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Search Results: 1 - 10 of 38 matches for " Monooxygenase "
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Form Follows Function: Structural and Catalytic Variation in the Class A Flavoprotein Monooxygenases
Karen Crozier-Reabe,Graham R. Moran
International Journal of Molecular Sciences , 2012, DOI: 10.3390/ijms131215601
Abstract: Flavoprotein monooxygenases (FPMOs) exhibit an array of mechanistic solutions to a common chemical objective; the monooxygenation of a target substrate. Each FPMO efficiently couples reduction of a flavin cofactor by NAD(P)H to oxygenation of the target substrate via a (hydro)peroxyflavin intermediate. This purpose of this review is to describe in detail the Class A flavoprotein hydroxylases (FPMO) in the context of the other FPMO classes (B–F). Both one and two component FPMOs are found in nature. Two-component enzymes require, in addition to the monooxygenase, the involvement of a reductase that first catalyzes the reduction of the flavin by NAD(P)H. The Class A and B FPMOs are single-component and manage to orchestrate the same net reaction within a single peptide. The Class A enzymes have, by some considerable margin, the most complete research record. These enzymes use choreographed movements of the flavin ring that facilitate access of the organic substrates to the active site, provide a means for interaction of NADPH with the flavin, offer a mechanism to sequester the dioxygen reduction chemistry from solvent and a means to release the product. The majority of the discrete catalytic events of the catalytic cycle can be observed directly in exquisite detail using spectrophotometric kinetic methods and many of the key mechanistic conclusions are further supported by structural data. This review attempts to compile each of the key observations made for both paradigm and newly discovered examples of Class A FPMOs into a complete catalytic description of one enzymatic turnover.
Integrated One-Pot Enrichment and Immobilization of Styrene Monooxygenase (StyA) Using SEPABEAD EC-EA and EC-Q1A Anion-Exchange Carriers
Reto Ruinatscha,Rohan Karande,Katja Buehler,Andreas Schmid
Molecules , 2011, DOI: 10.3390/molecules16075975
Abstract: A straightforward one-pot procedure combining enrichment and immobilization of recombinantely expressed FADH2 dependent styrene monooxygenase (StyA) directly from Escherichia coli cell extracts was investigated. Sepabeads EC-EA and EC-Q1A anion-exchange carriers were employed to non-covalently adsorb StyA from the cell extracts depending on basic parameters such as varying initial protein concentrations and pH. The protein fraction of the cell extract contained around 25% StyA. At low initial protein concentrations (2.5 mg mL?1) and pH 6, the enzyme could be enriched up to 52.4% on Sepabeads EC-EA and up to 46.0% on Sepabeads EC-Q1A, accounting for an almost complete StyA adsorption from the cell extracts. Higher initial protein concentrations were necessary to exploit the high loading capacity of the beads. At 20 mg mL?1, up to 37.6% of the theoretical bead loading capacity could be utilized for StyA binding using Sepabeads EC-EA, and 34.0% using Sepabeads EC-Q1A. For both carriers, protein leakage under reaction conditions could be reduced to less than 2%. During assays, the FADH2 cofactor necessary for StyA activity was supplied by the NADH-FAD reductase component styrene monooxygenase B (StyB). StyA immobilized on Sepabeads EC-Q1A displayed twice as high styrene epoxidation rates (0.2 U mgStyA?1) as compared to Sepabeads EC-EA. This activity could be increased to 0.7 U mgStyA?1 by co-immobilizing StyB on Sepabeads EC-Q1A, which corresponds to 33% of the soluble StyA activity.
Metabolism and Pharmacokinetics of the Anti-Tuberculosis Drug Ethionamide in a Flavin-Containing Monooxygenase Null Mouse
Amy L. Palmer,Virginia L. Leykam,Andrew Larkin,Sharon K. Krueger,Ian R. Phillips,Elizabeth A. Shephard,David E. Williams
Pharmaceuticals , 2012, DOI: 10.3390/ph5111147
Abstract: Multiple drug resistance (MDR) in Mycobacterium tuberculosis (mTB), the causative agent for tuberculosis (TB), has led to increased use of second-line drugs, including ethionamide (ETA). ETA is a prodrug bioactivated by mycobacterial and mammalian flavin-containing monooxygenases (FMOs). FMO2 is the major isoform in the lungs of most mammals, including primates. In humans a polymorphism exists in the expression of FMO2. FMO2.2 (truncated, inactive) protein is produced by the common allele, while the ancestral allele, encoding active FMO2.1, has been documented only in individuals of African and Hispanic origin, at an incidence of up to 50% and 7%, respectively. We hypothesized that FMO2 variability in TB-infected individuals would yield differences in concentrations and ratios of ETA prodrug and metabolites. In this study we assessed the impact of the FMO2 genetic polymorphism on the pharmacokinetics of ETA after administration of a single oral dose of ETA (125 mg/kg) to wild type and triple Fmo1/2/4-null mice, measuring levels of prodrug vs. metabolites in plasma collected from 0 to 3.5 h post-gavage. All mice metabolized ETA to ETA S-oxide (ETASO) and 2-ethyl-4-amidopyridine (ETAA). Wild type mice had higher plasma concentrations of metabolites than of parent compound ( p = 0.001). In contrast, Fmo1/2/4-null mice had higher plasma concentrations of parent compound than of metabolites ( p = 0.0001). Thus, the human FMO2 genotype could impact the therapeutic efficacy and/or toxicity of ETA.
Flavin-Containing Monooxygenase (FMO) Protein Expression and Its Activity in Rat Brain Microvascular Endothelial Cells  [PDF]
Eiichi Sakurai, Yukari Ueda, Yukari Mori, Yasuhumi Shinmyouzu, Eiko Sakurai
Pharmacology & Pharmacy (PP) , 2013, DOI: 10.4236/pp.2013.41001
Abstract: The aim of this study was to examine whether flavin-containing monooxygenase (FMO) protein was expressed in cultured rat brain microvascular endothelial cells (BMECs), which constitute the blood-brain barrier (BBB), and whether N-oxide from the tertiary amine, d-chlorpheniramine, was formed by FMO in rat BMECs. BMECs were isolated and cultured from the brains of three-week-old male Wistar rats. The expression of FMO1, FMO2 and FMO5 proteins was confirmed in rat BMECs by western blotting analysis using polyclonal anti-FMO antibodies, but FMO3 and FMO4 proteins were not found in the rat BBB. Moreover, N-oxide of d-chlorpheniramine was formed in rat BMECs. The intrinsic clearance value for N-oxidation at pH 8.4 was higher than that at pH 7.4. Inhibition of N-oxide formation by methimazole was found to be the best model of competitive inhibition yielding an apparent Ki value of 0.53 μmol/L, suggesting that N-oxidation was catalyzed by FMOs in rat BMECs. Although FMO activity in rat BMECs was lower than that in SD rat normal hepatocytes (rtNHeps), we suggest that rat BMECs enzymes can convert substrates of exogenous origin for detoxification, indicating that BMECs are an important barrier for metabolic products besides hepatic cells.
New Alleles of Rice ebisu dwarf (d2) Mutant Show both Brassinosteroid-Deficient and -Insensitive Phenotypes  [PDF]
Tomoaki Sakamoto, Yoichi Morinaka, Hidemi Kitano, Shozo Fujioka
American Journal of Plant Sciences (AJPS) , 2012, DOI: 10.4236/ajps.2012.312208
Abstract: ebisu dwarf (d2) is a mutant caused by mutation in a rice brassinosteroid biosynthetic enzyme gene, CYP90D2/D2, thereby conferring a brassinosteroid-deficient dwarf phenotype. Three newly isolated d2 alleles derived from a Nippon- bare mutant library (d2-3, d2-4, and d2-6) produced more severe dwarf phenotypes than the previously characterized null allele from a Taichung 65 mutant library, d2-1. Linkage analysis and a complementation test clearly indicated that the mutant phenotypes in d2-6 were caused by defects in CYP90D2/D2, and exogenous treatment with brassinolide, a bioactive brassinosteroid, rescued the dwarf phenotype of three Nipponbare-derived d2 mutants. However, the content of endogenous bioactive brassinosteroid, castasterone, and the expression of brassinosteroid-response genes indicated that partial suppression of the brassinosteroid response in addition to a brassinosteroid deficiency has occurred in the Nipponbare-derived d2 mutants. Based on these results, we discuss the possibility that wild-type Nipponbare has some defects in an unknown factor or factors related to the brassinosteroid response in rice.
Flavin-Dependent Enzymes in Cancer Prevention
Danuta Wojcieszyńska,Katarzyna Hupert-Kocurek,Urszula Guzik
International Journal of Molecular Sciences , 2012, DOI: 10.3390/ijms131216751
Abstract: Statistical studies have demonstrated that various agents may reduce the risk of cancer’s development. One of them is activity of flavin-dependent enzymes such as flavin-containing monooxygenase (FMO) GS-OX1, FAD-dependent 5,10-methylenetetrahydrofolate reductase and flavin-dependent monoamine oxidase. In the last decade, many papers concerning their structure, reaction mechanism and role in the cancer prevention were published. In our work, we provide a more in-depth analysis of flavin-dependent enzymes and their contribution to the cancer prevention. We present the actual knowledge about the glucosinolate synthesized by flavin-containing monooxygenase (FMO) GS-OX1 and its role in cancer prevention, discuss the influence of mutations in FAD-dependent 5,10-methylenetetrahydrofolate reductase on the cancer risk, and describe FAD as an important cofactor for the demethylation of histons. We also present our views on the role of riboflavin supplements in the prevention against cancer.
Structural and Catalytic Differences between Two FADH2-Dependent Monooxygenases: 2,4,5-TCP 4-Monooxygenase (TftD) from Burkholderia cepacia AC1100 and 2,4,6-TCP 4-Monooxygenase (TcpA) from Cupriavidus necator JMP134
Robert P. Hayes,Brian N. Webb,Arun Kumar Subramanian,Mark Nissen,Andrew Popchock,Luying Xun,ChulHee Kang
International Journal of Molecular Sciences , 2012, DOI: 10.3390/ijms13089769
Abstract: 2,4,5-TCP 4-monooxygenase (TftD) and 2,4,6-TCP 4-monooxygenase (TcpA) have been discovered in the biodegradation of 2,4,5-trichlorophenol (2,4,5-TCP) and 2,4,6-trichlorophenol (2,4,6-TCP). TcpA and TftD belong to the reduced flavin adenine dinucleotide (FADH 2)-dependent monooxygenases and both use 2,4,6-TCP as a substrate; however, the two enzymes produce different end products. TftD catalyzes a typical monooxygenase reaction, while TcpA catalyzes a typical monooxygenase reaction followed by a hydrolytic dechlorination. We have previously reported the 3D structure of TftD and confirmed the catalytic residue, His289. Here we have determined the crystal structure of TcpA and investigated the apparent differences in specificity and catalysis between these two closely related monooxygenases through structural comparison. Our computational docking results suggest that Ala293 in TcpA (Ile292 in TftD) is possibly responsible for the differences in substrate specificity between the two monooxygenases. We have also identified that Arg101 in TcpA could provide inductive effects/charge stabilization during hydrolytic dechlorination. The collective information provides a fundamental understanding of the catalytic reaction mechanism and the parameters for substrate specificity. The information may provide guidance for designing bioremediation strategies for polychlorophenols, a major group of environmental pollutants.
Actividad enzimática de metanótrofos marinos y su uso potencial en biorremediación
Inostroza,Agar A; Pantoja,Silvio; González,Rodrigo R;
Gayana (Concepción) , 2011, DOI: 10.4067/S0717-65382011000200002
Abstract: methanotrophs are microorganisms with an important role in the biogeochemistry of methane in the ocean. the most important enzyme involved in methane catabolism is the soluble methane monooxygenase (smmo), being also active on polycyclic aromatic compounds and other oil-derived substances considered pollutants. sediment samples were incubated in a specific liquid nitrate mineral salt (nms) medium and in presence of methane as a unique source of carbon to determine smmo activity of the methanotrophs. samples were obtained from an area with a visible emanation of methane (mocha island) and two areas without emanation (coronel bay and gulf of arauco). the microorganisms present in the incubations of surface sediments from mocha island have a two-fold smmo activity than the activities obtained in cultures performed under the same conditions but using samples collected from areas without emanation. moreover, an opposite relationship between smmo activity and methane concentration was detected in the sediment column, suggesting a positive effect of methane emission on smmo activity of the methanotroph community of mocha island. considering an area of impact of methane emission in mocha island of 400 m2, the maximun potential smmo activity could daily transform ca. 43 kg of naphthalene, thus providing basis for the development of technologies for microbial detoxification of polycyclic aromatic molecules.
Properties of (μ-Oxo)di-iron complexes and catalytic activity toward cyclohexane oxidation
Parrilha, Gabrieli L.;Ferreira, Sarah S.;Fernandes, Christiane;Silva, Giselle C.;Carvalho, Nakédia M. F.;Antunes, O. A. C.;Drago, Valderes;Bortoluzzi, Adailton J.;Horn Jr., Adolfo;
Journal of the Brazilian Chemical Society , 2010, DOI: 10.1590/S0103-50532010000400004
Abstract: we report herein the synthesis and characterization of two dinuclear μ-oxo iron compounds obtained through the reactions of feso4?7h2 o and fecl3?6h2 o with 1-(bis-pyridin-2-ylmethyl-amino)-3-chloropropan-2-ol (l5), which resulted in the compounds [(so4)(l5)fe(μ-o)fe(l5)(so4 )]?6h2o, 1, and [cl(l5)fe(μ-o)fe(l5)cl]cl2? 2h2o, 2. the electronic spectra of both compounds show absorption bands only in the uv range. the electrochemical analysis showed that the dinuclear unit is more stable under reduction in compound 1 than in compound 2, while the m?ssbauer spectroscopy revealed that the monodentate ligands (sulfate and chloride) have a significant influence on the m?ssbauer parameters determined for 1 and 2, particularly on the quadrupole splitting values. both compounds were studied as catalysts in reactions of cyclohexane oxidation, using h2o2 and t-buooh as oxidants, in a substrate:oxidant:catalyst ratio of 1000:1000:1. cyclohexanol, cyclohexanone, cyclohexyl hydroperoxide, t-butyl cyclohexyl peroxide and adipic acid were formed during the process. the experiments revealed that compound 2 is, in general, more active than compound 1 in promoting cyclohexane oxidation.
MICAL, the Flavoenzyme Participating in Cytoskeleton Dynamics
Maria A. Vanoni,Teresa Vitali,Daniela Zucchini
International Journal of Molecular Sciences , 2013, DOI: 10.3390/ijms14046920
Abstract: MICAL (from the Molecule Interacting with CasL) indicates a family of recently discovered cytosolic, multidomain proteins, which uniquely couple an N-terminal FAD-containing monooxygenase-like domain to typical calponine homology, LIM and coiled-coil protein-interaction modules. Genetic and cell biology approaches have demonstrated an essential role of the catalytic activity of the monooxygenase-like domain in transducing the signal initiated by semaphorins interaction with their plexin receptors, which results in local actin cytoskeleton disassembly as part of fundamental processes that include differentiation, migration and cell-cell contacts in neuronal and non-neuronal cell types. This review focuses on the structure-function relations of the MICAL monooxygenase-like domain as they are emerging from the available in vitro studies on mouse, human and Drosophila MICAL forms that demonstrated a NADPH-dependent actin depolymerizing activity of MICAL. With Drosophila MICAL forms, actin depolymerization was demonstrated to be associated to conversion of Met44 to methionine sulfone through a postulated hydroxylating reaction. Arguments supporting the concept that MICAL effect on F-actin may be reversible will be discussed.
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