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Abstract: Enhanced detoxification is one of the important mechanisms for insecticide resistance. Most research in this field to date has focused on the role of cytochrome P450s. Our previous work revealed that flavin‐dependent monooxygenases (FMOs) were involved in metabolic resistance of Spodoptera exigua. In the present study we investigated the molecular characteristics, expression patterns and oxidative activities of SeFMO on insecticides. Three FMO genes, which encode proteins with the typical FMO motifs, were cloned from S. exigua. The oxidative activities of eukaryotically expressed SeFMO enzymes were verified with the model substrate of FMO. Importantly, the SeFMOs had significantly higher oxidative activities on metaflumizone and lambda‐cyhalothrin than on model substrates and other insecticides tested. The three SeFMOs were mainly expressed in the midgut, fat body and Malpighian tubules. The tissues responsible for xenobiotic metabolism and their expression characteristics were similar to those of P450s acting as detoxification genes. The study also revealed that the expression of SeFMOs could be induced by insecticide exposure, and that SeFMOs were over‐expressed in a metaflumizone‐resistant strain of S. exigua. These results suggest that SeFMOs are important insecticide detoxifying enzymes, and that over‐expression of FMO genes may be one of the mechanisms for metabolic resistance in S. exigua. [ABSTRACT FROM AUTHOR]

Abstract: Glucosinolates, a class of specialized metabolites specific to the order Brassicales, have diverse bioactivities that are largely dependent on the structures of their side chains. Flavin‐containing monooxygenases (FMOs) encoded by the FMOGS‐OX genes have been found to catalyze side‐chain modifications during the synthesis of methionine‐derived aliphatic glucosinolates. Seven FMOGS‐OX genes have been identified in Arabidopsis Heynh., but the evolution of these genes in the Brassicaceae, a family including many economically important vegetables, is poorly understood. In this study, the phylogenetic and syntenic relationships of the FMOGS‐OX genes belonging to 12 sequenced Brassicaceae species were analyzed. Our results showed that the FMOGS‐OX genes included two tandem arrays, the FMOGS‐OX2‐4 group (group A) and the FMOGS‐OX5‐7 group (group B). The evolutionary histories of the FMOGS‐OX groups A and B were similar across the Brassicaceae, but two lineage‐specific evolutionary routes developed after these two separate species lineages diverged from Aethionema arabicum (L.) Andrz. ex DC. In the lineage I route, FMOGS‐OX gene copies tended to increase due to frequent tandem duplication events in most species and a whole genome triplication in Camelina sativa (L.) Crantz. In the lineage II route, gene copies decreased due to deletion events. Combining these results with those of previous studies, we speculated that the FMOGS‐OX genes were derived from an ancestral gene with a broad expression distribution and a broad range of substrates, which then underwent subfunctionalization to generate progeny limited in either spatial expression or substrate structure. Furthermore, the absence of FMOGS‐OX5 substrates in some FMOGS‐OX5‐containing species may suggest neofunctionalization of these genes. [ABSTRACT FROM AUTHOR]

This paper describes the stabilization of flavin-dependent monooxygenases under reaction conditions, using an engineered formulation of additives (the natural cofactors NADPH and FAD, and superoxide dismutase and catalase as catalytic antioxidants). This way, a 103- to 104-fold increase of the half-life was reached without resource-intensive directed evolution or structure-dependent protein engineering methods. The stabilized enzymes are highly valued for their synthetic potential in biotechnology and medicinal chemistry (enantioselective sulfur, nitrogen and Baeyer-Villiger oxidations; oxidative human metabolism), but widespread application was so far hindered by their notorious fragility. Our technology immediately enables their use, does not require structural knowledge of the biocatalyst, and creates a strong basis for the targeted development of improved variants by mutagenesis. [ABSTRACT FROM AUTHOR]

A nicotinamide adenine dinucleotide (NADH)-dependent redox-neutral convergent cascade composed of a recently discovered type II flavin-containing monooxygenase (FMO−E) and horse liver alcohol dehydrogenase (HLADH) has been established. Two model reaction cascades were analyzed for the synthesis of γ-butyrolactone and chiral bicyclic lactones. In the former cascade, all substrates were converted into one single product γ-butyrolactone with high atom efficiency. More than 130 mM γ-butyrolactone were obtained when applying 100 mM cyclobutanone and 50 mM 1,4-butanediol in this cascade. In the second cascade where bicyclo[4.2.0]octan-7-one and cis-1,2-cyclohexanedimethanol were coupled, the ketone substrate was converted to the corresponding normal lactone with an ee value of 89-74% (3a S, 7a S) by FMO−E alone and the abnormal lactone with an ee value of >99% (3a R, 7a S) was formed by both HLADH and FMO−E. [ABSTRACT FROM AUTHOR]

Trimethylamine (TMA) and trimethylamine N-oxide (TMAO) are widespread in the ocean and are important nitrogen source for bacteria. TMA monooxygenase (Tmm), a bacterial flavin-containing monooxygenase (FMO), is found widespread in marine bacteria and is responsible for converting TMA to TMAO. However, the molecular mechanism of TMA oxygenation by Tmm has not been explained. Here, we determined the crystal structures of two reaction intermediates of a marine bacterial Tmm ( RnTmm) and elucidated the catalytic mechanism of TMA oxidation by RnTmm. The catalytic process of Tmm consists of a reductive half-reaction and an oxidative half-reaction. In the reductive half-reaction, FAD is reduced and a C4a-hydroperoxyflavin intermediate forms. In the oxidative half-reaction, this intermediate attracts TMA through electronic interactions. After TMA binding, NADP+ bends and interacts with D317, shutting off the entrance to create a protected micro-environment for catalysis and exposing C4a-hydroperoxyflavin to TMA for oxidation. Sequence analysis suggests that the proposed catalytic mechanism is common for bacterial Tmms. These findings reveal the catalytic process of TMA oxidation by marine bacterial Tmm and first show that NADP+ undergoes a conformational change in the oxidative half-reaction of FMOs. [ABSTRACT FROM AUTHOR]

Tuberculosis is a global health problem especially with the emergence of drug-resistant Mycobacterium tuberculosis strains, creating an urgent need to identify new drug targets. The mycobacterial cell wall is an attractive target for chemotherapeutic agents. Gene products of mymA operon are known to be required for the maintenance of cell wall and play an important role in persistence, thus making them important drug targets. This study was undertaken to biochemically characterize the MymA as a flavin-containing monooxygenase (FMO). Our results established its enzymatic activity in vitro and found that the mycobacterial FMO requires NADPH and FAD as cofactors, similar to other characterized bacterial FMOs. The enzyme follows Michaelis-Menten kinetics to catalyze substrates such as trimethylamine and thiourea. We also propose that MymA could be one of the targets of the antituberculosis drug, isoniazid (INH), which is a cell wall inhibitor. Molecular docking studies revealed that INH targeted NADPH-binding site of the MymA. Further, experimental validation revealed that INH inhibits MymA with the IC50 value of 4.9 μ m. Thus, this study characterizes for the first time that MymA is a mycobacterial FMO, which may be a target of INH. [ABSTRACT FROM AUTHOR]

Flavin‐containing monooxygenase 1 (FMO1) largely remains to be characterized in cynomolgus macaque kidney. Immunoblotting showed expression of cynomolgus FMO1 in kidneys where activities of FMO1 (benzydamine N‐oxygenation) were detected. No sex differences were observed in their contents or activities. These results suggest the functional role of cynomolgus FMO1 in kidney. [ABSTRACT FROM AUTHOR]

S-Alk(en)yl- l-cysteine sulfoxides are cysteine-derived secondary metabolites highly accumulated in the genus Allium. Despite pharmaceutical importance, the enzymes that contribute to the biosynthesis of S-alk-(en)yl- l-cysteine sulfoxides in Allium plants remain largely unknown. Here, we report the identification of a flavin-containing monooxygenase, As FMO1, in garlic ( Allium sativum), which is responsible for the S-oxygenation reaction in the biosynthesis of S-allyl- l-cysteine sulfoxide (alliin). Recombinant As FMO1 protein catalyzed the stereoselective S-oxygenation of S-allyl- l-cysteine to nearly exclusively yield ( RC SS)- S-allylcysteine sulfoxide, which has identical stereochemistry to the major natural form of alliin in garlic. The S-oxygenation reaction catalyzed by As FMO1 was dependent on the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and flavin adenine dinucleotide (FAD), consistent with other known flavin-containing monooxygenases. As FMO1 preferred S-allyl- l-cysteine to γ-glutamyl- S-allyl- l-cysteine as the S-oxygenation substrate, suggesting that in garlic, the S-oxygenation of alliin biosynthetic intermediates primarily occurs after deglutamylation. The transient expression of green fluorescent protein ( GFP) fusion proteins indicated that As FMO1 is localized in the cytosol. As FMO1 mRNA was accumulated in storage leaves of pre-emergent nearly sprouting bulbs, and in various tissues of sprouted bulbs with green foliage leaves. Taken together, our results suggest that As FMO1 functions as an S-allyl- l-cysteine S-oxygenase, and contributes to the production of alliin both through the conversion of stored γ-glutamyl- S-allyl- l-cysteine to alliin in storage leaves during sprouting and through the de novo biosynthesis of alliin in green foliage leaves. [ABSTRACT FROM AUTHOR]

Plants use various mechanisms to cope with environmental stresses, which often threaten genome integrity. In Arabidopsis, DNA double-strand breaks (DSBs) reduce root meristem size in a SOG1-dependent manner. SOG1 is a key transcription factor controlling the response to DNA damage. However, the underlying mechanism remains largely unknown. In this study, we found that treatment with the DSB inducer zeocin increased the accumulation of H2O2 in root tips. Chromatin immunoprecipitation analysis showed that SOG1 directly binds to the promoter of FMO1, which encodes a flavin-containing monooxygenase and is associated with the production of reactive oxygen species (ROS), H2O2 in particular. Indeed, zeocin induced the expression of FMO1 in a SOG1-dependent manner, and neither the sog1 nor the fmo1 knockout mutant exhibited higher H2O2 accumulation in root tips. Consequently, both sog1 and fmo1 could tolerate exposure to zeocin, in terms of root growth and the maintenance of the meristem size. However, transgenic plants over-expressing FMO1 also accumulated H2O2 in response to zeocin exposure, suggesting that other ROS-synthesis genes are also involved in the regulation of ROS production. We conclude that SOG1-mediated regulation of ROS homeostasis plays a key role in the reduction of root meristem size under DNA stress conditions. [ABSTRACT FROM AUTHOR]

Within a newly identified set of flavin-containing monooxygenases (FMOs) from Rhodococcus jostii RHA1, we have identified three monooxygenases (FMO-E, FMO-F, and FMO-G) that are effective in catalyzing Baeyer-Villiger oxidations. These type II FMOs display relaxed coenzyme specificity by accepting both NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) and NADH (reduced form of nicotinamide adenine dinucleotide), as a coenzyme, which is a novel and attractive feature among biocatalysts capable of conducting Baeyer-Villiger oxidations. We purified FMO-E and determined that the Michaelis constants for both coenzymes were in the micromolar range, whereas the activity was highest for NADH. By using the stopped-flow technique, formation of a peroxyflavin-enzyme intermediate was observed, which indicated that type II FMOs follow a catalytic mechanism similar to that of other class B flavoprotein monooxygenases. A set of cyclobutanones and cyclohexanones were used to probe the regio- and enantioselectivity of all three recombinant monooxygenases. The biocatalysts readily accepted small cyclic ketones, which enabled the conversion of previously poorly accepted substrates by other monooxygenases (especially norcamphor), and exhibited excellent and unique regio- and enantioselectivities. Sequence analysis revealed that type II FMOs that act as Baeyer-Villiger monooxygenases contain a unique N-terminal domain. Sequence conservation in this protein domain can be used to identify new NADH-dependent Baeyer-Villiger monooxygenases, which would facilitate future biocatalyst discovery efforts. [ABSTRACT FROM AUTHOR]

Auxin has been regarded as the main signal molecule coordinating the growth and ripening of fruits in strawberry, the reference genomic system for Rosaceae. The mechanisms regulating auxin biosynthesis in strawberry are largely elusive. Recently, we demonstrated that two YUCCA genes are involved in flower and fruit development in cultivated strawberry. Here, we show that the woodland strawberry ( Fragaria vesca L.) genome harbors nine loci for YUCCA genes and eight of them encode functional proteins. Transcription pattern in different plant organs was different for all eight FvYUCs. Functionality of the FvYUC6 gene was studied in transgenic strawberry overexpressing FvYUC6, which showed typical high-auxin phenotypes. Overexpression of FvYUC6 also delayed flowering and led to complete male sterility in F. vesca. Additionally, specific repression of FvYUC6 expression by RNA interference significantly inhibited vegetative growth and reduced plant fertility. The development of leaves, roots, flowers, and fruits was greatly affected in FvYUC6-repressed plants. Expression of a subset of auxin-responsive genes was well correlated with the changes of FvYUC6 transcript levels and free indole-3-acetic acid levels in transgenic strawberry. These observations are consistent with an important role of FvYUC6 in auxin synthesis, and support a main role of the gene product in vegetative and reproductive development in woodland strawberry. [ABSTRACT FROM AUTHOR]

Abstract: The flavin-dependent monooxygenase TetX confers resistance to all clinically relevant tetracyclines, including the recently approved, broad-spectrum antibiotic tigecycline (Tygacil®) which is a critical last-ditch defense against multidrug-resistant pathogens. TetX represents the first resistance mechanism against tigecycline, which circumvents both the tet-gene encoded resistances, relying on active efflux of tetracyclines, and ribosomal protection proteins. The alternative enzyme-based mechanism of TetX depends on regioselective hydroxylation of tetracycline antibiotics to 11a-hydroxy-tetracyclines. Here, we report the X-ray crystallographic structure determinations at 2.1Å resolution of native TetX from Bacteroides thetaiotaomicron and its complexes with tetracyclines. Our crystal structures explain the extremely versatile substrate diversity of the enzyme and provide a first step towards the rational design of novel tetracycline derivatives to counter TetX-based resistance prior to emerging clinical observations. [Copyright &y& Elsevier]

Flavin monooxygenases (FMOs) play critical roles in plant growth and development by synthesizing auxin and other signaling molecules. However, the structure and function relationship within plant FMOs is not understood. Here we defined the important residues and domains of the Arabidopsis YUC1 FMO, a key enzyme in auxin biosynthesis. We previously showed that simultaneous inactivation of YUC1 and its homologue YUC4 caused severe defects in vascular and floral development. We mutagenized the yuc4 mutant and screened for mutants with phenotypes similar to those of yuc1 yuc4 double mutants. Among the isolated mutants, five of them contained mutations in the YUC1 gene. Interestingly, the mutations identified in the new yuc1 alleles were concentrated in the two GXGXXG motifs that are highly conserved among the plant FMOs. One such motif presumably binds to flavin adenine dinucleotide (FAD) cofactor and the other binds to nicotinamide adenine dinucleotide phosphate (NADPH). We also identified the Ser139 to Phe conversion in yuc1, a mutation that is located between the two nucleotide‐binding sites. By analyzing a series of yuc1 mutants, we identified key residues and motifs essential for the functions of YUC1 FMO. [ABSTRACT FROM AUTHOR]

Capolongo, F., Santi, A., Anfossi, P., Montesissa, C. Benzydamine as a useful substrate of hepatic flavin-containing monooxygenase activity in veterinary species. J. vet. Pharmacol. Therap. doi: 10.1111/j.1365-2885.2009.01145.x. Benzydamine (BZ), a weak base and an indazole derivative with analgesic and antipyretic properties used in human and veterinary medicine, is metabolized in human, rat, cattle and rabbit to a wide range of metabolites. One of the main metabolites, BZ N-oxide (BZ-NO), is produced in the liver and brain by flavin-containing monooxigenases (FMOs), by liver and brain enzymes. To evaluate the suitability of BZ as an FMO probe in veterinary species, BZ metabolism was studied in vitro using liver microsomes from bovine, rabbit and swine. Kinetic parameters, Km and Vmax, of BZ-NO production, were evaluated to corroborate the pivotal role of FMOs. Inhibition studies were carried out by heat inactivation and by specific FMO chemical inhibitors: trimethylamine and methimazole. The results confirmed the presence of FMO activity in the liver and the role of BZ as a suitable marker of FMO enzyme activities for the veterinary species considered. [ABSTRACT FROM AUTHOR]

A rice mutant with rolling leaf, namely γ- rl, was obtained from M2 progenies of a native indica rice stable strain Qinghuazhan (QHZ) from mutagenesis of dry seeds by γ-rays. Genetic analysis using the F2 population from a cross between this mutant and QHZ indicated the mutation was controlled by a single recessive gene. In order to map the locus for this mutation, another F2 population with 601 rolling leaf plants was constructed from a cross between γ- rl and a japonica cultivar 02428. After primary mapping with SSR (simple sequence repeats) markers, the mutated locus was located at the short arm of chromosome 3, flanked by RM6829 and RM3126. A number of SSR, InDel (insertion/deletion) and SNP (single nucleotide polymorphism) markers within this region were further developed for fine mapping. Finally, two markers, SNP121679 and InDel422395, were identified to be flanked to this locus with genetic distances of 0.08 cM and 0.17 cM respectively, and two SNP markers, SNP75346 and SNP110263, were found to be co-segregated with this locus. These results suggested that this locus was distinguished from all loci for the rolling leaf mutation in rice reported so far, and thus renamed rl10(t). By searching the rice genome database with closely linked markers using BLAST programs, an e-physical map covering rl10(t) locus spanning about a 50 kb region was constructed. Expression analysis of the genes predicted in this region showed that a gene encoding putative flavin-containing monooxygenase (FMO) was silenced in γ- rl, thus this is the most likely candidate responsible for the rolling leaf mutation. [ABSTRACT FROM AUTHOR]

In this review, the major enzyme systems involved in vivo in the oxidative metabolism of xenobiotic amines in humans are discussed, i.e. the monooxygenases [cytochrome P450 system (CYPs) and flavin-containing monooxygenases (FMOs)] and the amine oxidases (AOs). Concerning the metabolism of xenobiotic amines (drugs in particular) by monoamine oxidases (MAOs), this aspect has been largely neglected in the past. An exception is the extensive investigation carried out on the inhibition of the metabolism of tyramine, when tyramine-containing food is ingested by subjects taking inhibitors of MAO A or of both MAO A and B. Moreover, investigations in humans on the metabolism of drug amines on the market by AOs, such as semicarbazide-sensitive amine oxidases (SSAOs) and polyamine oxidases (PAOs), are practically nonexistent, with the exception of amlodipine. In contrast to MAOs, monooxygenases (CYP isoenzymes more than FMOs) have been extensively investigated concerning their involvement in the metabolism of xenobiotics. It is possible that the contribution of AOs to the overall metabolism of xenobiotic amines in humans is underestimated or erroneously estimated, as most investigations of drug metabolism are performed using in vitro test systems optimized for CYP activity, such as liver microsomes, and most investigations of drug metabolism in vivo in humans carry out only the identification of the final, stable metabolites. However, for some drugs on the market, the involvement of MAOs in their in vivo metabolism in humans has been demonstrated recently, among these drugs citalopram, sertraline and the triptans are examples that can be mentioned. [ABSTRACT FROM AUTHOR]

The small component of 4-hydroxyphenylacelate 3-monoxygenase (HpaC) is an NADH oxidoreductase containing a flavin molecule as a cofactor. HpaC reduces a flavin molecule and reduced flavin is subsequently supplied to the large component of 4-hydroxyphenylacetate 3-monooxygenase (HpaB). The HpaC protein from Thermus thermophilus HB8 has been overexpressed in Escherichia coli and crystallized. During purification, the cluted HpaC protein solutions were separated into colourless and yellow-coloured fractions (i.e. apo-HpaC and HpaC-flavin complex, respectively). Crystals of apoHpaC grown in 5%(v/v) isopropyl alcohol. 0.1 M HEPES-NaOH pH 7.0. 40%(w/v) polyethylene glycol (PEG) 4000 and 10%(v/v) glycerol diffracted X-rays to a resolution of 1.85 Å. whereas crystals of the HpaC-flavin complex grown in 20%(w/v) PEG 1000, 10%(w/v) PEG 8000 and 10%(v/v) glycerol diffracted X-rays to a resolution of 1.3 Å. Both crystals belong to the monoclinic system, space group P2[SUB1], with similar unit-cell parameters. Selenomethionyl protein crystals of the HpaC-flavin complex grown under similar conditions to the native crystals diffracted X-rays to a resolution of 1.8 Å. They also belong to the monoclinic space group P2[SUB1]. but are not isomorphous to crystals of the HpaC-flavin complex of the native protein. MAD data for structure determination were successfully collected using these crystals. [ABSTRACT FROM AUTHOR]

Aims To determine the FMO and P450 isoform selectivity for metabolism of benzydamine and caffeine, two potential in vivo probes for human FMO. Methods Metabolic incubations were conducted at physiological pH using substrate concentrations of 0.01-10 mM with either recombinant human FMOs, P450s or human liver microsomes serving as the enzyme source. Products of caffeine and benzydamine metabolism were analysed by reversed-phase h.p.l.c, with u.v. and fluorescence detection. Results CYP1A2, but none of the human FMOs, catalysed metabolism of caffeine. In contrast, benzydamine was a substrate for human FMO1, FMO3, FMO4 and FMO5. Apparent K[sub m] values for benzydamine N-oxygenation were 60±8 μM, 80±8 μM, >3 mM and >2 mM, for FMO1, FMO3, FMO4 and FMO5, respectively. The corresponding V[sub max] values were 46±2 min[sup -1], 36±2 min[sup -1], -75 min[sup -1] and <1 min[sup minus;1]. Small quantities of benzydamine N-oxide were also formed by CYPs 1A1, 1A2, 2C19, 2D6 and 3A4. Conclusions FMO1 and FMO3 catalyse benzydamine N-oxygenation with the highest efficiency. However, it is likely that the metabolic capacity of hepatic FMO3 is a much greater contributor to plasma levels of the N-oxide metabolite in vivo than is extrahepatic FMO1. Therefore, benzydamine, but not caffeine, is a potential in vivo probe for human FMO3. [ABSTRACT FROM AUTHOR]

The side chain of Tyr222 in p-hydroxybenzoate hydroxylase interacts with the carboxy moiety of the substrate. Studies on the Tyr222→Phe mutant, [F222]p-hydroxybenzoate hydroxylase, have shown that disruption of this interaction hampers the hydroxylation of 4-hydroxybenzoate. Tyr222 is possibly involved in flavin motion, which may facilitate the exchange of substrate and product during catalysis. To elucidate the function of Tyr222 in more detail, in the present study the substrate and product during catalysis. To elucidate the function of Tyr222 in more detail, in the present study the substrate and effector specificity of the Tyr222→Ala mutant, [A222]p-hydroxybenzoate hydroxylase, was investigated. Replacement of Tyr222 by Ala impairs the binding of the physiological substrate 4-hydroxybenzoate and the substrate analog 4-aminobenzoate. With these compounds, hydroxylase tightly interacts with 2,4-dihydroxybenzoate and 2-hydroxy-4-aminobenzoate. Crystallographic data [Schreuder, H.A., Mattevi, A., Oblomova, g., Kalk, K. H., Hol, W.G.J., van der Bolt, F.J.T. & van Berkel, W.J.H. (1994) Biochemsitry 33, 10161-10170] suggest that this is due to motion of the flavin ring out of the active site, allowing hydrogen-bond interaction between the 2-hydroxy group of the substrate analogs and N3 of the flaving. [A222]p-Hydroxybenzoate hydroxylase produces about 0.6 mol 2,3,4-trihydroxybenzoate from 2,4-dihydroxybenzoate/mol NADPH oxidized. This indicates that reduction of the Tyr222→Ala mutant shifts the equilibrium of flavin conformers towards the productive 'in' position. [A222]p-Hydroxybenzoate hydroxylase converts 2-fluoro-4-hydroxybenzoate to 2-fluor-3,4-dihydroxybenzoate. The regioselectivity of hydroxylation suggests that [A222]p-hydroxybenzoate hydroxylase binds the fluorinated substrate in the same orientation as wild-type. Spectral studies suggest that wild-type and [A222]p-hydroxybenzoate hydroxylase bind 2-fluoro-4-hydroxybenzoate in the phenolate form with the flavin ring preferring the 'out' conformation. Despite activation of the fluorinated substrate and in contrast to the wild-type enzyme. [A222]p-hydroxybenzoate hydroxylase largely produces hydrogen peroxide. The effector specificity of p-hydroxybenzoate hydroxylase is not changed by the Tyr222→Ala replacement. This supports the idea that the effector specificity is mainly dictated by the protein-substrate interactions at the re-side of the flavin ring. [ABSTRACT FROM AUTHOR]

We have previously described the isolation and sequencing of cDNA clones encoding flavin-containing monooxygenases (FMOs) 1 and 4 of man [Dolphin, C., Shephard, E. A., Povey, S., Palmer, C. N. A., Ziegler, D. M., Ayesh, R., Smith, R. L. & Phillips, I. R. (1991) J. Biol. Chem. 266, 12379–12385; Dolphin, C., Shephard, E. A., Povey, S., Smith, R. L. & Phillips, I. R. (1992) Biochem. J. 287, 261–267]. We present heine the isolation of a cDNA for FMO3 of man. The sequence of this cDNA and the amino acid sequence deduced from it differ substantially from those previously reported for this member of the FMO family of man. In addition, we have investigated, by quantitative RNase protection assays, the expression in severed foetal and adult human tissues of genes encoding FMO1, FMO3 and FMO4. Our results demonstrate that, in the adult, FMO1 is expressed in kidney but not in liver, whereas in the foetus it is expressed in both organs. The lack of expression of FM01 in adult human liver is in marked contrast to the situation in other mammals, such as pig and rabbit, in which FMO1 constitutes a major form of the enzyme in the liver of the adult animal. The mRNA encoding FMO3 is abundant in adult liver and is also present, in low abundance, in some foetal tissues. Thus, FMO1 and FMO3 are both subject to developmental and tissue-specific regulation, with a developmental switch in the expression of the genes taking place in the liver. FMO4 mRNA is present in low abundance in several foetal and adult tissues and thus the corresponding gene appears to be expressed constitutively. [ABSTRACT FROM AUTHOR]

This study describes the regioselective hydroxylation and the rates of conversion of a series of fluorinated phenol derivatives by phenol hydroxylase from the yeast Trichosporon cutaneum. The natural logarithm of the kcat value for the conversion of the phenolic substrates correlates with the calculated energy of the reactive electrons in the highest occupied molecular orbital of the substrate (r = 0.85). This observation supports the hypothesis that at physiological pH (7.6) and 25°C, in the absence of monovalent anions, the nucleophilic attack of the electrons in the highest occupied molecular orbital of the substrate on the C(4a)-hydroperoxyflavin enzyme intermediate is of major importance in determining the overall rate of catalysis. Results from 19F-NMR analysis of the incubation mixtures demonstrate for phenols with two identical ortho substituents, that the ortho position which becomes preferentially hydroxylated is the one with the highest density of the reactive electrons in the highest occupied molecular orbital. A halogen substituent at a meta position decreases the chances for hydroxylation at the adjacent ortho position further khan expected on the basis of the calculated reactivity. This result indicates a contribution of a protein/substrate dipolar interaction, influencing the time-averaged orientation of the substrate with respect to the reactive C(4a)-hydroperoxyflavin intermediate. [ABSTRACT FROM AUTHOR]

A new variant of aerobic benzoate degradation has been found in a denitrifying bacterium in which benzoyl-CoA is the first intermediate [Altenschmidt, U., Oswald, B., Steiner, E., Herrmann, H. & Fuchs, G. (1993) New aerobic benzoate oxidation pathway via benzoyl-coenzyme A and 3-hydroxybenzoyl-coenzyme A in a denitrifying Pseudomonas sp, J. Bacteriol. 175, 4851–4858)]. The initial reaction is catalyzed by benzoate-CoA ligase (AMP-forming), converting benzoate into benzoyl-CoA. The next step is 3-hydroxylation of benzoyl-CoA to 3-hydroxybenzoyl-CoA catalyzed by a flavin-nucleotide-dependent monooxygenase, benzoyl-CoA 3-monooxygenase. This novel enzyme has been purified and studied. It is specific for NADPH and requires the presence of a flavin nucleotide for activity; both FAD or FMN function similarly well as cofactor. Only benzoyl-CoA, but not benzoate, is hydroxylated. The protein is a monomer of Mr 65000 and is induced when cells are grown aerobically with benzoate. 3-Hydroxybenzoyl-CoA is further hydroxylated para to the hydroxyl group affording 2,5-dihydroxybenzoate (gentisate). This reaction requires another monooxygenase, 3-hydroxybenzoyl-CoA 6-monooxygenase, which is unspecific with respect to the pyridine nucleotide. Cells contain a second 6-monooxygenase activity which acts on free 3-hydroxybenzoate. Based on these and other data, the outlines of the new aerobic benzoate pathway have been deduced. The proposed intermediates are benzoyl-CoA, 3-hydroxybenzoyl-CoA, gentisate, maleylpyruvate, fumarylpyruvate and fumarate plus pyruvate. [ABSTRACT FROM AUTHOR]

Analysis of the partially refined X-ray crystallographic structure of the active site of p-hydroxybenzoate hydroxylase shows clearly that the enzyme is complementary to a flavin C4,C4a-dioxetane derivative. A mechanism is proposed based on such an intermediate as the oxenoid transferring agent. A non-enzymatic analogue of this reaction is not known in organic chemistry. A possible evolutionary pathway for such a non-imitable enzyme is discussed. [ABSTRACT FROM AUTHOR]

Previous work identified two common ethnically-dependent allelic variants of human flavin-containing monooxygenase 2 (FMO2) that encode protein with monooxygenase activity that could occur in combination with the functional FM02*1 allele (Q472 protein); the encoded proteins were S195L and N413K. To understand how these variants alter enzyme activity, we performed assays with the FMO substrate, methyl p-tolyl sulfide (MTS) under a range of conditions with S195L, N413K and Q472 produced by over-expression in Sf9 insect cells. MTS sulfoxidation was assayed by HPLC. All protein variants demonstrated the same pattern of response to the presence of magnesium (increased activity) and to heat (decreased activity). Increased activity was observed with Q472 and N413K in response to increasing pH and to cholate, while S195L had diminished activity. The major effect of the N413K substitution was to increase velocity compared with Q472, while S195L had reduced activity and altered patterns of response to incubation conditions. In contrast to Q472 and N413K, S195L activity was negligible unless NADPH was added before MTS. Also, the K,, for S195L is at least 10-fold higher than for Q472 and N413K. Although these allelic variants are expected to occur infrequently as mutations to the FM02*1 allele, they contribute to our overall understanding of mammalian FMO structure and function. [ABSTRACT FROM AUTHOR]

Rebeccamycin, an antitumor compound, is synthesized by L. aerocolonigenes from the precursor L-tryptophan [Biosci. Biotechnol. Biochem. (2003) 67: 127-38]. One step in the biosynthetic pathway involves the net eight-electron oxidation of chlorinated chromopyrrolic acid to generate the rebeccamycin aglycone, which is carried out by the enzymatic pair, RebP and RebC [PNAS (2005) 102: 461-6]. Substitution of RebC with StaC -- a homologue with 65% identity to RebC -- results in the production of the staurosporine aglycone in lieu of the rebeccamycin aglycone [JACS (2006) 128: 12289-98]. While both RebC and StaC are annotated as flavin monooxygenases, only RebC is able to bind flavin adenine dinucleotide [JACS (2006) 128: 12289-98]. The structure of RebC bound to flavin adenine dinucleotide (FAD) was recently solved with 1.8A resolution using phases from a samarium derivative. The structure of RebC bears much resemblance to phenol hydroxylase, a flavin-dependent monooxygenase. This suggests that RebC is also a flavin-dependent monooxygenase, which might react with a yet-unidentified product produced by RebP. Residues in the structure near FAD are potentially responsible for the differences in FAD-binding and activity found between RebC and StaC. We are using the crystal structure as a guide to investigate the properties of various RebC mutants, in hope of determining the structural basis for enzyme specificity. [ABSTRACT FROM AUTHOR]

FAD-dependent monooxygenases are divided into three classes, flavin-containing monooxygenases (FMO), N-hydroxylating monooxygenase (NMO), and Baeyer-Villiger monooxygenase (BVMO). In prokaryotes, no FMO proteins have been characterized unlike other members of NMO or BVFO classes. A gene from Methlophaga sp. Strain SK1, a restricted facultative methylotroph, of which expression on E. coli produced blue dye indigo was cloned. The deduced amino acid sequence of the gene showed similarity to the mammalian FMOs that convert xenobiotics to more hydrophilic metabolites. This is the first member of the FMO family characterized in prokaryotes. A crystal of recombinant FMO containing hexaHis-tag was obtained under the condition of 1.5 M ammonium sulfate and 5% isopropanol, and the crystal belongs to space group C2. We determined the crystal structure of the FMO by single-wavelength anomalous dispersion at 2.4 Å resolution. FMO is composed of two structural domains, small and large domains. Both domains contain a parallel β-sheet flanked by an antiparallel β-sheet and α helices. The small domain is inserted in the parallel β-sheet of the large domain, which includes C-terminal helices. There is a channel between two domains and a cofactor FAD was found on depressed surface of large domain in the channel. It suggests that the channel is the active site of the FMO. [ABSTRACT FROM AUTHOR]