trimer, a monomer, and a monomer, respectively. LigXd contains FAD as the prosthetic group and showed much higher reductase activity toward 2,6-dichlorophenolindophenol with NADH than with NADPH. A mixture of purified LigXa, LigXc, and LigXd converted DDVA into 2,2',3-trihydroxy-3'-methoxy-5,5'-dicarboxybiphenyl in the presence of NADH, indicating that DDVA O-demethylase is a three-component monooxygenase. This enzyme requires Fe(II) for its activity and is highly specific for DDVA, with a Km value of 63.5 µM and kcat of 6.1 s(-1). Genome searches in six other sphingomonads revealed genes similar to ligXc and ligXd (>58% amino acid sequence identities) with a limited number of electron transfer component genes, yet a number of diverse oxygenase component genes were found. This fact implies that these few electron transfer components are able to interact with numerous oxygenase components and the conserved LigXc and LigXd orthologs are important in sphingomonads.

A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6: purification and characterization.

Dicamba O-demethylase is a multicomponent enzyme that catalyzes the conversion of the herbicide 2-methoxy-3,6-dichlorobenzoic acid (dicamba) to 3,6-dichlorosalicylic acid (DCSA). The three components of the enzyme were purified and characterized. Oxygenase(DIC) is a homotrimer (alpha)3 with a subunit molecular mass of approximately 40 kDa. FerredoxinDIC and reductaseDIC are monomers with molecular weights of approximately 14 and 45 kDa, respectively. EPR spectroscopic analysis suggested the presence of a single [2Fe-2S](2+/1+) cluster in ferredoxinDIC and a single Rieske [2Fe-2S](2+; 1+) cluster within oxygenaseDIC. Consistent with the presence of a Rieske iron-sulfur cluster, oxygenaseDIC displayed a high reduction potential of E(m,7.0) = -21 mV whereas ferredoxinDIC exhibited a reduction potential of approximately E(m,7.0) = -171 mV. Optimal oxygenaseDIC activity in vitro depended on the addition of Fe2+. The identification of formaldehyde and DCSA as reaction products demonstrated that dicamba O-demethylase acts as a monooxygenase. Taken together, these data suggest that oxygenaseDIC is an important new member of the Rieske non-heme iron family of oxygenases.

Isolation and characterization of a veratrol:corrinoid protein methyl transferase from Acetobacterium dehalogenans.

From 3-methoxyphenol-grown cells of Acetobacterium dehalogenans, an inducible enzyme was purified that mediated the transfer of the methyl groups of veratrol (1,2-dimethoxybenzene) to a corrinoid protein enriched from the same cells. In this reaction, veratrol was converted via 2-methoxyphenol to 1,2-dihydroxybenzene. The veratrol:corrinoid protein methyl transferase, designated MTIver, had an apparent molecular mass of about 32 kDa. With respect to the N-terminal amino acid sequence and other characteristics, MTIver is different from the vanillate:corrinoid protein methyl transferase (MTIvan) isolated earlier from the same bacterium. For the methyl transfer from veratrol to tetrahydrofolate, two additional protein fractions were required, one of which contained a corrinoid protein. This protein was not identical with the corrinoid protein of the vanillate O-demethylase system. However, the latter corrinoid protein could also serve as methyl acceptor for the veratrol:corrinoid protein methyl transferase. MTIver catalyzed the demethylation of veratrol, 3,4-dimethoxybenzoate, 2-methoxyphenol, and 3-methoxyphenol. Vanillate (3-methoxy-4-hydroxybenzoate), 2-methoxybenzoate, or 4-methoxybenzoate could not serve as substrates.

Characterization of a three-component vanillate O-demethylase from Moorella thermoacetica.

The Moorella thermoacetica aromatic O-demethylase was characterized as an inducible three-component system with similarity to the methanogenic methanol, methylamine, and methanethiol methyltransferases and to the O-demethylase system from Acetobacterium dehalogenans. MtvB catalyzes methyl transfer from a phenylmethylether to the cobalt center of MtvC, a corrinoid protein. MtvA catalyzes transmethylation from MtvC to tetrahydrofolate, forming methyltetrahydrofolate. Cobalamin can substitute for MtvC.

DNA methylation is a reversible biological signal.

The pattern of DNA methylation plays an important role in regulating different genome functions. To test the hypothesis that DNA methylation is a reversible biochemical process, we purified a DNA demethylase from human cells that catalyzes the cleavage of a methyl residue from 5-methyl cytosine and its release as methanol. We show that similar to DNA methyltransferase, DNA demethylase shows CpG dinucleotide specificity, can demethylate mdCpdG sites in different sequence contexts, and demethylates both fully methylated and hemimethylated DNA. Thus, contrary to the commonly accepted model, DNA methylation is a reversible signal, similar to other physiological biochemical modifications.

A mammalian protein with specific demethylase activity for mCpG DNA.

DNA-methylation patterns are important for regulating genome functions, and are determined by the enzymatic processes of methylation and demethylation. The demethylating enzyme has now been identified: a mammalian complementary DNA encodes a methyl-CpG-binding domain, bears a demethylase activity that transforms methylated cytosine bases to cytosine, and demethylates a plasmid when the cDNA is translated or transiently transfected into human embryonal kidney cells in vitro. The discovery of this DNA demethylase should provide a basis for the molecular and developmental analysis of the role of DNA methylation and demethylation.

Isolation of O-demethylase, an ether-cleaving enzyme system of the homoacetogenic strain MC.

The O-demethylase of the methylotrophic homoacetogenic bacterium strain MC was purified to apparent homogeneity. The enzyme system consisted of four different components that were designated A, B, C, and D according to their elution sequence from the anionic-exchange chromatography column. All four components were essentially required for catalysis of the transfer of the methyl group from phenyl methyl ethers to tetrahydrofolate. According to gel filtration and SDS-PAGE, components A and B were monomers with apparent molecular masses of approximately 26 kDa (subunit 25 kDa) and 36 (subunit 41 kDa), respectively; component C appeared to be a trimeric protein (195 kDa, subunit 67 kDa); and component D was probably a dimer (64 kDa, subunit 30 kDa). Component A contained one corrinoid per monomer. In crude extracts, component D appeared to be the rate-limiting protein for the complete methyl transfer reaction. Additional requirements for the reaction were ATP and low-potential reducing equivalents supplied by either titanium(III) citrate or H2 plus hydrogenase purified from strain MC.

Product autofluorescence activation in the cytochrome P450 monooxygenase system.

The relative increase of the 7-ethoxycoumarin-O-deethylase and the 6,7-dimethoxycoumarin-O-demethylase activities were found 93 and 236% using a reconstituted cytochrome P4502B1:NADPH-P450 reductase system by adding to the reaction mixtures their own products. The assays were irradiated during the reactions with the excitation wavelength maximum of their products umbelliferone (lambda E = 365 nm) or scopoletin (lambda E 98 nm), respectively. Addition of the products to the reaction mixtures without irradiation (dark reaction) had no activating effect on the specific activities of the 7-ethoxycoumarin-O-deethylase or the 6,7-dimethoxycoumarin-O-demethylase. The relative increase of the specific activities is dependent on the excitation light intensities and was at maximum when the light intensity at the sample cuvette was 0.4 mW/cm2. The activation energies of the P4502B1-dependent 7-ethoxycoumarin-O-deethylation reaction obtained from Arrhenius plots with and without added umbelliferone and irradiation with lambda E = 365 nm are 14.7 kJ/mol and 33.5 kJ/mol, respectively, in the temperature range of 27-37 degrees C. The irradiation energy of the fluorescent product umbelliferone change the catalytic mechanism, which has a two times lower activation energy in the presence of the irradiated product umbelliferone. Umbelliferone and scopoletin have highest fluorescence intensities in the wavelength range of the blue light (440-480 nm). The photochemical action spectrum of the 7-ethoxycoumarin-O-deethylase of the P4502B1:reductase system is also found to be in the wavelength range of 420-470 nm. No activation effect was seen with irradiating light lower than 400 nm.(ABSTRACT TRUNCATED AT 250 WORDS)

Further observations on an ether-O-oxidase, formerly called alkyl etherase, from liver tissue.

1. The microsomal enzyme from liver previously called an "etherase" is now described more accurately as an ether-O-oxidase. It has been investigated further to free it from the membranes in aqueous solution and to try to define its physiological substrate. 2. After a variety of attempts with detergents, etc., the enzyme was obtained in impure solution from precipitation with 35-45% (NH4)2SO4 solution after a short digestion at room temperature. 3. When a suitably reinforced the enzyme in solution forms citrate from added ethyl ether, as it does in membranous form. This indicates the intermediary formation of acetyl CoA. 4. The enzyme in solution is unstable, though some activity remains after standing at 0degrees C for 2-3 days. Activity is lost rapidly by deep freezing, exposure to 2M-NaCl and at a pH more acid than pH 5-0. 5. The enzyme does not appear to be a known oxidase obtainable from liver microsomes; it is not for instance part of the inducible mixed oxygenase system, nor a peroxidase or catalase. 6. Since there were some similarities in stability with enzymes dealing with protozoal plasmalogens, or with lanosterol or cholesterol, we were led to explore these substrates in detail, with negative results. But a specimen of cholesterol oxidase from the branching bacterium Nocardia gave O-oxidation with diethylether. 7. The enzyme is present in the livers of all four animals examined, namely the rat, pig, guinea-pig and pigeon, but not in kidney or brain. 8. The enzyme takes up O2 with some compounds containing O-me groups. 9. The hypothesis is advanced that this normal oxidase in liver membranes exists to deal with some substances from plant sources which might prove toxic upon entering the circulation.

Mescaline-induced changes of brain-cortex ribosomes. Mescaline demethylase activity of brain-cortex soluble supernatant.

Brain-cortex slices demethylate mescaline and p-methoxyacetanilide, a reference O-demethylating substrate, though the rate of demethylation of mescaline is about one third that of the reference substrate. The demethylase activity is localized mostly in the soluble supernatant (105 000 x g). It is purified 47-fold with respect to the demethylation of mescaline by ammonium sulfate precipitation and DEAE cellulose chromatography. The partially purified demethylase, which is stable for 3-5 days at -5 degrees C in the presence of dithiothreitol and glutathione and is inhibited by p-chloromercuribenzoate, has maximal activity at pH between 7.2 and 8.0. It demethylates mescaline into 3,4-dimethoxy-5-hydroxyphenethylamine and 3,5-dimethoxy-4-hydroxyphenethylamine and some unidentified derivatives.

Bacterial attack on phenolic ethers. Purification and characterization of the components of the meta O-dealkylase of Pseudomonas fluorescens Tp.

The meta O-dealkylase of Pseudomonas fluorescens Tp has been resolved into two protein components, neither of which is a cytochrome. The substrate binding terminal oxidase has been purified and shown to be a non-haem iron protein of approximate molecular weight 118,000, consisting of two seemingly identical subunits, each of molecular weight 55,000. Binding of substrate by the terminal oxidase has been established by difference spectroscopy. The amino acid composition of the protein has also been determined. The NADH-dependent reductase of the system has been partly purified and appears to have a molecular weight of 80,000. The similarity between this and other bacterial O-dealkylases is discussed.

An affinity-column procedure for the purification of veratrate O-demethylase from fungi.

An affinity column procedure is reported for purifying veratrate O-demethylase from higher fungi. The procedure is based on the affinity of the fungal demethylases for veratrate, which was coupled to AH-Sepharose 4B. An over 300-fold purification of the enzyme from an Ascomycete (Chaetomium piluliferum), and a lower degree of purification (20-fold) from a Basidiomycete (Xerocomus badius), were obtained. The O-demethylases from higher fungi require NADH and oxygen. The enzyme activity is sensitive to exposure to oxygen. The pH optima are 5 for enzyme from Chaetomium, and 7 for demethylase from Xerocomus, respectively. The enzymes are not specific for veratrate. They also demethylate p- and m-anisate and 3,4-dimethoxycinnamate, but to a lower degree.