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1.  Mapping the conformational space accessible to catechol-O-methyltransferase 
Crystal structures of mouse, rat and human catechol-O-methyltransferase were determined in apo, semi-holo, holo and Michaelis forms under a variety of conditions. Domain swaps and large conformational changes in the active sites are observed, which testify to why this enzyme is a difficult drug target.
Methylation catalysed by catechol-O-methyltransferase (COMT) is the main pathway of catechol neurotransmitter deactivation in the prefrontal cortex. Low levels of this class of neurotransmitters are held to be causative of diseases such as schizophrenia, depression and Parkinson’s disease. Inhibition of COMT may increase neurotransmitter levels, thus offering a route for treatment. Structure-based drug design hitherto seems to be based on the closed enzyme conformation. Here, a set of apo, semi-holo, holo and Michaelis form crystal structures are described that define the conformational space available to COMT and that include likely intermediates along the catalytic pathway. Domain swaps and sizeable loop movements around the active site testify to the flexibility of this enzyme, rendering COMT a difficult drug target. The low affinity of the co-substrate S-adenosylmethionine and the large conformational changes involved during catalysis highlight significant energetic investment to achieve the closed conformation. Since each conformation of COMT is a bona fide target for inhibitors, other states than the closed conformation may be promising to address. Crystallographic data for an alternative avenue of COMT inhibition, i.e. locking of the apo state by an inhibitor, are presented. The set of COMT structures may prove to be useful for the development of novel classes of inhibitors.
PMCID: PMC4118827  PMID: 25084335
catechol-O-methyltransferase; neurotransmitter degradation; catecholamine metabolism; SAM; S-adenosylmethionine
2.  How Metal Substitution Affects the Enzymatic Activity of Catechol-O-Methyltransferase 
PLoS ONE  2012;7(10):e47172.
Catechol-O-methyltransferase (COMT) degrades catecholamines, such as dopamine and epinephrine, by methylating them in the presence of a divalent metal cation (usually Mg(II)), and S-adenosyl-L-methionine. The enzymatic activity of COMT is known to be vitally dependent on the nature of the bound metal: replacement of Mg(II) with Ca(II) leads to a complete deactivation of COMT; Fe(II) is slightly less than potent Mg(II), and Fe(III) is again an inhibitor. Considering the fairly modest role that the metal plays in the catalyzed reaction, this dependence is puzzling, and to date remains an enigma. Using a quantum mechanical / molecular mechanical dynamics method for extensive sampling of protein structure, and first principle quantum mechanical calculations for the subsequent mechanistic study, we explicate the effect of metal substitution on the rate determining step in the catalytic cycle of COMT, the methyl transfer. In full accord with experimental data, Mg(II) bound to COMT is the most potent of the studied cations and it is closely followed by Fe(II), whereas Fe(III) is unable to promote catalysis. In the case of Ca(II), a repacking of the protein binding site is observed, leading to a significant increase in the activation barrier and higher energy of reaction. Importantly, the origin of the effect of metal substitution is different for different metals: for Fe(III) it is the electronic effect, whereas in the case of Ca(II) it is instead the effect of suboptimal protein structure.
PMCID: PMC3466255  PMID: 23056605
3.  Methylation of sulfhydryl groups: a new function for a family of small molecule plant O-methyltransferases 
In plants, type I and II S-adenosyl-L-methionine-dependent O-methyltransferases (OMTs) catalyze most hydroxyl group methylations of small molecules. A homology-based RT-PCR strategy using Catharanthus roseus (Madagascar periwinkle) RNA previously identified six new type I plant OMT family members. We now describe the molecular and biochemical characterization of a seventh protein. It shares 56–58% identity with caffeic acid OMTs (COMTs), but it failed to methylate COMT substrates, and had no activity with flavonoids. However, the in vitro incubations revealed unusually high background levels without added substrates. A search for the responsible component revealed that the enzyme methylated dithiothreitol (DTT), the reducing agent added for enzyme stabilization. Unexpectedly, product analysis revealed that the methylation occurred on a sulfhydryl moiety, not on a hydroxyl group. Analysis of 34 compounds indicated a broad substrate range, with a preference for small hydrophobic molecules. Benzene thiol (Km 220 μM) and furfuryl thiol (Km 60 μM) were the best substrates (6–7-fold better than DTT). Small isosteric hydrophobic substrates with hydroxyl groups, like phenol and guaiacol, were also methylated, but the activities were at least 5-fold lower than with thiols. The enzyme was named C. roseus S-methyltransferase 1 (CrSMT1). Models based on the COMT crystal structure suggest that S-methylation is mechanistically identical to O-methylation. CrSMT1 so far is the only recognized example of an S-methyltransferase in this protein family. Its properties indicate that a few changes in key residues are sufficient to convert an OMT into a S-methyltransferase (SMT). Future functional investigations of plant methyltransferases should consider the possibility that the enzymes may direct methylation at sulfhydryl groups.
PMCID: PMC2860623  PMID: 16623883
Catharanthus roseus; S-methyltransferase; O-methyltransferase; evolution; protein modeling; homology-based cDNA cloning
4.  Serotonin-Induced Hypersensitivity via Inhibition of Catechol O-Methyltransferase Activity 
Molecular Pain  2012;8:25.
The subcutaneous and systemic injection of serotonin reduces cutaneous and visceral pain thresholds and increases responses to noxious stimuli. Different subtypes of 5-hydroxytryptamine (5-HT) receptors are suggested to be associated with different types of pain responses. Here we show that serotonin also inhibits catechol O-methyltransferase (COMT), an enzyme that contributes to modultion the perception of pain, via non-competitive binding to the site bound by catechol substrates with a binding affinity comparable to the binding affinity of catechol itself (Ki = 44 μM). Using computational modeling, biochemical tests and cellular assays we show that serotonin actively competes with the methyl donor S-adenosyl-L-methionine (SAM) within the catalytic site. Binding of serotonin to the catalytic site inhibits the access of SAM, thus preventing methylation of COMT substrates. The results of in vivo animal studies show that serotonin-induced pain hypersensitivity in mice is reduced by either SAM pretreatment or by the combined administration of selective antagonists for β2- and β3-adrenergic receptors, which have been previously shown to mediate COMT-dependent pain signaling. Our results suggest that inhibition of COMT via serotonin binding contributes to pain hypersensitivity, providing additional strategies for the treatment of clinical pain conditions.
PMCID: PMC3495668  PMID: 22500608
5.  Structure-function relationships of wheat flavone O-methyltransferase: Homology modeling and site-directed mutagenesis 
BMC Plant Biology  2010;10:156.
Wheat (Triticum aestivum L.) O-methyltransferase (TaOMT2) catalyzes the sequential methylation of the flavone, tricetin, to its 3'-methyl- (selgin), 3',5'-dimethyl- (tricin) and 3',4',5'-trimethyl ether derivatives. Tricin, a potential multifunctional nutraceutical, is the major enzyme reaction product. These successive methylations raised the question as to whether they take place in one, or different active sites. We constructed a 3-D model of this protein using the crystal structure of the highly homologous Medicago sativa caffeic acid/5-hydroxyferulic acid O-methyltransferase (MsCOMT) as a template with the aim of proposing a mechanism for multiple methyl transfer reactions in wheat.
This model revealed unique structural features of TaOMT2 which permit the stepwise methylation of tricetin. Substrate binding is mediated by an extensive network of H-bonds and van der Waals interactions. Mutational analysis of structurally guided active site residues identified those involved in binding and catalysis. The partly buried tricetin active site, as well as proximity and orientation effects ensured sequential methylation of the substrate within the same pocket. Stepwise methylation of tricetin involves deprotonation of its hydroxyl groups by a His262-Asp263 pair followed by nucleophilic attack of SAM-methyl groups. We also demonstrate that Val309, which is conserved in a number of graminaceous flavone OMTs, defines the preference of TaOMT2 for tricetin as the substrate.
We propose a mechanism for the sequential methylation of tricetin, and discuss the potential application of TaOMT2 to increase the production of tricin as a nutraceutical. The single amino acid residue in TaOMT2, Val309, determines its preference for tricetin as the substrate, and may define the evolutionary differences between the two closely related proteins, COMT and flavone OMT.
PMCID: PMC3017781  PMID: 20670441
6.  Identification, Characterization, and Ontogenic Study of a Catechol O-methyltransferase from Zebrafish 
To establish the zebrafish as a model for investigating the methylation pathway of drug metabolism, we embarked on the molecular cloning of the zebrafish catechol O-methyltransferase (COMT). By searching the GenBank database, a zebrafish nucleotide sequence encoding a putative COMT was identified. Based on the sequence information, we designed and synthesized oligonucleotides corresponding to its 5’- and 3’-coding regions of this zebrafish COMT. Using the first-strand cDNA reverse-transcribed from the total RNA isolated from a 3-month-old adult female zebrafish as the template, the cDNA encoding the zebrafish COMT was PCR-amplified. The recombinant zebrafish COMT protein was subsequently expressed in and purified from BL21 (DE3) Escherichia coli cells transformed with the pGEX-2TK expression vector harboring the zebrafish COMT cDNA. Upon enzymatic characterization, purified COMT displayed methylating activity toward dopamine, dopa, and catecholestrogens, as well as three representative catechol drugs, methyldopa, dobutamine, and isoproterenol. A reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed developmental stage-dependent expression of the zebrafish COMT during embryonic development and throughout the larval stage onto maturity. These results provide a foundation for investigating the involvement of COMT-mediated methylation in protection against the adverse effects of catechol drugs and other xenobiotic catechols during the developmental process.
PMCID: PMC3515678  PMID: 21371608
Catechol O-methyltransferase; developmental expression; methylation; molecular cloning; zebrafish
7.  A Hotspot of Inactivation: The A22S and V108M Polymorphisms Individually Destabilize the Active Site Structure of Catechol O-Methyltransferase† 
Biochemistry  2009;48(27):6450-6460.
Human catechol O-methyltransferase (COMT) contains three common polymorphisms (A22S, A52T, V108M), two of which (A22S, V108M) render the protein susceptible to deactivation by temperature or oxidation. We have performed multiple molecular dynamics simulations of the wild-type, A22S, A52T, and V108M COMT proteins to explore the structural consequences of these mutations. In total, we have amassed over 1.4 μs of simulation time representing the largest set of simulations detailing the effects of polymorphisms on a protein system to date. The A52T mutation had no significant effect on COMT structure in accord with experiment, thereby serving as a good negative control for the simulation set. Residues 22 (α2) and 108 (α5) interact with each other throughout the simulations and are located in a polymorphic hotspot ∼20Å from the active site. Introduction of either the larger Ser (22) or Met (108) tightens this interaction, pulling α2 and α5 towards each other and away from the protein core. The V108M polymorphism rearranges active-site residues in α5, β3 and α6, increasing the S-adenosylmethionine site solvent exposure. The A22S mutation reorients α2, moving critical catechol-binding residues away from the substrate-binding pocket. The A22S and V108M polymorphisms evolved independently in Northern European and Asian populations. While the decreased activities of both A22S and V108M COMT are associated with an increased risk for schizophrenia, the V108M induced destabilization is also linked with improved cognitive function. These results suggest that polymorphisms within this hotspot may have evolved to regulate COMT activity and that heterozygosity for either mutation may be advantageous.
PMCID: PMC2906713  PMID: 19435324
catechol O-methyltransferase; polymorphism; molecular dynamics; schizophrenia; breast cancer; drug development
8.  Inhibition of Human Catechol-O-Methyltransferase (COMT)-Mediated O-Methylation of Catechol Estrogens by Major Polyphenolic Components Present in Coffee1 
In the present study, we investigated the inhibitory effect of three catechol-containing coffee polyphenols, chlorogenic acid, caffeic acid and caffeic acid phenethyl ester, on the O-methylation of 2- and 4-hydroxyestradiol (2-OH-E2 and 4-OH-E2, respectively) catalyzed by the cytosolic catechol-O-methyltransferase (COMT) isolated from human liver and placenta. When human liver COMT was used as the enzyme, chlorogenic acid and caffeic acid each inhibited the O-methylation of 2-OH-E2 in a concentration-dependent manner, with IC50 values of 1.3–1.4 and 6.3–12.5 μM, respectively, and they also inhibited the O-methylation of 4-OH-E2, with IC50 values of 0.7–0.8 and 1.3–3.1 μM, respectively. Similar inhibition pattern was seen with human placental COMT preparation. Caffeic acid phenethyl ester had a comparable effect as caffeic acid for inhibiting the O-methylation of 2-OH-E2, but it had a weaker inhibition of the O-methylation of 4-OH-E2. Enzyme kinetic analyses showed that chlorogenic acid and caffeic acid inhibited the human liver and placental COMT-mediated O-methylation of catechol estrogens with a mixed mechanism of inhibition (competitive plus noncompetitive). Computational molecular modeling analysis showed that chlorogenic acid and caffeic acid can bind to human soluble COMT at the active site in a similar manner as the catechol estrogen substrates. Moreover, the binding energy values of these two coffee polyphenols are lower than that of catechol estrogens, which means that coffee polyphenols have higher binding affinity for the enzyme than the natural substrates. This computational finding agreed perfectly with our biochemical data.
PMCID: PMC2674329  PMID: 19095062
Coffee polyphenols; Catechol estrogens; 2-Hydroxyestradiol; 4-Hydroxyestradiol; Catechol-O-methyltransferase (COMT); Estrogen metabolism; Liver; Placenta; Enzyme inhibition; Computational modeling
9.  Is COMT a Susceptibility Gene for Schizophrenia? 
Schizophrenia Bulletin  2007;33(3):635-641.
Catechol-O-methyl transferase (COMT) is a catabolic enzyme involved in the degradation of a number of bioactive molecules; of principal interest to psychiatry, these include dopamine. The enzyme is encoded by the COMT gene. COMT is located (along with 47 other genes) in a fragment of chromosome 22q11 which when deleted results in a complex syndrome, the psychiatric manifestations of which include schizophrenia and other psychoses. These 2 observations have placed COMT near the top of a rather long list of plausible candidate genes for schizophrenia. The ability to test the hypothesis that COMT might be a susceptibility gene for schizophrenia has been simplified in principle by the existence of a valine-to-methionine (Val/Met) polymorphism which results respectively in high and low activity forms of the enzyme. Given the unequivocal effect of this polymorphism on the function of COMT, and the evidence for a critical role for dopamine in the pathophysiology and treatment of psychosis, there are strong prior expectations that Val/Met influences susceptibility to schizophrenia as well as other psychiatric phenotypes. Indeed the Val/Met polymorphism has become the most widely studied polymorphism in psychiatry. In this review, we consider the evidence for and against the involvement of COMT in schizophrenia. The current data allow us to virtually exclude a simple relationship between schizophrenia and the Val/Met variant previously thought to dominate COMT function. However, recent data suggest a more complex pattern of genetic regulation of COMT function beyond that attributable to the Val/Met locus. Moreover, it is also clear that there is a complex nonlinear relationship between dopamine availability and brain function. These 2 factors, allied to phenotypic complexity within schizophrenia, make it difficult to draw strong conclusions regarding COMT in schizophrenia. Nevertheless, emerging research that takes greater account of all these levels of complexity is beginning to provide tantalizing, but far from definitive, support for the view that COMT influences susceptibility to at least some forms of psychosis.
PMCID: PMC2526139  PMID: 17412710
schizophrenia; COMT; prefrontal cortex; association; cognition; psychosis
10.  Association of Single Nucleotide Polymorphisms in CYP1B1 and COMT Genes with Breast Cancer Susceptibility in Indian Women 
Disease markers  2009;27(5):203-210.
Cytochrome P450 1B1 (CYP1B1) and catechol-$O$-methyltransferase (COMT) enzymes play critical roles in estrogen metabolism. Alterations in the catalytic activity of CYP1B1 and COMT enzymes have been found associated with altered breast cancer risk in postmenopausal women in many populations. The substitution of leucine (Leu) to valine (Val) at codon 432 increases the catalytic activity of CYP1B1, however, substitution of Val to methionine (Met) at codon 158 decreases the catalytic activity of COMT. The present study was performed to evaluate the associations of CYP1B1 Leu432Val and/or COMT Val158Met polymorphisms with total, premenopausal and postmenopausal breast cancer risks in Indian women. COMT and CYP1B1 polymorphisms in controls and breast cancer patients were analyzed employing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed by gel electrophoresis. Although CYP1B1 and COMT genotypes did not exhibit statistically significant association with breast cancer risks when analyzed individually, COMT wild type (Val158Val) in combination with CYP1B1 heterozygous variant (Leu432Val) [OR: 0.21; 95% CI (0.05–0.82), p value; 0.021] and COMT heterozygous variant (Val158Met) in combination with CYP1B1 wild type (Leu432Leu) [OR: 0.29; 95% CI (0.08–0.96), p value; 0.042] showed significant protective association with premenopausal breast cancer risk. The results demonstrate that CYP1B1 wild type in combination with COMT heterozygous or their inverse combination offer protection against breast cancer in premenopausal Indian women.
PMCID: PMC3835283  PMID: 20037207
Breast cancer; genetic polymorphism; CYP1B1; COMT
11.  Down-regulation of the caffeic acid O-methyltransferase gene in switchgrass reveals a novel monolignol analog 
Down-regulation of the caffeic acid 3-O-methyltransferase EC (COMT) gene in the lignin biosynthetic pathway of switchgrass (Panicum virgatum) resulted in cell walls of transgenic plants releasing more constituent sugars after pretreatment by dilute acid and treatment with glycosyl hydrolases from an added enzyme preparation and from Clostridium thermocellum. Fermentation of both wild-type and transgenic switchgrass after milder hot water pretreatment with no water washing showed that only the transgenic switchgrass inhibited C. thermocellum. Gas chromatography–mass spectrometry (GCMS)-based metabolomics were undertaken on cell wall aqueous extracts to determine the nature of the microbial inhibitors.
GCMS confirmed the increased concentration of a number of phenolic acids and aldehydes that are known inhibitors of microbial fermentation. Metabolomic analyses of the transgenic biomass additionally revealed the presence of a novel monolignol-like metabolite, identified as trans-3, 4-dimethoxy-5-hydroxycinnamyl alcohol (iso-sinapyl alcohol) in both non-pretreated, as well as hot water pretreated samples. iso-Sinapyl alcohol and its glucoside were subsequently generated by organic synthesis and the identity of natural and synthetic materials were confirmed by mass spectrometric and NMR analyses. The additional novel presence of iso-sinapic acid, iso-sinapyl aldehyde, and iso-syringin suggest the increased activity of a para-methyltransferase, concomitant with the reduced COMT activity, a strict meta-methyltransferase. Quantum chemical calculations were used to predict the most likely homodimeric lignans generated from dehydration reactions, but these products were not evident in plant samples.
Down-regulation of COMT activity in switchgrass resulted in the accumulation of previously undetected metabolites resembling sinapyl alcohol and its related metabolites, but that are derived from para-methylation of 5-hydroxyconiferyl alcohol, and related precursors and products; the accumulation of which suggests altered metabolism of 5-hydroxyconiferyl alcohol in switchgrass. Given that there was no indication that iso-sinapyl alcohol was integrated in cell walls, it is considered a monolignol analog. Diversion of substrates from sinapyl alcohol to free iso-sinapyl alcohol, its glucoside, and associated upstream lignin pathway changes, including increased phenolic aldehydes and acids, are together associated with more facile cell wall deconstruction, and to the observed inhibitory effect on microbial growth. However, iso-sinapyl alcohol and iso-sinapic acid, added separately to media, were not inhibitory to C. thermocellum cultures.
PMCID: PMC3524654  PMID: 22998926
trans-3; 4-Dimethoxy-5-hydroxycinnamyl alcohol; iso-Sinapyl alcohol; Monolignol; Switchgrass; Bioenergy; Recalcitrance; Caffeic acid O-methyltransferase; Transgenic
12.  Effect of Tolcapone on Brain Activity During a Variable Attentional Control Task: A Double-Blind, Placebo-Controlled, Counter-Balanced Trial in Healthy Volunteers 
CNS drugs  2013;27(8):663-673.
Attention is the capacity to flexibly orient behaviors and thoughts towards a goal by selecting and integrating relevant contextual information. The dorsal cingulate (dCC) and prefrontal (PFC) cortices play critical roles in attention. Evidence indicates that catechol-O-methyltransferase (COMT) modulates dopaminergic tone in the PFC and dCC.
In this study, we explored the effect of tolcapone, a CNS penetrant COMT inhibitor that increases cortical dopamine levels, on brain activity during a Variable Attentional Control (VAC) task.
Study Design
We performed a double-blinded, placebo-controlled, counter-balanced trial with tolcapone (Tasmar, tablets, 100 mg three times a day for 1 day and then 200 mg three times a day for 6 days; identifier: NCT00044083).
The study was conducted in the Clinical Center of the National Institute of Mental Health from 2005 to 2009. Patients Twenty healthy volunteers (11 males; mean age = 32.7 years) with good imaging and performance data on both arms of the study were investigated.
Participants underwent 3T blood-oxygen-level-dependent (BOLD) functional magnetic resonance imaging (fMRI) while performing the event-related VAC task, which varies attention over three levels of load: LOW, INT (intermediate), and HIGH.
Main Outcome Measure
Changes in behavioral data and individual contrast images were analyzed using ANOVA with drug and task load as co-factors.
There was a significant main effect of increasing task load, with resulting decreased accuracy and increased reaction time. While there was no significant effect of tolcapone on these behavioral measures, the neuroimaging data showed a significant effect on load-related changes in dCC, with significantly lower dCC activation on tolcapone compared with placebo. Further, neural activity in dCC correlated positively with COMT enzyme activity (i.e., lower COMT activity and presumably more dopamine was associated with lower activation in dCC, i.e., more efficient information processing).
Our results show that pharmacological reduction of COMT activity modulates the engagement of attentional mechanisms, selectively enhancing the efficiency of dCC processing in healthy volunteers, reflected as decreased activity for the same level of performance.
PMCID: PMC4135358  PMID: 23794107
13.  The Influence of Metabolic Syndrome, Physical Activity, and Genotype on Catechol-O-Methyl Transferase Promoter-Region Methylation in Schizophrenia 
The pharmacogenomics journal  2012;13(3):264-271.
The catechol-o-methyl transferase (COMT) 158Val/Met variant has been suggested to play a role in COMT function. Epigenetic regulation of COMT may further influence the prevalence of metabolic syndrome in these patient populations. This study examined the correlation between COMT promoter methylation and metabolic syndrome in schizophrenia patients receiving atypical antipsychotic (AAP) therapy. DNA was extracted from peripheral blood samples of schizophrenia subjects screened for metabolic syndrome. Pyrosequencing was used to analyze two methylation sites of the COMT-s promoter region. Associations between AAP use, lifestyle variables, metabolic syndrome, and COMT genotype with peak methylation values were analyzed. Data are reported in 85 subjects. Methylation on CpG site 1 had a mean of 79.08% (± 4.71) and 12.43% (±1.19) on site 2. COMT genotype proved to be an indicator of COMT methylation status on site 1 (F(2,84) = 5.78, p=0.0044) and site 2 (F(2,84), p=0.027). A significant negative correlation between physical activity and COMT promoter region methylation was found in Val/Val homozygous patients (Site 1: p=0.013 and Site 2: p=0.019). Those homozygous for Met/Met showed a positive correlation between promoter site methylation and physical activity (Site 1: p=0.027, Site 2: p=0.005), and between CpG site methylation and metabolic syndrome (Site 1: p=0.002; Site 2: p=0.001). The results of this study suggest COMT promoter region methylation is largely influenced by COMT genotype and that physical activity plays a significant role in epigenetic modulation of COMT.
PMCID: PMC3663896  PMID: 22391769
COMT; Metabolic Syndrome; Methylation; Schizophrenia; Activity; Genotype
14.  Departure from multiplicative interaction for catechol-O-methyltransferase genotype and active/passive exposure to tobacco smoke among women with breast cancer 
Women with homozygous polymorphic alleles of catechol-O-methyltransferase (COMT-LL) metabolize 2-hydroxylated estradiol, a suspected anticarcinogenic metabolite of estrogen, at a four-fold lower rate than women with no polymorphic alleles (COMT-HH) or heterozygous women (COMT-HL). We hypothesized that COMT-LL women exposed actively or passively to tobacco smoke would have higher exposure to 2-hydroxylated estradiol than never-active/never passive exposed women, and should therefore have a lower risk of breast cancer than women exposed to tobacco smoke or with higher COMT activity.
We used a case-only design to evaluate departure from multiplicative interaction between COMT genotype and smoking status. We identified 502 cases of invasive incident breast cancer and characterized COMT genotype. Information on tobacco use and other potential breast cancer risk factors were obtained by structured interviews.
We observed moderate departure from multiplicative interaction for COMT-HL genotype and history of ever-active smoking (adjusted odds ratio [aOR] = 1.6, 95% confidence interval [CI]: 0.7, 3.8) and more pronounced departure for women who smoked 40 or more years (aOR = 2.3, 95% CI: 0.8, 7.0). We observed considerable departure from multiplicative interaction for COMT-HL genotype and history of ever-passive smoking (aOR = 2.0, 95% CI: 0.8, 5.2) or for having lived with a smoker after age 20 (aOR = 2.8, 95% CI: 0.8, 10).
With greater control over potential misclassification errors and a large case-only population, we found evidence to support an interaction between COMT genotype and tobacco smoke exposure in breast cancer etiology.
PMCID: PMC1373621  PMID: 16417624
15.  Comprehensive interrogation of CpG island methylation in the gene encoding COMT, a key estrogen and catecholamine regulator 
The catechol-O-methyltransferase (COMT) enzyme has been widely studied due to its multiple roles in neurological functioning, estrogen biology, and methylation metabolic pathways. Numerous studies have investigated variation in the large COMT gene, with the majority focusing on single nucleotide polymorphisms (SNPs). This body of work has linked COMT genetic variation with a vast array of conditions, including several neurobehavioral disorders, pain sensitivity, and multiple human cancers. Based on COMT’s numerous biological roles and recent studies suggesting that methylation of the COMT gene impacts COMT gene expression, we comprehensively interrogated methylation in over 200 CpG dinucleotide sequences spanning the length of the COMT gene.
Using saliva-derived DNA from a non-clinical sample of human subjects, we tested for associations between COMT CpG methylation and factors reported to interact with COMT genetic effects, including demographic factors and alcohol use. Finally, we tested associations between COMT CpG methylation state and COMT gene expression in breast cancer cell lines. We interrogated >200 CpGs in 13 amplicons spanning the 5’ UTR to the last exon of the CpG dinucleotide-rich COMT gene in n = 48 subjects, n = 11 cell lines and 1 endogenous 18S rRNA control.
With the exception of the CpG island in the 5’UTR and 1st exon, all other CpG islands were strongly methylated with typical dynamic ranges between 50-90%. In the saliva samples, methylation of multiple COMT loci was associated with socioeconomic status or ethnicity. We found associations between methylation at numerous loci and genotype at the functional Val 158 Met SNP (rs4680), and most of the correlations between methylation and demographic and alcohol use factors were Val 158 Met allele-specific. Methylation at several of these loci also associated with COMT gene expression in breast cancer cell lines.
We report the first comprehensive interrogation of COMT methylation. We corroborate previous findings of variation in COMT methylation with gene expression and the Val 158 Met genotype, and also report novel associations with socioeconomic status (SES) and ethnicity at several methylated loci. These results point to novel mechanisms for COMT regulation, which may have broad therapeutic implications.
PMCID: PMC3910242  PMID: 24460628
16.  Phylogenetic, Molecular, and Biochemical Characterization of Caffeic Acid o-Methyltransferase Gene Family in Brachypodium distachyon 
Caffeic acid o-methyltransferase (COMT) is one of the important enzymes controlling lignin monomer production in plant cell wall synthesis. Analysis of the genome sequence of the new grass model Brachypodium distachyon identified four COMT gene homologs, designated as BdCOMT1, BdCOMT2, BdCOMT3, and BdCOMT4. Phylogenetic analysis suggested that they belong to the COMT gene family, whereas syntenic analysis through comparisons with rice and sorghum revealed that BdCOMT4 on Chromosome 3 is the orthologous copy of the COMT genes well characterized in other grass species. The other three COMT genes are unique to Brachypodium since orthologous copies are not found in the collinear regions of rice and sorghum genomes. Expression studies indicated that all four Brachypodium COMT genes are transcribed but with distinct patterns of tissue specificity. Full-length cDNAs were cloned in frame into the pQE-T7 expression vector for the purification of recombinant Brachypodium COMT proteins. Biochemical characterization of enzyme activity and substrate specificity showed that BdCOMT4 has significant effect on a broad range of substrates with the highest preference for caffeic acid. The other three COMTs had low or no effect on these substrates, suggesting that a diversified evolution occurred on these duplicate genes that not only impacted their pattern of expression, but also altered their biochemical properties.
PMCID: PMC3562662  PMID: 23431288
17.  Catechol-O-methyltransferase: characteristics, polymorphisms and role in breast cancer 
Catechol estrogens are carcinogenic, probably because of their estrogenicity and potential for further oxidative metabolism to reactive quinones. Estrogenic quinones cause oxidative DNA damage as well as form mutagenic depurinating adenine and guanine adducts. O-Methylation by catechol-O-methyltransferase (COMT) blocks their estrogenicity and prevents their oxidation to quinones. A single gene encodes both membrane bound (MB) and soluble (S) forms of COMT. The COMT gene contains 34 single nucleotide polymorphisms (SNPs). The valine108 (S-COMT)/158 (MB-COMT) SNP encodes a low activity form of COMT and has been widely studied as a putative risk factor for breast cancer, with inconsistent results. Investigations of two other SNPs in the promoter of MB-COMT that may affect its expression have also provided mixed results. Future studies on the role of COMT in breast cancer should incorporate measurement of biomarkers that reflect COMT activity and its protective effects.
PMCID: PMC3665426  PMID: 23734165
18.  Comt1 Genotype and Expression Predicts Anxiety and Nociceptive Sensitivity in Inbred Strains of Mice 
Genes, brain, and behavior  2010;9(8):933-946.
Catechol-O-methyltransferase (COMT) is an ubiquitously expressed enzyme that maintains basic biologic functions by inactivating catechol substrates. In humans, polymorphic variance at the COMT locus has been associated with modulation of pain sensitivity (Andersen & Skorpen, 2009) and risk for developing psychiatric disorders (Harrison & Tunbridge, 2008). A functional haplotype associated with increased pain sensitivity was shown to result in decreased COMT activity by altering mRNA secondary structure-dependent protein translation (Nackley et al., 2006). However, the exact mechanisms whereby COMT modulates pain sensitivity and behavior remain unclear and can be further studied in animal models. We have assessed Comt1 gene expression levels in multiple brain regions in inbred strains of mice and have discovered that Comt1 is differentially expressed among the strains, and this differential expression is cis-regulated. A B2 Short Interspersed Element (SINE) was inserted in the 3′UTR of Comt1 in 14 strains generating a common haplotype that correlates with gene expression. Experiments using mammalian expression vectors of full-length cDNA clones with and without the SINE element demonstrate that strains with the SINE haplotype (+SINE) have greater Comt1 enzymatic activity. +SINE mice also exhibit behavioral differences in anxiety assays and decreased pain sensitivity. These results suggest that a haplotype, defined by a 3′ UTR B2 SINE element, regulates Comt1 expression and some mouse behaviors.
PMCID: PMC2975805  PMID: 20659173
19.  COMT Val158Met Genotype Determines the Direction of Cognitive Effects Produced by Catechol-O-Methyltransferase Inhibition 
Biological Psychiatry  2012;71(6):538-544.
Catechol-O-methyltransferase (COMT) metabolizes dopamine. The COMT Val158Met polymorphism influences its activity, and multiple neural correlates of this genotype on dopaminergic phenotypes, especially working memory, have been reported. COMT activity can also be regulated pharmacologically by COMT inhibitors. The inverted-U relationship between cortical dopamine signaling and working memory predicts that the effects of COMT inhibition will differ according to COMT genotype.
Thirty-four COMT Met158Met (Met-COMT) and 33 COMT Val158Val (Val-COMT) men were given a single 200-mg dose of the brain-penetrant COMT inhibitor tolcapone or placebo in a randomized, double-blind, between-subjects design. They completed the N-back task of working memory and a gambling task.
In the placebo group, Met-COMT subjects outperformed Val-COMT subjects on the 2- back, and they were more risk averse. Tolcapone had opposite effects in the two genotype groups: it worsened N-back performance in Met-COMT subjects but enhanced it in Val-COMT subjects. Tolcapone made Met-COMT subjects less risk averse but Val-COMT subjects more so. In both tasks, tolcapone reversed the baseline genotype differences.
Depending on genotype, COMT inhibition can enhance or impair working memory and increase or decrease risky decision making. To our knowledge, the data are the clearest demonstration to date that the direction of effect of a drug can be influenced by a polymorphism in its target gene. The results support the inverted-U model of dopamine function. The findings are of translational relevance, because COMT inhibitors are used in the adjunctive treatment of Parkinson's disease and are under evaluation in schizophrenia and other disorders.
PMCID: PMC3314969  PMID: 22364739
Catechol-o-methyltransferase; decision making; pharmacogenetics; polymorphism; tolcapone; working memory
20.  Hypomethylation of MB-COMT promoter is a major risk factor for schizophrenia and bipolar disorder 
Human molecular genetics  2006;15(21):3132-3145.
The variability in phenotypic presentations and the lack of consistency of genetic associations in mental illnesses remain a major challenge in molecular psychiatry. Recently, it has become increasingly clear that altered promoter DNA methylation could play a critical role in mediating differential regulation of genes and in facilitating short-term adaptation in response to the environment. Here, we report the investigation of the differential activity of membrane-bound catechol-O-methyltransferase (MB-COMT) due to altered promoter methylation and the nature of the contribution of COMT Val158Met polymorphism as risk factors for schizophrenia and bipolar disorder by analyzing 115 post-mortem brain samples from the frontal lobe. These studies are the first to reveal that the MB-COMT promoter DNA is frequently hypomethylated in schizophrenia and bipolar disorder patients, compared with the controls (methylation rate: 26 and 29 versus 60%; P = 0.004 and 0.008, respectively), particularly in the left frontal lobes (methylation rate: 29 and 30 versus 81%; P = 0.003 and 0.002, respectively). Quantitative gene-expression analyses showed a corresponding increase in transcript levels of MB-COMT in schizophrenia and bipolar disorder patients compared with the controls (P = 0.02) with an accompanying inverse correlation between MB-COMT and DRD1 expression. Furthermore, there was a tendency for the enrichment of the Val allele of the COMT Val158Met polymorphism with MB-COMT hypomethylation in the patients. These findings suggest that MB-COMT over-expression due to promoter hypomethylation and/or hyperactive allele of COMT may increase dopamine degradation in the frontal lobe providing a molecular basis for the shared symptoms of schizophrenia and bipolar disorder.
PMCID: PMC2799943  PMID: 16984965
21.  Catechol-O-Methyltransferase Polymorphism and Endometriosis 
Purpose: Catechol-O-methyltransferase (COMT) inactivates the estradiol metabolites, 2-hydroxy and 4-hydroxy catechols, which have been implicated in the pathogenesis of endometriosis. A COMT valine to methionine polymorphism (G-to-A) in exon 4 of the COMT gene is polymorphic in the human population, with 25% of Caucasians being homozygous for the low-activity allele (COMT-L) of the enzyme. In a case-control study we investigated whether this COMT polymorphism is associated with endometriosis.
Methods: Polymerase chain reaction was performed to analyze the COMT genotype among women with surgically and histologically confirmed endometriosis (study group; n = 91) and in women without evidence of endometriosis confirmed by laparoscopy or laparotomy (control group; n = 92).
Results: Allele frequencies for the low-activity allele (COMT-L) among women with endometriosis and controls were 0.50 and 0.50, respectively (p = 0.999; odds ratio = 1.0, 95% CI: 0.66–1.51).
Conclusions: Our results suggest that the valine to methionine polymorphism in exon 4 of the COMT gene is not associated with the risk of endometriosis compared to a surgical control population.
PMCID: PMC3455752  PMID: 12168735
Endometriosis; COMT; polymorphism; catecholestrogens; Caucasian population
22.  Opposite effects of tolcapone on amphetamine-disrupted startle gating in low vs. high COMT-expressing rat strains 
Differential sensitivity to the prepulse inhibition (PPI)-disruptive effects of dopamine agonists in Sprague–Dawley (SD) vs. Long Evans (LE) rats is heritable, reflects differential activation of DA signaling, and is associated with differences in the brain expression of specific genes, including those of the catecholamine catabolic enzyme, catechol-O-methyltransferase (COMT). In humans, both basal and drug-modified PPI differs significantly between individuals with polymorphisms conferring low- vs. high-activity of COMT. We used the COMT inhibitor, tolcapone, to assess the role of COMT activity in regulating the differential effects of the dopamine releaser, amphetamine (AMPH), on PPI in SD and LE rats.
Acoustic startle and PPI were assessed in SD and LE male rats after pretreatment with tolcapone (vehicle vs. 30 mg/kg ip) and treatment with AMPH(vehicle vs. 4.5 mg/kg sc), using 10–120 ms prepulse intervals.
After tolcapone, AMPH significantly potentiated PPI in LE rats, and significantly disrupted PPI in SD rats. These patterns could not be explained by drug effects on pulse alone startle magnitude.
The impact of COMT inhibition on AMPH-modified PPI was categorically different in strains exhibiting low vs. high levels of forebrain Comt expression, consistent with reports in humans that tolcapone has opposite effects on PPI among individuals with polymorphisms conferring low vs. high COMT activity. The present model provides a basis for understanding the mechanisms by which the effects of COMT inhibition on sensorimotor gating – and potentially, related neurocognitive and clinical functions – under hyperdopaminergic states are dependent on an individual's basal levels of COMT activity.
PMCID: PMC3760336  PMID: 23567203
Amphetamine; Dopamine; Catechol-O-methyltransferase; Prepulse inhibition; Schizophrenia; Strain — tolcapone
23.  Lack of Association Between COMT and Working Memory in a Population-Based Cohort of Healthy Young Adults 
Neuropsychopharmacology  2013;38(7):1253-1263.
The Val158Met polymorphism of the catechol-O-methyltransferase (COMT) gene is an important regulator of dopamine in the prefrontal cortex, an area critical to working memory. Working memory deficits are present in several psychiatric disorders, and there is wide variation in working memory capacity in the normal population. Association studies of COMT and working memory in healthy volunteers have yielded inconsistent results, possibly because of small sample sizes. Here we examine COMT in relation to N-Back working memory task performance in a large population-based cohort of young adults. We predicted individuals with one or two copies of the Met allele would perform better, and that this relationship would be more evident in males than females. Participants (N=1857–2659) tested at 18 years of age, were enrolled in the Avon Longitudinal Study of Parents and Children (ALSPAC). We used multiple regression to examine effects of sex and COMT genotype on N-Back hits, false positives, discriminability (d'), and reaction time while controlling for important covariates. COMT genotype did not predict hits or d'. There was a nominally significant interaction between COMT and sex on false positives, but this was not in the predicted direction, and was not significant after controlling for covariates. COMT genotype was not related to working memory in this large population-based cohort. It is possible COMT is not meaningfully associated with working memory in healthy young adults, or that COMT effects are detectable only in assessments reflecting neural processes underlying cognition, such as fMRI, rather than in behavioral performance.
PMCID: PMC3656369  PMID: 23337869
dopamine; cognition; learning & memory; neurogenetics; COMT genotype; executive functioning; ALSPAC; COMT genotype; executive functioning; working memory; genetics; ALSPAC
24.  Breast Cancer Risk Reduction and Membrane-Bound Catechol O-Methyltransferase Genetic Polymorphisms 
Cancer research  2008;68(14):5997-6005.
Catechol O-methyltransferase (COMT)-catalyzed methylation of catecholestrogens has been proposed to play a protective role in estrogen-induced genotoxic carcinogenesis. We have taken a comprehensive approach to test the hypothesis that genetic variation in COMT might influence breast cancer risk. Fifteen COMT SNPs selected on the basis of in-depth resequencing of the COMT gene were genotyped in 1482 DNA samples from a Mayo Clinic breast cancer case-control study. Two common SNPs in the distal promoter for membrane-bound (MB) COMT, rs2020917 and rs737865, were associated with breast cancer risk reduction in premenopausal women in the Mayo Clinic study, with allele-specific odds ratios of 0.70 (95% CI = 0.52–0.95) and 0.68 (95% CI = 0.51–0.92), respectively. These two SNPs were then subjected to functional genomic analysis and were genotyped in an additional 3683 DNA samples from two independent case-control studies (GENICA and GESBC). Functional genomic experiments showed that these SNPs could up-regulate transcription and that they altered DNA-protein binding patterns. Furthermore, substrate kinetic and exon array analyses suggested a role for MB-COMT in catecholestrogen inactivation. The GENICA results were similar to the Mayo case-control observations, with ORs of 0.85 (95% CI = 0.72–1.00) and 0.85 (95% CI = 0.72–1.01) for the two SNPs. No significant effect was observed in the GESBC study. These studies demonstrated that two SNPs in the COMT distal promoter were associated with breast cancer risk reduction in 2 of 3 case-control studies, compatible with the results of functional genomic experiments, suggesting a role for MB-COMT in breast cancer risk.
PMCID: PMC2518124  PMID: 18632656
Catechol O-methyltransferase; COMT; MB-COMT; S-COMT; breast cancer risk; genetic polymorphism; SNPs; functional genomics
25.  Structural Mechanism of S-Adenosyl Methionine Binding to Catechol O-Methyltransferase 
PLoS ONE  2011;6(8):e24287.
Methyltransferases possess a homologous domain that requires both a divalent metal cation and S-adenosyl-L-methionine (SAM) to catalyze its reactions. The kinetics of several methyltransferases has been well characterized; however, the details regarding their structural mechanisms have remained unclear to date. Using catechol O-methyltransferase (COMT) as a model, we perform discrete molecular dynamics and computational docking simulations to elucidate the initial stages of cofactor binding. We find that COMT binds SAM via an induced-fit mechanism, where SAM adopts a different docking pose in the absence of metal and substrate in comparison to the holoenzyme. Flexible modeling of the active site side-chains is essential for observing the lowest energy state in the apoenzyme; rigid docking tools are unable to recapitulate the pose unless the appropriate side-chain conformations are given a priori. From our docking results, we hypothesize that the metal reorients SAM in a conformation suitable for donating its methyl substituent to the recipient ligand. The proposed mechanism enables a general understanding of how divalent metal cations contribute to methyltransferase function.
PMCID: PMC3164188  PMID: 21904625

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