Crystal Structure of Catechol O-Methyltransferase Complexed with Nitecapone.

Catechol O-methyltransferase (COMT) is known as an important drug-target protein in the field of Parkinson's disease. All clinically approved COMT inhibitors bring a 5-substituted-3-nitrocatechol ring as a pharmacophore, and they bind to COMT with S-adenosylmethionine (SAM) and an Mg2+ ion to form a quaternary complex (COMT/SAM/Mg2+/inhibitor). However, structural information about such quaternary complexes is only available for a few inhibitors. Here, a new crystal structure of COMT complexed with nitecapone (5), SAM and Mg2+ is revealed. Comparison of the structures of these complexes indicates that conformation of the catechol binding pocket is almost constant regardless of structure of the inhibitors. The only restriction of the side chain of inhibitors (i.e., the substituent at the 5-position of 3-nitrocatechol) seems to be that it does not make steric repulsion with COMT. However, recent crystallographic and biochemical studies suggest that COMT is a flexible protein, and its conformational flexibility seems crucial for its catalytic process. Based on this information, implications of these quaternary inhibitor complexes were investigated. Met 40 in the α2α3-loop makes atomic contacts with SAM or S-adenosylhomocysteine and the 3-position of the catechol inhibitor. This interaction seems to play a critical role in the affinity of the inhibitor and to stabilize the COMT/SAM/Mg2+/nitrocatechol inhibitor complex by fixing the flexible α2α3-loop.

Recovery of biological active catechol-O-methyltransferase isoforms from Q-sepharose.

The development of new catechol-O-methyltransferase inhibitors has led to an improvement in the treatment of Parkinson's disease. However, despite the fact that the soluble isoform has been extensively investigated, few studies have been published concerning membrane isoform chromatographic recovery and bioactivity levels. In this work, chromatographic profiles of both catechol-O-methyltransferase isoforms were compared using quaternary amine as a ligand to evaluate its activity levels and recovery rates. Results show that both proteins required different conditions for adsorption; the soluble isoform adsorption was performed at low ionic strength, while the membrane isoform required increasing linear salt gradient. However, the application of 0.5% Triton X-100 promoted membrane isoform adsorption even at low ionic strength. Indeed, chromatographic conditions of both isoforms became similar when detergents were applied. The developed methods also appear to be highly effective in bioactivity recovery, presenting rates of 107% for soluble protein and 67 and 91% for membrane isoform without and with detergents, respectively. The chromatographic strategies with and without detergents resulted in a 4.3- and sevenfold purification, respectively, corresponding to specific activity values of 331 and 496 nmol/h/mg. Thus, the use of Q-sepharose as anion exchanger was effective in the recovery of both enzymes, which is a requirement for further kinetic and pharmacological trials.

Performance of hydrophobic interaction ligands for human membrane-bound catechol-O-methyltransferase purification.

Despite of membrane catechol-O-methyltransferase (MBCOMT, EC 2.1.1.6) physiological importance on catecholamines' O-methylation, no studies allowed their total isolation. Therefore, for the first time, we compare the performance of three hydrophobic adsorbents (butyl-, epoxy-, and octyl-Sepharose) in purification of recombinant human COMT (hMBCOMT) from crude Brevibacillus choshinensis cell lysates to develop a sustainable chromatographic process. Hydrophobic matrices were evaluated in terms of selectivity and hMBCOMT's binding and elution conditions. Results show that hMBCOMT's adsorption was promoted on octyl and butyl at ≤375 mM NaH2 PO4, while on epoxy higher concentrations (>850 mM) were required. Additionally, hMBCOMT's elution was promoted on epoxy, butyl, and octyl using respectively 0.1-0.5, 0.25-1, and 1% of Triton X-100. On butyl media, a stepwise strategy using 375 and 0 mM NaH2PO4, followed by three elution steps at 0.25, 0.7 and 1% Triton X-100, allowed selective hMBCOMT isolation. In conclusion, significant amounts of MBCOMT were purified with high selectivity on a single chromatography procedure, despite its elution occurs on multiple peaks. Although successful applications of hydrophobic interaction chromatography in purification of membrane proteins are uncommon, we proved that traditional hydrophobic matrices can open a promising unexplored field to fulfill specific requirements for kinetic and pharmacological trials.

Detoxication of structurally diverse polycyclic aromatic hydrocarbon (PAH) o-quinones by human recombinant catechol-O-methyltransferase (COMT) via O-methylation of PAH catechols.

Polycyclic aromatic hydrocarbons (PAH) are environmental and tobacco carcinogens. Metabolic activation of intermediate PAH trans-dihydrodiols by aldo-keto reductases (AKRs) leads to the formation of electrophilic and redox-active o-quinones. We investigated whether O-methylation by human recombinant soluble catechol-O-methyltransferase (S-COMT) is a feasible detoxication step for a panel of structurally diverse PAH-catechols produced during the redox-cycling process. Classes of PAH non-K-region o-quinones (bay region, methylated bay region, and fjord region o-quinones) produced by AKRs were employed in the studies. PAH o-quinones were reduced to the corresponding catechols by dithiothreitol under anaerobic conditions and then further O-methylated by human S-COMT in the presence of S-[³H]adenosyl-l-methionine as a methyl group donor. The formation of the O-methylated catechols was detected by HPLC-UV coupled with in-line radiometric detection, and unlabeled products were also characterized by LC-MS/MS. Human S-COMT was able to catalyze O-methylation of all of the PAH-catechols and generated two isomeric metabolites in different proportions. LC-MS/MS showed that each isomer was a mono-O-methylated metabolite. ¹H NMR was used to assign the predominant positional isomer of benzo[a]pyrene-7,8-catechol as the O-8-monomethylated catechol. The catalytic efficiency (k(cat)/K(m)) varied among different classes of PAH-catechols by 500-fold. The ability of S-COMT to produce two isomeric products from PAH-catechols was rationalized using the crystal structure of the enzyme. We provide evidence that O-8-monomethylated benzo[a]pyrene-7,8-catechol is formed in three different human lung cell lines. It is concluded that human S-COMT may play a critical role in the detoxication of PAH o-quinones generated by AKRs.

Assessment of COMT isolation by HIC using a dual salt system and low temperature.

Sodium citrate (SC) and low temperatures between 7 and 5 degrees C are effective in suppressing aggregation of proteins and may be beneficial to be included during a purification process. In this work, we analyzed the application of dual salt system, ammonium sulfate (AS) and SC on binding and elution conditions of recombinant hSCOMT on typical HIC sorbents. Specifically in butyl and octyl supports, the use of, respectively, 300 mM AS/200 mM SC and 25 mM AS/25 mM SC in the loading buffer resulted in complete binding of COMT. Elution was obtained by decreasing the ionic strength to 0 M of salt. For the delineate goal, it also favorably increased the support chain length while a consequent decrease in the dual ionic strength was observed for hSCOMT retention. In the presence of dual salt systems octyl media exhibited classic HIC behavior, good protein selectivity, an excellent purification factor and reduced denaturation effects of hSCOMT observed with higher salt concentrations. Also the inclusion of temperature control during the elution step appears to be advantageous for greater activity recovery without enzyme aggregation. In fact, these results could allow the prediction of most stabilizing conditions for this termolabile enzyme on the chromatographic stage, regarding salt types and therefore effectiveness to improve HIC selectivity and desirable purity on the target fractions.

The V108M mutation decreases the structural stability of catechol O-methyltransferase.

The human gene for catechol O-methyltransferase has a common single-nucleotide polymorphism that results in substitution of methionine (M) for valine (V) 108 in the soluble form of the enzyme (s-COMT). 108M s-COMT loses enzymatic activity more rapidly than 108V s-COMT at physiological temperature, and the 108M allele has been associated with increased risk of breast cancer and several neuropsychiatric disorders. We used circular dichroism (CD), dynamic light scattering, and fluorescence spectroscopy to examine how the 108V/M polymorphism affects the stability of the purified, recombinant protein to heat and guanidine hydrochloride (GuHCl). COMT contains two tryptophan residues, W143 and W38Y, which are located in loops that border the S-adenosylmethionine (SAM) and catechol binding sites. We therefore also studied the single-tryptophan mutants W38Y and W143Y in order to dissect the contributions of the individual tryptophans to the fluorescence signals. The 108V and 108M proteins differed in the stability of both the tertiary structure surrounding the active site, as probed by the fluorescence yields and emission spectra, and their global secondary structure as reflected by CD. With either probe, the midpoint of the thermal transition of 108M s-COMT was 5 to 7 degrees C lower than that of 108V s-COMT, and the free energy of unfolding at 25 degrees C was smaller by about 0.4 kcal/mol. 108M s-COMT also was more prone to aggregation or partial unfolding to a form with an increased radius of hydration at 37 degrees C. The co-substrate SAM stabilized the secondary structure of both 108V and 108M s-COMT. W143 dominates the tryptophan fluorescence of the folded protein and accounts for most of the decrease in fluorescence that accompanies unfolding by GuHCl. While replacing either tryptophan by tyrosine was mildly destabilizing, the lower stability of the 108M variant was retained in all cases.

Structural and biochemical characterization of Rv0187, an O-methyltransferase from Mycobacterium tuberculosis.

Catechol O-methyltransferase (COMT) is widely distributed in nature and installs a methyl group onto one of the vicinal hydroxyl groups of a catechol derivative. Enzymes belonging to this family require two cofactors for methyl transfer: S-adenosyl-l-methionine as a methyl donor and a divalent metal cation for regiospecific binding and activation of a substrate. We have determined two high-resolution crystal structures of Rv0187, one of three COMT paralogs from Mycobacterium tuberculosis, in the presence and absence of cofactors. The cofactor-bound structure clearly locates strontium ions and S-adenosyl-l-homocysteine in the active site, and together with the complementary structure of the ligand-free form, it suggests conformational dynamics induced by the binding of cofactors. Examination of in vitro activities revealed promiscuous substrate specificity and relaxed regioselectivity against various catechol-like compounds. Unexpectedly, mutation of the proposed catalytic lysine residue did not abolish activity but altered the overall landscape of regiospecific methylation.

A new approach on the purification of recombinant human soluble catechol-O-methyltransferase from an Escherichia coli extract using hydrophobic interaction chromatography.

Catechol-O-methyltransferase (COMT) is a significant target in protein engineering due to its role not only in normal brain function but also to its possible involvement in some human disorders. In this work, a new approach was employed for the purification of recombinant human soluble COMT (hSCOMT) using hydrophobic interaction chromatography, as the main isolation method, from an Escherichia coli culture broth. A simplified overall process flow is proposed. Indeed, with an optimized heterologous expression system for recombinant hSCOMT production, such as E. coli, it was possible to produce and recover the active monomeric enzyme directly from the cell crude culture broth either by a freeze/thaw or ultrasonication lysis step. The recombinant enzyme present in the bacterial soluble fraction, exhibited similar affinity for epinephrine (K(m) 276 [215; 337] microM) and the methyl donor (S-adenosyl-L-methionine, SAMe) (K(m) 36 [30; 41]microM) as human SCOMT. After the precipitation step by 55% of ammonium sulphate, a HIC step on the butyl-sepharose resin was found to be highly effective in selectively eluting a range of contaminating key proteins present in the concentrate soluble extract. Consequently, the partially purified eluate from HIC could then be loaded and polished by gel filtration in order to increase the process efficiency. The final product appeared as a single band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The procedure resulted in a global 10.9-fold purification with a specific activity of 5500 nmol/h/mg of protein. The widespread applicability of the process, here described, to different COMT sources could make this protocol highly useful for all studies requiring purified and active COMT proteins.

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.

Immunoaffinity purification and partial amino acid sequence analysis of catechol-O-methyltransferase from pig liver.

Monoclonal antibodies (mAbs) against the soluble form (S-COMT) of catechol-O-methyltransferase (COMT, EC 2.1.1.6) were produced using a purified preparation of the enzyme from pig liver as antigen. The selected monoclonal antibodies recognized the enzyme with different capacities. One of them (Co60-1B/7) showed a significant cross reaction with S-COMT from rat and human liver. A protein band of 23 kDa was recognized by the mAbs on Western blots of the soluble fraction of pig liver. The mAbs were also able to recognize the membrane-bound form of the enzyme, which was found to be mainly localized in the microsomal fraction of pig and rat liver as well as of the human hepatoma cell line Hep G2. The protein bands detected in microsomes had a molecular mass of 26 kDa in pig and rat liver and displayed a slightly higher molecular mass (29 kDa) in the Hep G2 cell line. A single step method for the immunoaffinity purification of pig liver S-COMT was developed by using a Sepharose 4B column to which the mAb Co54-5F/8 was covalently coupled. Acid elution conditions were optimized to obtain the enzyme in active form with a good yield. SDS-PAGE analysis of the purified preparation revealed a single protein band with a molecular mass of 23 kDa with 154-fold enrichment in enzyme activity over the starting material. Since the N-terminus was blocked, purified enzyme preparations were cleaved with trypsin. Two fragments of 22 and 33 amino acids in length could be sequenced by Edman degradation.

Expression of enzymatically active rat liver and human placental catechol-O-methyltransferase in Escherichia coli; purification and partial characterization of the enzyme.

To produce sufficient amounts of recombinant catechol-O-methyltransferase (COMT) for structural and functional studies the coding regions of the rat liver and human placental COMT genes have been introduced into a bacterial expression vector pKEX14. Recombinant COMT was produced in Escherichia coli up to 10% of total bacterial protein after the induction of the T7 RNA polymerase gene with isopropyl-beta-D-thiogalactopyranoside. Both the rat and human enzymes were enzymatically active, soluble and reacted with anti-COMT antiserum in Western blotting. Both enzymes were purified from E. coli cells and partially characterized by determining their specific activity, apparent molecular weight and pI.

Rapid assay for catechol-O-methyltransferase activity by high-performance liquid chromatography-fluorescence detection.

A rapid assay for measuring the activities of catechol-O-methyltransferase (COMT) is described. The method is based on high-performance liquid chromatography (HPLC)-fluorescence detection, and includes on-line extraction of catecholamines with a precolumn, separation of norepinephrine (NE) and normetanephrine (NMN) on an ODS column, electrochemical oxidation, and post-column fluorogenic derivatization using ethylenediamine. The method took less than 25 min for one sample, which is half that of the previous method and the sensitivity was similar. The intra-day assay precisions were 0.52-1.6%, and the inter-day assay precisions were 3.6-5.8% for rat liver and cerebral cortex (n = 5). The method is suitable for the rapid measurement of COMT activities of many biological samples.

Catechol O-methyltransferase pharmacogenetics: photoaffinity labelling and western blot analysis of human liver samples.

The level of catechol O-methyltransferase (COMT) activity and COMT thermal stability in human tissue are controlled by a common genetic polymorphism. We studied individual hepatic biopsy samples shown previously to have phenotypically high, low or intermediate COMT activities and thermal stabilities to test the hypothesis that the molecular mass (M(r)) and/or isoelectric point (pI) of the enzyme might differ in tissue from subjects with different presumed genotypes for the COMT genetic polymorphism. COMT was partially purified from each hepatic tissue sample by sequential ion exchange and gel filtration chromatography, and photoaffinity labelling was performed with [3H-methyl]-S-adenosyl-L-methionine ([3H-methyl]-Ado-Met), the methyl donor for the COMT enzymatic reaction. Two-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE) analysis of individual samples consistently showed the presence of three [3H-methyl]-Ado-Met photoaffinity labelled proteins with pI values of 5.4, 5.5 and 5.7, all three of which had M(r) values of approximately 27.1 kDa. The same pattern was observed in all samples irrespective of COMT phenotype. Western blot analysis of 2-D SDS-PAGE gels performed with rabbit polyclonal antibodies to partially purified human kidney COMT showed a pattern similar to that found during photoaffinity labelling. Once again, the same pattern was found in all samples irrespective of COMT phenotype. Therefore, neither photoaffinity labelling nor Western blot analysis revealed differences in either M(r) or pI of cytoplasmic COMT in hepatic tissue from subjects selected on the basis of different phenotypic expression of the COMT genetic polymorphism.

Purification and partial characterization of rat liver soluble catechol-O-methyltransferase.

The rat liver soluble catechol-O-methyltransferase (EC 2.1.1.6.) has been purified utilizing a combination of conventional chromatography and HPLC. The purified enzyme has a molecular mass of 25 kDa, a pI of 5.1, and exists in two forms which differ in the nature of their intramolecular disulfide bonds. This difference causes these two protein forms to behave differently in reversed phase chromatography.

[Purification and properties of rat kidney catechol-O-methyltransferase]. / Purification et propriétés de la catéchol-O-méthyltransférase du rein de rat

The S-adenosyl-methionine: catechol-O-methyltransferase (EC 2.1.1.6) from rat kidney was purified about 650 fold as compared with the homogenate and the result of disc electrophoresis presented. The purification involved extraction, precipitation at pH 5, ammonium sulfate fractionation, Chromatographies on Biogel 0.5 m, Ultrogel AcA 44 and DE Sephadex A 50. Affinity chromatography was tried but unsuccessful. The enzyme exhibited two pH optima at 7.9 and 9.6 with a minimum at about 8.9. The COMT had a temperature optimum of 50 degrees C, with activation energy of 23.1 Kcal/Mole between 25-35 degrees C, 18.9 Kcal/mole between 35-45 degrees C and the Q10 within the range of 25-35 degrees amounted to 3.5. The molecular weight was estimated to be 21500+/-1000 daltons from its behavior on Ultrogel AcA 44 and the pH1 determined by electrofocalisation was near 5.50. The time of half life of the best purified enzymatic extract was found to be 2 h 10 min. at -20 degrees C. At basic pH the instability of the enzyme was increased. Since O-methylation required the presence of divalent cations, our results show that apparent Michaelis constants for Mg++ and Mn++ were respectively 0.50 X 10(-3) M and 0.33 X 10(-5) M. The study of their Hill's number indicated that there was only one point of fixation on the enzyme. The Km value determined by Florini and Vestling's method were 2.5 X 10(-4) M and 11.9 X 10(-5) M for epinephrine and S-adenosyl-methionine respectively. All results were discussed with respect to other investigations.

Catechol-O-methyltransferase as a target for melanoma destruction?

Catechols may interfere in melanogenesis by causing increased levels of toxic quinones. Several catechols and known inhibitors of the enzyme catechol-O-methyltransferase (COMT) were therefore tested for their toxicity towards a pigmented melanoma cell line, UCLA-SO-(M14). The inhibition of thymidine incorporation as a result of exposure to the compounds was measured. All agents were compared to 4-hydroxyanisole (4HA), a depigmenting agent extensively studied as an antimelanoma drug. The compounds were also tested on the epithelial cell line, CNCM-I-(221) in the presence and absence of tyrosinase. All the compounds were more effective than 4HA towards the M14-cells at either 10(-4) M or 10(-5) M. The toxicity of 4HA towards the 221-cells was shown to be completely dependent on the presence of tyrosinase. Effects of the test agents on the 221-cells were also observed in the absence of tyrosinase. Although some of them were shown to be good substrates for tyrosinase only small changes in toxicity were observed as a result of the presence of the enzyme in comparison with 4HA. No direct correlation of the toxicity of the agents and COMT inhibition was observed. The possible mode of action of the compounds through inhibition of COMT and interference in melanogenesis is discussed together with other possibilities and factors involved.

Behaviour and properties of catechol-O-methyltransferase from human placenta.

A procedure is reported for the purification of human placental catechol-O-methyltransferase. The preparation is apparently homogeneous and behaves as a monomer with an approximate Mr of 23,000. The sequence of the first 21 amino acid residues from the N-terminal end of the protein is reported. The activity of the enzyme is strongly influenced by the nature of the buffer in which it is assayed.

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.

Characterization of non-nitrocatechol pan and isoform specific catechol-O-methyltransferase inhibitors and substrates.

Reduced dopamine neurotransmission in the prefrontal cortex has been implicated as causal for the negative symptoms and cognitive deficit associated with schizophrenia; thus, a compound which selectively enhances dopamine neurotransmission in the prefrontal cortex may have therapeutic potential. Inhibition of catechol-O-methyltransferase (COMT, EC 2.1.1.6) offers a unique advantage, since this enzyme is the primary mechanism for the elimination of dopamine in cortical areas. Since membrane bound COMT (MB-COMT) is the predominant isoform in human brain, a high throughput screen (HTS) to identify novel MB-COMT specific inhibitors was completed. Subsequent optimization led to the identification of novel, non-nitrocatechol COMT inhibitors, some of which interact specifically with MB-COMT. Compounds were characterized for in vitro efficacy versus human and rat MB and soluble (S)-COMT. Select compounds were administered to male Wistar rats, and ex vivo COMT activity, compound levels in plasma and cerebrospinal fluid (CSF), and CSF dopamine metabolite levels were determined as measures of preclinical efficacy. Finally, novel non-nitrocatechol COMT inhibitors displayed less potent uncoupling of the mitochondrial membrane potential (MMP) compared to tolcapone as well as nonhepatotoxic entacapone, thus mitigating the risk of hepatotoxicity.

A novel prokaryotic expression system for biosynthesis of recombinant human membrane-bound catechol-O-methyltransferase.

Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. While large amounts of purified proteins are required for pharmaceutical and crystallization attempts, there is an unmet need for the development of novel heterologous membrane protein overexpression systems. Specifically, we tested the application of Brevibacillus choshinensis cells for the biosynthesis of human membrane bound catechol-O-methyltransferase (hMBCOMT). In terms of the upstream stage moderate to high expression was obtained for complex media formulation with a value near 45 nmol/h/mg for hMBCOMT specific activity achieved at 20 h culture with 37°C and 250 rpm. Subsequently, the efficiency for reconstitution of hMBCOMT is markedly null in the presence of ionic detergents, such as sodium dodecyl sulphate (SDS). In general, for non-ionic and zwiterionic detergents, until a detergent critic micellar concentration (CMC) of 1.0 mM, hMBCOMT shows more biological activity at lower detergent concentrations while for detergent CMC higher than 1 mM, higher detergent concentrations seem to be ideal for hMBCOMT solubilization. Indeed, from the detergents tested, the non-ionic digitonin at 0.5% (w/v) appears to be the most suitable for hMBCOMT solubilization.