Studies in vitro on the involvement of O-sulphate esters in the formation of O-methylated 3,4-dihydroxybenzoic acid by rat liver.

The involvement of O-sulphate esters in the directed O-methylation was investigated in vitro with a dialysed "high-speed' supernatant from rat liver as the enzyme preparation and the catechol compound 3,4-dihydroxybenzoic acid as the substrate. The enzyme reactions involved were studied separately with the O-methylated and O-sulphated derivatives. The rate of hydrolysis by arylsulphatase was 14.5 nmol/min per mg of protein for 3-methoxy-4-sulphonyloxybenzoic acid and 10.1 nmol/min per mg of protein for 4-methoxy-3-sulphonyloxybenzoic acid. The sulphotransferase activity towards the guaiacols 4-hydroxy-3-methoxybenzoic acid and 3-hydroxy-4-methoxybenzoic acid was 570pmol of 4-O-sulphated and 350pmol of 3-O-sulphated product formed/min per mg of protein. The 3-O- and 4-O-sulphate esters of 3,4-dihydroxybenzoic acid could not serve as substrates for the catechol O-methyltransferase reaction. When either ester was incubated in the presence of S-adenosyl-L-methionine, but without the arylsulphatase inhibitor KH2PO4, 3,4-dihydroxybenzoic acid was formed, which was subsequently O-methylated in a meta/para ratio of 4.6. It is concluded that O-methylation can precede O-sulphation but that O-sulphation prevents further metabolism by O-methylation. Also O-sulphate esters do not have a directing effect on O-methylation. From the study of the simultaneous action of sulphotransferase and catechol O-methyltransferase on 3,4-dihydroxybenzoic acid we conclude that O-sulphation and O-methylation proceed independently of each other under the assay conditions used, both directed preferentially to the 3-hydroxy group.     

Regioselectivity of catechol O-methyltransferase. The effect of pH on the site of O-methylation of fluorinated norepinephrines

Selectivity of catechol O-methyltransferase has been examined for the three ring-fluorinated norepinephrines to elucidate the role of acidity of the phenolic groups in their methylation. Substitution of fluorine at the 5-position of norepinephrine reverses the selectivity of catechol O-methyltransferase so that p-O-methylation predominates. The 5-fluoro substituent also causes the pKa of the p-hydroxyl group to decrease substantially. In contrast, 2- and 6-fluoronorepinephrines are methylated predominantly at the m-hydroxyl group. These results suggest that acidity of a phenolic group can play an important role in its ability to be methylated by catechol O-methyltransferase. Percentages of p-O-methylation of norepinephrine and its fluorinated derivatives increase with pH. This relative increase in p-O-methylation appears to accompany ionization of a group with pKa of 8.6, 7.7, 7.9, and 8.4 for norepinephrine and its 2-, 5-, and 6-fluoro derivative, respectively. These pKa values are the same as or similar to the pKa values of a phenolic hydroxyl group of these substrates. 3,4-Dihydroxybenzyl alcohol and its 5-fluoro derivative are O-methylated by catechol O-methyltransferase to form p- and m-O-methyl products in approximately 1:1 and 4:1 ratios, respectively, at all pH values. Based on the above results, a catechol-binding site model for catechol O-methyltransferase is proposed in which the two phenolic hydroxyl groups of catechol substrates are postulated to be approximately equally spaced from the methyl group of the cosubstrate S-adenosylmethionine.     

The effect of bulk hydrogen ion concentration upon the apparent kinetic parameters of purified pig liver catechol O-methyltransferase

In order to investigate the pH dependence of catechol O-methyltransferase (S-adenosyl-L-methionine:catechol O-methyltransferase, EC 2.1.1.6), kinetic parameters have been determined for the highly purified enzyme from pig liver over the pH range 6.75-8.20 using the substrates S-adenosylmethionine (AdoMet) and 3,4-dihydroxyphenylacetic acid (DOPAC). The Km for AdoMet was found to be invariant with pH while the Km for DOPAC decreased sharply with increasing pH. The group responsible for the latter has a pK of approx. 7.1. The logarithmic (Dixon) plot of Km against pH for both substrates and that of Vmax/Km against pH for DOPAC mirror the kinetic behaviour revealed by linear plots. However, for other parameters, linear graphs indicate peaks too narrow to be explicable by a simple kinetic mechanism, whereas logarithmic plots of these parameters produce graphs apparently not reflecting this behaviour. We conclude that these results are not the products of random error or artefactual data analysis but are too complex to be explicable by a simple model of kinetic behaviour. Possible explanations (adherence of catechol O-methyltransferase to a higher-order mechanism or a dual mode of substrate binding) are advanced.     

Properties of catechol O-methyltransferases from brain and liver of rat and human.

Kinetic and electrophoretic properties of catechol O-methyltransferases (EC 2.1.1.6) from brain and liver were studied. The enzyme of either rat or human tissues exhibited a single molecular form when subjected to electrophoresis at pH7.9. At pH9 a second, apparently oxidized, form was detected. Isoelectric-focusing experiments also indicated only one enzyme form, which was identical from extracts of brain and liver of each species (pI = 5.2 for rat, 5.5 for human). Similarities between brain and liver catechol O-methyltransferase of a given species were also demonstrated by kinetic parameters, meta/para ratios of products, and inhibitor potencies. Human catechol O-methyltransferase exhibited lower Km values than did the rat enzyme for S-adenosyl-L-methionine, dopamine and dihydroxybenzoic acid. Adrenochrome inhibited both rat and human enzyme. It was concluded (1) that only a single enzyme form could be demonstrated in the physiological pH region; (2) that catechol O-methyltransferase of brain could not be distinguished from the liver enzyme of the same species; and (3) that species differences exist between the enzymes of rat and human tissues.     

Identification of the enzymatic active site of tobacco caffeoyl-coenzyme A O-methyltransferase by site-directed mutagenesis.

Animal catechol O-methyltransferases and plant caffeoyl-coenzyme A O-methyltransferases share about 20% sequence identity and display common structural features. The crystallographic structure of rat liver catechol O-methyltransferase was used as a template to construct a homology model for tobacco caffeoyl-coenzyme A O-methyltransferase. Integrating substrate specificity data, the three-dimensional model identified several amino acid residues putatively involved in substrate binding. These residues were mutated by a polymerase chain reaction method and wild-type and mutant enzymes were each expressed in Escherichia coli and purified. Substitution of Arg-220 with Thr resulted in the total loss of enzyme activity, thus indicating that Arg-220 is involved in the electrostatic interaction with the coenzyme A moiety of the substrate. Changes of Asp-58 to Ala and Gln-61 to Ser were shown to increase K(m) values for caffeoyl coenzyme A and to decrease catalytic activity. Deletions of two amino acid sequences specific for plant enzymes abolished activity. The secondary structures of the mutants, as measured by circular dichroism, were essentially unperturbed as compared with the wild type. Similar changes in circular dichroism spectra were observed after addition of caffeoyl coenzyme A to the wild-type enzyme and the substitution mutants but not in the case of deletion mutants, thus revealing the importance of these sequences in substrate-enzyme interactions.     

Interaction of silver nanoparticles with catechol O-methyltransferase: Spectroscopic and simulation analyses

Catechol O-methyltransferase, an enzyme involved in the metabolism of catechol containing compounds, catalyzes the transfer of a methyl group between S-adenosylmethionine and the hydroxyl groups of the catechol. Furthermore it is considered a potential drug target for Parkinson’s disease as it metabolizes the drug levodopa. Consequently inhibitors of the enzyme would increase levels of levodopa. In this study, absorption, fluorescence and infrared spectroscopy as well as computational simulation studies investigated human soluble catechol O-methyltransferase interaction with silver nanoparticles. The nanoparticles form a corona with the enzyme and quenches the fluorescence of Trp143. This amino acid maintains the correct structural orientation for the catechol ring during catalysis through a static mechanism supported by a non-fluorescent fluorophore–nanoparticle complex. The enzyme has one binding site for AgNPs in a thermodynamically spontaneous binding driven by electrostatic interactions as confirmed by negative ΔG and ΔH and positive ΔS values. Fourier transform infrared spectroscopy within the amide I region of the enzyme indicated that the interaction causes relaxation of its β?structures, while simulation studies indicated the involvement of six polar amino acids. These findings suggest AgNPs influence the catalytic activity of catechol O-methyltransferase, and therefore have potential in controlling the activity of the enzyme.     

Kinetics and inhibition studies of catechol O-methyltransferase from the yeast Candida tropicalis.

The Kms for esculetin and S-adenosyl-L-methionine for catechol O-methyltransferase from the yeast Candida tropicalis were 6.2 and 40 microM, respectively. S-Adenosyl-L-homocysteine was a very potent competitive inhibitor with respect to S-adenosyl-L-methionine, with a Ki of 6.9 microM. Of the catechol-related inhibitors, purpurogallin, with a Ki of 0.07 microM, showed the greatest inhibitory effect. Sulfhydryl group-blocking reagents, such as thiol-oxidizing 2-iodosobenzoic acid and mercaptide-forming p-chloromercuribenzoic acid, provided evidence for sulfhydryl groups in the active site of the enzyme. Yeast catechol O-methyltransferase is a metal-dependent enzyme and requires Mg2+ for full activity. Zn2+ and Mn2+ but not Ca2+ were able to substitute for Mg2+. Mn2+ showed optimal enzyme activation at concentrations 50- to 100-fold lower than those of Mg2+.     

Crystallization and preliminary X-ray investigation of a recombinant form of rat catechol O-methyltransferase.

Rat catechol O-methyltransferase cDNA was introduced into an E. coli expression vector pKEX14, which utilizes the inducible T7 promoter. Active and soluble recombinant catechol O-methyltransferase was produced in bacteria and purified to electrophoretic homogeneity by chromatographic procedures. The purified enzyme has been crystallized by the method of vapor diffusion using polyethylene glycol as precipitant. The space group is P3(1)21 or P3(2)21 with a = b = 51.3 A and c = 168.5 A and one molecule in the asymmetric unit. The crystals diffract beyond 3.2 A and are suitable for three-dimensional X-ray structure determination.     

A rapid and sensitive radioenzymatic assay for catechol estrogens in tissues

A rapid and sensitive radioenzymatic assay for measuring catechol estrogens in tissue has been developed. This assay is based on converting the relatively labile catechol estrogens to stable O-methylated derivatives by the enzyme catechol-O-methyl-transferase. Using a radioactive methyl donor of high specific activity (³H-S-adenosylmethionine) and solvent extraction with non-polar solvents a sensitivity of 25 pg can be achieved. The specificity of this assay was confirmed by thin layer chromatography and mass spectral analysis of the reaction products. Catechol estrogens in rat liver are significantly decreased after ovariectomy and markedly increased by treatment with estrogen.     

A general liquid chromatography/mass spectroscopy-based assay for detection and quantitation of methyltransferase activity

Methyltransferases form a large class of enzymes, most of which use S-adenosylmethionine as the methyl donor. In fact, S-adenosylmethionine is second only to ATP in the variety of reactions for which it serves as a cofactor. Several methods to measure methyltransferase activity have been described, most of which are applicable only to specific enzymes and/or substrates. In this work we describe a sensitive liquid chromatography/mass spectroscopy-based methyltransferase assay. The assay monitors the conversion of S-adenosylmethionine to S-adenosylhomocysteine and can be applied to any methyltransferase and substrate of interest. We used the well-characterized enzyme catechol O-methyltransferase to demonstrate that the assay can monitor activity with a variety of substrates, can identify new substrates, and can be used even with crude preparation of enzyme. Furthermore, we demonstrate the utility of the assay for kinetic characterization of enzymatic activity.     

Separation methods for catechol O-methyltransferase activity assay: physiological and pathophysiological relevance

Catechol O-methyltransferase (COMT) transfers a methyl group from S-adenosyl-L-methionine to the catechol substrate in the presence of magnesium. After the characterisation of COMT more than four decades ago, a wide variety of COMT enzyme assays have been introduced. COMT activity analysis usually consists of the handling of the sample and incubation followed by separation and detection of the reaction products. Several of these assays are validated, reliable and sensitive. Besides the studies of the basic properties of COMT, the activity assay has also been applied to explore the relation of COMT to various disease states or disorders. In addition, COMT activity analysis has been applied clinically since COMT inhibitors have been introduced as adjuvant drugs in the treatment of Parkinson's disease.     

Cloning of a gene encoding hydroxyquinol 1,2-dioxygenase that catalyzes both intradiol and extradiol ring cleavage of catechol.

Two Escherichia coli transformants with catechol 1,2-dioxygenase activity were selected from a gene library of the benzamide-assimilating bacterium Arthrobacter species strain BA-5-17, which produces four catechol 1,2-dioxygenase isozymes. A DNA fragment isolated from one transformant contained a complete open reading frame (ORF). The deduced amino acid sequence of the ORF shared high identity with hydroxyquinol 1,2-dioxygenase. An enzyme expressed by the ORF was purified to homogeneity and characterized. When hydroxyquinol was used as a substrate, the purified enzyme showed 6.8-fold activity of that for catechol. On the basis of the sequence identity and substrate specificity of the enzyme, we concluded that the ORF encoded hydroxyquinol 1,2-dioxygenase. When catechol was used as a substrate, cis,cis-muconic acid and 2-hydroxymuconic 6-semialdehyde, which were products by the intradiol and extradiol ring cleavage activities, respectively, were produced. These results showed that the hydroxyquinol 1,2-dioxygenase reported here was a novel dioxygenase that catalyzed both the intradiol and extradiol cleavage of catechol.     

Substrate profiles and expression of caffeoyl coenzyme A and caffeic acid O-methyltransferases in secondary xylem of aspen during seasonal development

Seasonal expression of caffeoyl-CoA O-methyltransferase (EC 2.1.1.104) was analyzed in aspen developing secondary xylem in parallel with caffeate O-methyltransferase (EC 2.1.1.68). Enzyme activity and mRNA levels for both enzymes peaked in the middle of the growing season. These results strongly suggest that both forms of O-methyltransferase were actively participating in lignin precursor biosynthesis during the growing season. To determine the role of each enzyme form, xylem extracts from two days in the growing season were assayed with four substrates: caffeoyl-CoA, 5-hydroxyferuloyl-CoA, caffeate acid and 5-hydroxyferulic acid. Recombinant forms of caffeoyl-CoA and caffeate O-methyltransferase were also assayed with these substrates. The recombinant enzymes have different substrate specificity with the caffeoyl-CoA O-methyltransferase being essentially specific for CoA ester substrates with a preference for caffeoyl-CoA, while caffeate O-methyltransferase utilized all four substrates with a preference for the free acid forms. We suggest that caffeoyl-CoA O-methyltransferase is likely to be responsible for biosynthesis of lignin precursors in the guaiacyl pathway and may represent a more primitive enzyme form leftover from very early land plant evolution. Caffeate O-methyltransferase is more likely to be responsible for lignin precursor biosynthesis in the syringyl pathway, especially since it can catalyze methylation of 5-hydroxyferuloyl-CoA quite effectively. This latter enzyme form then may be considered a more recently evolved component of the lignin biosynthetic pathways of the evolutionarily advanced plants such as angiosperms.     

Photoaffinity labeling of catechol O-methyltransferase with 8-azido-S-adenosylmethionine

An in vitro system using an enzyme extract containing ATP:L-methionine S-adenosyltransferase from Escherichia coli MRE 600 cells was used to synthesize 8-azido-S-adenosyl-L-methionine from methionine and 8-azidoadenosine 5'-triphosphate. In the absence of ultraviolet light and analog can serve as a methyl donor for porcine catechol O-methyltransferase. Photolysis of 8-azido-S-adenosyl[35S]methionine in the presence of catechol O-methyltransferase results in covalent incorporation. Addition of either authentic S-adenosylmethionine or S-adenosylhomocysteine, but not adenosine 5'-monophosphate, to the photolysis reaction mixture eliminates the photoincorporation. These results indicate that the incorporation is occurring at the S-adenosylmethionine binding site in the catechol O-methyltransferase.     

Purification and characterization of rat heart and brain catechol methyltransferase.

In an effort to detect the similarities and differences in the properties of rat heart, brain and liver catechol methyltransferase (S-adenosyl-L-methionine:catechol O-methyltransferase, EC 2.1.1.6), we have determined the cellular distribution of this enzyme activity and extensively purified the soluble and microsomal enzymes present in these tissues. Purification of soluble heart (688-fold) and brain enzymes (240-fold) were achieved using an affinity chromatographic system. The properties of these enzymes were compared with respect to their molecular weights, substrate specificities, inhibitor specificities and immunological properties. The characteristics of the enzyme active sites were investigated using various methyl acceptor substrates and various analogs of S-adenosylmethionine as methyl donors. A series of analogs of S-adenosylhomocysteine was also evaluated as inhibitors of these enzymes. The immunological properties of the purified soluble and microsomal enzymes from heart and brain were investigated using an antibody isolated from rabbits which had been immunized with the soluble rat liver enzyme. In general the properties of catechol methyltransferases isolated from heart and brain were similar to the properties of the enzyme isolated from liver. Some minor differences in substrate and inhibitor specificities were observed which might suggest slight differences in the active sites of these enzymes.     

Inhibition of Catechol-O-Methyltransferase by 6,7-Dihydroxy-3,4-Dihydroisoquinolines Related to Dopamine: Demonstration Using Liquid Chromatography and a Novel Substrate for O-Methylation

We report that 6,7-dihydroxy-3,4-dihydroisoquinolines related to dopamine are potent inhibitors of catechol-O-methyltransferase (COMT), but are not apparent substrates for the enzyme in vitro or in vivo. Three dihydroxy (catecholic) dihydroisoquinolines, including the 1-benzyl (DesDHP) and the 1-methyl (DSAL) analogs, were found to inhibit COMT activity in rat liver supernatant more effectively than the well-known inhibitor, tropolone. Inhibition of O-methylation was uncompetitive with substrate, and O-methylated products of the catecholic dihydroisoquinolines were undetectable. For these in vitro studies, a facile liquid chromatographic assay was developed utilizing as a site-specific substrate, 1-methyl-6,7-dihydroxy-tetrahydroisoquinoline-1-carboxylate (salsolinol-1-carboxylate). This catechol produces only one phenolic product isomer when incubated with liver supernatant and S-adenosylmethionine. Following central injection of DSAL in rats, inhibition of brain COMT in vivo was indicated by the reduced brain levels of homovanillic acid, but not of 3,4-dihydroxyphenylacetic acid. Furthermore, O-methylated DSAL metabolites could not be detected in brain by liquid or gas chromatography. We suggest that 6,7-dihydroxy-dihydroisoquinolines are "nonmethylatable" COMT inhibitors because they exist as quinoidal tautomers resembling pyridones or tropolones rather than as catechols. Quinoid formation is supported by the fluorescence and ultraviolet spectra for DSAL and its O-methyl derivatives. The experiments reveal a new class of COMT inhibitors that may be of pharmacological and mechanistic value. Additionally, 3,4-dihydroisoquinolines could arise endogenously via oxidation of the 1,2,3,4-tetrahydroisoquinolines which are ingested or produced from cellular catecholamine condensations. However, it is unlikely that dihydroisoquinoline (e.g., DSAL) concentrations necessary to inhibit COMT significantly would be attained via endogenous pathways.     

Isolation of the low-molecular-mass form of catechol O-methyltransferase from rat liver and immunocytochemical localization of the enzyme in the glycogen compartment

The low-molecular-mass form of two distinct catechol O-methyltransferase activities (S-adenosyl-L-methionine: catechol O-methyltransferase, COMT, EC 2.1.1.6) has been purified to homogeneity from rat liver using 40-70% ammonium sulfate precipitation, gel filtration on Sephadex G-100, adsorption on hydroxyapatite C and ion-exchange chromatography on DEAE-Sepharose CL-6B. The relative molecular mass Mr, determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis is 22 400 +/- 500. Irradiation of the enzyme in the presence of 8-azido-[methyl-3H]AdoMet results in the specific labeling of the catalytic site of the enzyme. Photolabeling was successful with crude COMT preparations and with the isolated enzyme. Immunocytochemical studies present new information about the localization of the low-molecular-mass form in the liver parenchyma. Subcellularly COMT immunoreactivity could be attributed exclusively to the compartment with glycogen granules. Nucleus, mitochondria and endoplasmic reticulum showed no immunostaining.     

Extradiol cleavage of 3-substituted catechols by an intradiol dioxygenase, pyrocatechase, from a Pseudomonad.

Pyrocatechase (catechol 1,2-oxidoreductase (decyclizing), EC 1.13.11.1), a ferric ion-containing dioxygenase from Pseudomonas arvilla C-1, catalyzes the intradiol cleavage of catechol with insertion of 2 atoms of molecular oxygen to form cis,cis-muconic acid. The enzyme also catalyzed the oxidation of various catechol derivatives, including 4-methylcatechol, 4-chlorocatechol, 4-formylcatechol (protocatechualdehyde), 4,5-dichlorocatechol, 3,5-dichlorocatechol, 3-methylcatechol, 3-methoxycatechol, and 3-hydroxycatechol (pyrogallol). All of these substrates gave products having an absorption maximum at around 260 nm, which is characteristic of cis,cis-muconic acid derivatives. However, when 3-methylcatechol was used as substrate, the product formed showed two absorption maxima at 390 and 260 nm. These two absorption maxima were found to be attributable to two different products, 2-hydroxy-6-oxo-2,4-heptadienoic acid and 5-carboxy-2-methyl-2,4-pentadienoic acid (2-methylmuconic acid). The former was produced by the extradiol cleavage between the carbon atom carrying the hydroxyl group and the carbon atom carrying the hydroxyl group and the carbon atom carrying the methyl group; the latter by an intradiol cleavage between two hydroxyl groups. Since these products were unstable, they were converted to and identified as 6-methylpyridine-2-carboxylic acid and 2-methylmuconic acid dimethylester, respectively. Similarly, 3-methoxycatechol gave two products, namely, 2-hydroxy-5-methoxycarbonyl-2,4-pentadienoic acid and 5-carboxy-2-methoxy-2,4-pentadienoic acid (2-methoxymuconic acid). With 3-methylcatechol as substrate, the ratio of intradiol and extradiol cleavage activities of Pseudomonas pyrocatechase during purification was almost constant and was about 17. The final preparation of the enzyme was homogeneous when examined by disc gel electrophoresis and catalyzed both reactions simultaneously with the same ratio as during purification. All attempts to resolve the enzyme into two components with separate activities, including inactivation of the enzyme with urea or heat, treatment with sulfhydryl-blocking reagents or chelating agents, and inhibition of the enzyme with various inhibitors, proved unsuccessful. These results strongly suggest that Pseudomonas pyrocatechase is a single enzyme, which catalyzes simultaneously both intradiol and extradiol cleavages of some 3-substituted catechols.     

Characterization of SafC, a catechol 4-O-methyltransferase involved in saframycin biosynthesis

Members of the saframycin/safracin/ecteinascidin family of peptide natural products are potent antitumor agents currently under clinical development. Saframycin MX1, from Myxococcus xanthus, is synthesized by a nonribosomal peptide synthetase, SafAB, and an O-methyltransferase, SafC, although other proteins are likely involved in the pathway. SafC was overexpressed in Escherichia coli, purified to homogeneity, and assayed for its ability to methylate a variety of substrates. SafC was able to catalyze the O-methylation of catechol derivatives but not phenols. Among the substrates tested, the best substrate for SafC was L-dihydroxyphenylalanine (L-dopa), which was methylated specifically in the 4'-O position (k(cat)/K(m) = 5.5 x 10(3) M(-1) s(-1)). SafC displayed less activity on other catechol derivatives, including catechol, dopamine, and caffeic acid. The more labile l-5'-methyldopa was an extremely poor substrate for SafC (k(cat)/K(m) = approximately 2.8 x 10(-5) M(-1) s(-1)). L-dopa thioester derivatives were also much less reactive than L-dopa. These results indicate that SafC-catalyzed 4'-O-methylation of L-dopa occurs prior to 5'-C-methylation, suggesting that 4'-O-methylation is likely the first committed step in the biosynthesis of saframycin MX1. SafC has biotechnological potential as a methyltransferase with unique regioselectivity.     

Production of three O-methhylated esculetins with Escherichia coli expressing O-methyltransferase from poplar

O-Methyltransferase, POMT-9 was expressed in Escherichia coli. HPLC analysis of reaction products revealed three peaks corresponding to isoscopoletin, scopoletin, and scoparone, and their structures were determined using NMR. Biotransformation of esculetin with E. coli expressing POMT-9 generated scopoletin, isoscopoletin, and scoparone at 30.3, 21, and 31 microM respectively. POMT-9 is the first O-methyltransferase that produces three different O-methylated products.