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1. Benzydamine is used clinically as a nonsteroidal anti-inflammatory drug in oral rinses and is employed in preclinical research as a flavin-containing monooxygenase (FMO) probe substrate. In this study, plasma concentrations of benzydamine and its primaryN-oxide andN-demethylated metabolites were investigated in control TK-NOG mice, in humanized-liver mice, and in mice whose liver cells had been ablated with ganciclovir. 2. Following oral administration of benzydamine (10 mg/kg) in humanized-liver TK-NOG mice, plasma concentrations of benzydamineN-oxide were slightly higher than those of demethyl benzydamine. In contrast, in control and ganciclovir-treated TK-NOG mice, concentrations of demethyl benzydamine were slightly higher than those of benzydamineN-oxide. 3. Simulations of human plasma concentrations of benzydamine and itsN-oxide were achieved using simplified physiologically based pharmacokinetic models based on data from control TK-NOG mice and from reported benzydamine concentrations after low-dose administration in humans. Estimated clearance rates based on data from humanized-liver and ganciclovir-treated TK-NOG mice were two orders magnitude high. 4. The pharmacokinetic profiles of benzydamine were different for control and humanized-liver TK-NOG mice. Humanized-liver mice are generally accepted human models; however, drug oxidation in mouse kidney might need to be considered when probe substrates undergo FMO-dependent drug oxidation in mouse liver and kidney. [ABSTRACT FROM PUBLISHER]

Flavin cofactors are widely used by enzymes to catalyze a broad range of chemical reactions. Traditionally, flavins in enzymes are regarded as redox centers, which enable enzymes to catalyze the oxidation or reduction of substrates. However, a new class of flavoenzyme has emerged over the past quarter century in which the flavin functions as a catalytic center in a non-redox reaction. Here we introduce the unifying concept of flavin hot spots to understand and categorize the mechanisms and reactivities of both traditional and noncanonical flavoenzymes. The major hot spots of reactivity include the N5, C4a, and C4O atoms of the isoalloxazine, and the 2′ hydroxyl of the ribityl chain. The role of hot spots in traditional flavoenzymes, such as monooxygenases, is briefly reviewed. A more detailed description of flavin hot spots in noncanonical flavoenzymes is provided, with a focus on UDP-galactopyranose mutase, where the N5 functions as a nucleophile that attacks the anomeric carbon atom of the substrate. Recent results from mechanistic enzymology, kinetic crystallography, and computational chemistry provide a complete picture of the chemical mechanism of UDP-galactopyranose mutase. [ABSTRACT FROM AUTHOR]

A click chemistry approach based on the reaction between alkynylflavins and mono(6-azido-6-deoxy)-β-cyclodextrin has proven to be a useful tool for the synthesis of flavin-cyclodextrin conjugates studied as monooxygenase mimics in enantioselective sulfoxidations. [ABSTRACT FROM AUTHOR]

Abstract: Pyrrolizidine alkaloids are secondary metabolites that are produced by certain plants as a chemical defense against herbivores. They represent a promising system to study the evolution of pathways in plant secondary metabolism. Recently, a specific gene of this pathway has been shown to have originated by duplication of a gene involved in primary metabolism followed by diversification and optimization for its specific function in the defense machinery of these plants. Furthermore, pyrrolizidine alkaloids are one of the best-studied examples of a plant defense system that has been recruited by several insect lineages for their own chemical defense. In each case, this recruitment requires sophisticated mechanisms of adaptations, e.g., efficient excretion, transport, suppression of toxification, or detoxification. In this review, we briefly summarize detoxification mechanism known for pyrrolizidine alkaloids and focus on pyrrolizidine alkaloid N-oxidation as one of the mechanisms allowing insects to accumulate the sequestered toxins in an inactivated protoxic form. Recent research into the evolution of pyrrolizidine alkaloid N-oxygenases of adapted arctiid moths (Lepidoptera) has shown that this enzyme originated by the duplication of a gene encoding a flavin-dependent monooxygenase of unknown function early in the arctiid lineage. The available data suggest several similarities in the molecular evolution of this adaptation strategy of insects to the mechanisms described previously for the evolution of the respective pathway in plants. [Copyright &y& Elsevier]

Abstract: A bacterial flavin-containing monooxygenase (bFMO) catalyses the oxygenation of indole to produce indigoid compounds. In the reductive half of the indole oxygenation reaction, NADPH acts as a reducing agent, and NADP+ remains at the active site, protecting bFMO from reoxidation. Here, the crystal structures of bFMO and bFMO in complex with NADP+, and a mutant bFMOY207S, which lacks indole oxygenation activity, with and without indole are reported. The crystal structures revealed overlapping binding sites for NADP+ and indole, suggestive of a double-displacement reaction mechanism for bFMO. In biochemical assays, indole inhibited NADPH oxidase activity, and NADPH in turn inhibited the binding of indole and decreased indoxyl production. Comparison of the structures of bFMO with and without bound NADP+ revealed that NADPH induces conformational changes in two active site motifs. One of the motifs contained Arg-229, which participates in interactions with the phosphate group of NADPH and appears be a determinant of the preferential binding of bFMO to NADPH rather than NADH. The second motif contained Tyr-207. The mutant bFMOY207S exhibited very little indoxyl producing activity; however, the NADPH oxidase activity of the mutant was higher than the wild-type enzyme. It suggests a role for Y207, in the protection of hydroperoxyFAD. We describe an indole oxygenation reaction mechanism for bFMO that involves a ping-pong-like interaction of NADPH and indole. [Copyright &y& Elsevier]

Abstract: The FMN-dependent two-component monooxygenase systems catalyze a diverse range of reactions. These two-component systems are composed of an FMN reductase enzyme and a monooxygenase enzyme that catalyze the oxidation of various substrates. The role of the reductase is to supply reduced flavin to the monooxygenase enzyme, while the monooxygenase enzyme utilizes the reduced flavin to activate molecular oxygen. Unlike flavoproteins with a tightly or covalently bound prosthetic group, these enzymes catalyze the reductive and oxidative half-reaction on two separate enzymes. An interesting feature of these enzymes is their ability to transfer reduced flavin from the reductase to the monooxygenase enzyme. This review covers the reported mechanistic and structural properties of these enzyme systems, and evaluates the mechanism of flavin transfer. [Copyright &y& Elsevier]

This work describes for the first time the identification of a reaction intermediate, C4a- hydroperoxyflavin, during the oxidative half-reaction of a flavoprotein oxidase, pyranose 2-oxidase (P2O) from Trametes multicolor, by using rapid kinetics. The reduced P2O reacted with oxygen with a forward rate constant of 5.8 × 104 M-1 s-1 and a reverse rate constant of 2 s-1, resulting in the formation of a C4a-hydroperoxyflavin intermediate which decayed with a rate constant of 18 s-1. The absorption spectrum of the intermediate resembled the spectra of flavin-dependent monooxygenases. A hydrophobic cavity formed at the re side of the flavin ring in the closed state structure of P2O may help in stabilizing the intermediate. [ABSTRACT FROM AUTHOR]

Abstract: Flavin-containing monooxygenases (FMOs) are microsomal enzymes that catalyze the NADPH-dependent oxidation of a large number of sulfur-, selenium-, and nitrogen-containing compounds. Five active isoforms (FMO1–5) have been identified and shown to be differently expressed in various mammalian tissues. Previous work from this laboratory has shown l-methionine to be S-oxidized by rat, rabbit and human FMO1–4, with FMO3 exhibiting the highest stereoselectivity for the formation of the d-diastereomer of methionine sulfoxide. In this report, we describe new studies aimed at determining if N-acetyl-l-methionine and peptides containing l-methionine can be substrates for FMOs. Experiments were carried out using either rabbit liver microsomes or human cDNA-expressed FMOs. The results show that while N-acetyl-l-methionine and peptides with a modified methionine amino group may not function as substrates for FMOs, peptides containing a free N-terminal methionine may act as FMO substrates. With human cDNA-expressed FMO1, FMO3, and FMO5, both FMO1 and FMO3 exhibited activity with the active peptides whereas FMO5 was inactive. With FMO3, the activity measured with methionine was similar (1 mM) or higher (5 mM) than the activity measured with H-Met-Val-OH and H-Met-Phe-OH. With FMO1, H-Met-Phe-OH and methionine exhibited similar activities whereas activity with H-Met-Val-OH was much lower. Collectively, the results show that FMOs can oxidize peptides containing a free N-terminal methionine. Thus, the role of FMOs in the oxidation of methionine in larger peptides or proteins warrants further investigation. [Copyright &y& Elsevier]

By cofactor redesign, self-sufficient monooxygenases could be prepared. Tight binding of N-alkylated flavins to riboflavin-binding protein results in the creation of artificial flavoenzymes capable of H2O2-driven enantioselective sulfoxidations. By altering the flavin structure, opposite enantioselectivities could be achieved, in accordance with the binding mode predicted by in silicoflavin-protein docking of the unnatural flavin cofactors. The study shows that cofactor redesign is a viable approach to create artificial flavoenzymes with unprecedented activities. [ABSTRACT FROM AUTHOR]

The article discusses published research that describes the crystal structure of the Baeyer-Villiger monooxygenase MtmOIV and the binding of flavin adenine dinucleotide (FAD) and premithramycin in the active site. This pertains to a reaction associated with streptomycetes, wherein an oxidation process initiates decarboxylation. Specifically discussed are the residues that allow the enzyme to accommodate the saccharide chains.

An abstract of the article "In Vitro Sulfoxidation of Phorate and Mithiocarb by Human P450 and FMO Isoforms" is presented. The study reveals that cytochrome P450 and flavin containing monooxygenases (FMO) are the primary enzyme systems responsible for catalyzing the oxidation of xenobiotics. It was found out that phorate metabolism to phorate sulfoxide and methiocarb metabolism to methiocarb sulfoxide by human liver were driven by P450 as compared to FMO.