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Microbial remediation has become one of the promising ways to eliminate polycyclic aromatic hydrocarbons (PAHs) pollution due to its efficient enzyme metabolism system. Catechol 1,2-dioxygenase (C12O) is a crucial rate-limiting enzyme in the degradation pathway of PAHs in Achromobacter xylosoxidans DN002 that opens the benzene ring through the ortho-cleavage pathway. However, little attention has been given to explore the interaction mechanism of relevant enzyme–substrate. This study aims to investigate the binding interaction between C12O of strain DN002 and catechol by means of a molecular biological approach combined with homology modeling, molecular docking, and multiple spectroscopies. The removal rate of catechol in the mutant strain of cat A deletion was only 12.03%, compared to the wild-type strain (54.21%). A Ramachandran plot of active site regions of the primary amino acid sequences in the native enzyme showed that 93.5% sequences were in the most favored regions on account of the results of homology modeling, while an additional 6.2% amino acid sequences were found in conditionally allowed regions, and 0.4% in generously allowed regions. The binding pocket of C12O with catechol was analyzed to obtain that the catalytic trimeric group of Tyr164-His224-His226 was proven to be great vital for the ring-opening reaction of catechol by molecular docking. In the native enzyme, binding complexes were spontaneously formed by hydrophobic interactions. Binding constants and thermodynamic potentials from fluorescence spectra indicated that catechol effectively quenched the intrinsic fluorescence of C12O in the C12O/catechol complex via conventional static and dynamic quenching mechanisms of C12O. The results of ultraviolet and visible (UV) spectra, synchronous fluorescence, and circular dichroism (CD) spectra revealed conspicuous changes in the local conformation, and site-directed mutagenesis confirmed the role of predicted key residues during catalysis, wherein His226 had a significant effect on catechol utilization by C12O. This is the first report to reveal interactions of C12O with substrate from the molecular docking results, providing the mechanistic understanding of representative dioxygenases involved in aromatic compound degradation, and a solid foundation for further site modifications as well as strategies for the directed evolution of this enzyme.

(2012) PEM Anchorage on Titanium Using Catechol Grafting. PLoS ONE 7(11): e50326. doi:10.1371/journal.pone.0050326 Citation: Marie H, Barrere A, Schoenstein F, Chavanne M-H, Grosgogeat B, Mora L (2013) Correction: PEM Anchorage on Titanium Using Catechol Grafting.

Nordihydroguaiaretic acid (NDGA) is a major lignan metabolite found in Larrea spp., which are widely used in South America to treat various diseases. In breast tissue, estradiol is metabolized to the catechol estrogens such as 4-hydroxyestradiol (4-OHE2), which have been proposed to be cancer initiators potentially involved in mammary carcinogenesis. Catechol-O-methyltransferase (COMT) catalyzes the O-methylation of catechol estrogens to their less toxic methoxy derivatives, such as 4-O-methylestradiol (4-MeOE2). The present study investigated the novel biological activities of NDGA in relation to COMT and the effects of COMT inhibition by NDGA on 4-OHE2-induced cyto- and genotoxicity in MCF-7 human breast cancer cells. Two methoxylated metabolites of NDGA, 3-O-methylNDGA (3-MNDGA) and 4-O-methyl NDGA (4-MNDGA), were identified in the reaction mixture containing human recombinant COMT, NDGA, and cofactors. Km values for the COMT-catalyzed metabolism of NDGA were 2.6 µM and 2.2 µM for 3-MNDGA and 4-MNDGA, respectively. The COMT-catalyzed methylation of 4-OHE2 was inhibited by NDGA at an IC50 of 22.4 µM in a mixed-type mode of inhibition by double reciprocal plot analysis. Molecular docking studies predicted that NDGA would adopt a stable conformation at the COMT active site, mainly owing to the hydrogen bond network. NDGA is likely both a substrate for and an inhibitor of COMT. Comet and apurinic/apyrimidinic site quantitation assays, cell death, and apoptosis in MCF-7 cells showed that NDGA decreased COMT-mediated formation of 4-MeOE2 and increased 4-OHE2-induced DNA damage and cytotoxicity. Thus, NDGA has the potential to reduce COMT activity in mammary tissues and prevent the inactivation of mutagenic estradiol metabolites, thereby increasing catechol estrogen-induced genotoxicities.

Enzymatic browning because of polyphenol oxidases (PPOs) contributes to the color quality of fruit and vegetable (FV) products. Physical and chemical methods have been developed to inhibit the activity of PPOs, and several synthetic chemical compounds are commonly being used as PPO inhibitors in FV products. Recently, there has been an emphasis on consumer-oriented innovations in the food industry. Consumers tend to urge the use of natural and environment-friendly PPO inhibitors. The purpose of this review is to summarize the mechanisms underlying the anti-browning action of chemical PPO inhibitors and current trends in the research on these inhibitors. Based on their mechanisms of action, chemical inhibitors can be categorized as antioxidants, reducing agents, chelating agents, acidulants, and/or mixed-type PPO inhibitors. Here, we focused on the food ingredients, dietary components, food by-products, and waste associated with anti-browning activity.

We investigated the mechanism underlying the coexistence of the phenol-degrading bacteria Cupriavidus sp. strain P-10 and Comamonas thiooxydans strain R2 using a chemostat culture supplemented with phenol as the sole carbon and energy source. Quantitative PCR (qPCR) targeting the genes encoding the large subunit of phenol hydroxylase dmpN and aphN for strains P-10 and R2, respectively, revealed the coexistence of the two strains even under phenol-limiting conditions. According to the reverse transcription qPCR targeting dmpN , aphN , catechol 2,3-dioxygenase genes ( dmpB and aphB ), and catechol 1,2-dioxygenase gene ( catA ) of strain P-10, both strains transcribed these genes, suggesting that phenol was metabolized individually. However, mathematical simulations demonstrated that the two strains never coexisted because competitive exclusion occurred at any phenol sharing ratio. The simulation revealed that strain P-10 outcompeted strain R2 when it occupied more than 64% of the phenol. These results suggest that phenol sharing, with fluctuations in the ratio within an appropriate range, is indispensable for coexistence under resource-limiting conditions. The phenol concentration in the chemostat remained spatially heterogeneous for 15 s. If coexistence was accomplished through the phenol-sharing, either strain P-10 or R2 could invade another single-strain chemostat culture using phenol, even when inoculated at a significantly lower cell density than the resident strain. Strains P-10 and R2 successfully invaded and coexisted with other strains in the chemostat culture. These results suggest that fluctuation of the limiting substrate sharing ratio enabled stable coexistence of bacterial strains under competitive conditions. Key point • Phenol concentration was spatiotemporally heterogeneous in the chemostat culture

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.

Human catechol O-methyltransferase (COMT) has emerged as a model for understanding enzyme-catalyzed methyl transfer from S-adenosylmethionine (AdoMet) to small-molecule catecholate acceptors. Mutation of a single residue (tyrosine 68) behind the methyl-bearing sulfonium of AdoMet was previously shown to impair COMT activity by interfering with methyl donor–acceptor compaction within the activated ground state of the wild type enzyme [J. Zhang, H. J. Kulik, T. J. Martinez, J. P. Klinman, Proc. Natl. Acad. Sci. U.S.A. 112, 7954–7959 (2015)]. This predicts the involvement of spatially defined protein dynamical effects that further tune the donor/acceptor distance and geometry as well as the electrostatics of the reactants. Here, we present a hydrogen/deuterium exchange (HDX)-mass spectrometric study of wild type and mutant COMT, comparing temperature dependences of HDX against corresponding kinetic and cofactor binding parameters. The data show that the impaired Tyr68Ala mutant displays similar breaks in Arrhenius plots of both kinetic and HDX properties that are absent in the wild type enzyme. The spatial resolution of HDX below a break point of 15–20 °C indicates changes in flexibility across ∼40% of the protein structure that is confined primarily to the periphery of the AdoMet binding site. Above 20 °C, Tyr68Ala behaves more like WT in HDX, but its rate and enthalpic barrier remain significantly altered. The impairment of catalysis by Tyr68Ala can be understood in the context of a mutationally induced alteration in protein motions that becomes manifest along and perpendicular to the primary group transfer coordinate.

PurposeTolcapone is an efficacious catechol-O-methyltransferase inhibitor for Parkinson’s disease (PD). However, safety issues hampered its use in clinical practice. We aimed to provide evidence of safety and efficacy of tolcapone by a systematic literature review to support clinicians’ choices in the use of an enlarging PD therapeutic armamentarium.MethodsWe searched PubMed for studies on PD patients treated with tolcapone, documenting the following outcomes: liver enzyme, adverse events (AEs), daily Off-time, levodopa daily dose, unified Parkinson’s disease rating scale (UPDRS) part-III, quality of life (QoL), and non-motor symptoms. FAERS and EudraVigilance databases for suspected AEs were interrogated for potential additional cases of hepatotoxicity.ResultsThirty-two studies were included, for a total of 4780 patients treated with tolcapone. Pertaining safety, 0.9% of patients showed liver enzyme elevation > 2. Over 23 years, we found 7 cases of severe liver injury related to tolcapone, 3 of which were fatal. All fatal cases did not follow the guidelines for liver function monitoring. FAERS and EudraVigilance database search yielded 61 reports of suspected liver AEs possibly related to tolcapone.Pertaining efficacy, the median reduction of hours/day spent in Off was 2.1 (range 1–3.2), of levodopa was 108.9 mg (1–251.5), of “On” UPDRS-III was 3.6 points (1.1–6.5). Most studies reported a significant improvement of QoL and non-motor symptoms.ConclusionLiterature data showed the absence of relevant safety concerns of tolcapone when strict adherence to hepatic function monitoring is respected. Given its high efficacy on motor fluctuations, tolcapone is probably an underutilized tool in the therapeutic PD armamentarium.

Alcohol Use Disorder (AUD) is characterized by loss of control over drinking. Behavioral control is mediated, in part, by cortical dopamine signaling. Inhibition of catechol-O-methyltransferase (COMT), the enzyme primarily responsible for cortical dopamine inactivation, may increase cortical dopamine, especially among individuals with genetically mediated lower dopaminergic tone, such as COMT rs4680 (val158met) val-allele homozygotes. This study was a randomized, placebo-controlled, pharmacogenetic trial of the COMT inhibitor tolcapone. Ninety non-treatment-seeking AUD individuals were prospectively genotyped for rs4680 and randomized to tolcapone (200 mg t.i.d.) or placebo for 8 days. At baseline and on day 7, peripheral COMT activity was assayed, and participants completed an fMRI alcohol cue-reactivity task; on day 8, they completed a bar-lab paradigm. Primary outcomes were: (1) natural drinking during the medication period; (2) alcohol self-administration in the bar lab; and (3) alcohol cue-elicited cortical (right inferior frontal gyrus [rIFG]) and ventral striatal activation. At baseline, the rs4680 val-allele had an additive effect on COMT activity. Tolcapone, relative to placebo, reduced COMT activity in all genotype groups. COMT genotype moderated tolcapone’s effect on drinking during the medication period and in the bar lab, such that tolcapone, relative to placebo, reduced drinking only among val-allele homozygotes. Tolcapone did not affect cue-elicited ventral striatal activation but reduced rIFG activation; less rIFG activation on day 7 was associated with less drinking during the medication period. Taken together, these data suggest that COMT inhibition may reduce drinking specifically among individuals genetically predisposed to excessive COMT activity and potentially low cortical dopamine tone.ClinicalTrials.gov identifier: NCT02949934 https://clinicaltrials.gov/ct2/show/NCT02949934

Inhibitors of catechol- O -methyltransferase (COMT) are commonly used as an adjunct to levodopa in patients with Parkinson’s disease (PD) for the amelioration of wearing-off symptoms. This narrative review aims to discuss the role of COMT inhibitors on peripheral levodopa metabolism and continuous brain delivery of levodopa, and to describe their metabolic properties. Oral application of levodopa formulations with a dopa decarboxylase inhibitor (DDI) results in fluctuating levodopa plasma concentrations, predominantly due to the short half-life of levodopa and its slowing of gastric emptying. Following transport across the blood–brain barrier and its metabolic conversion to dopamine, these peripheral ‘ups and downs’ of levodopa are reflected in fluctuating dopamine levels in the synaptic cleft between presynaptic and postsynaptic dopaminergic neurons of the nigrostriatal system. As a result, pulsatile postsynaptic dopaminergic stimulation takes place and results in the occurrence of motor complications, such as wearing-off and dyskinesia. More continuous plasma behaviour was observed after the combination of levodopa/DDI formulations with COMT inhibitors. These compounds also weaken a levodopa/DDI-related homocysteine increase, as biomarker for an impaired methylation capacity, which is involved in an elevated oxidative stress exposure. These findings favour the concept of chronic levodopa/DDI application with concomitant inhibition of COMT and monoamine oxidase, since deamination of dopamine via this enzyme also generates free radicals. This triple combination is suggested as standard levodopa application in patients with PD who need levodopa, if they will tolerate it.