Molecular Cloning, Sequencing, and Expression inEscherichia coliof Mouse Flavin-Containing Monooxygenase 3 (FMO3): Comparison with the Human Isoform

Falls, J. Greg; Cherrington, Nathan J.; Clements, Kieran M.; Philpot, Richard M.; Levi, Patricia E.; Rose, Randy L.; Hodgson, Ernest

doi:10.1006/abbi.1997.0322

Cited by 30 publications

(27 citation statements)

References 45 publications

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“…Whereas human FMO3-mediated catalysis of clozapine N-oxidation was similar to values reported previously by Tugnait et al (1997) (data not shown), dFMO3-mediated N-oxidation did not display saturability (maximal observed activity of 1.5 nmol/min/nmol of enzyme). Comparisons of recombinant FMO3 from mouse and human have demonstrated relatively similar kinetics of methimazole S-oxidation, and methimazole S-oxidation by these two isoforms can be inhibited by a variety of known N-and S-containing substrates to a similar extent as well (Falls et al, 1997). In addition, Stevens et al (2003) have demonstrated that the K m values for N-oxidation of imipramine by dFMO1 (4.7 M) and hFMO1 (7.8 M) are also relatively similar.…”

Section: Expression and Characterization Of Dog Fmo3mentioning

confidence: 91%

Expression and Characterization of Functional Dog Flavin-Containing Monooxygenase 3

Lickteig

Riley²,

Melton³

et al. 2009

Drug Metab Dispos

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ABSTRACT:Mammalian flavin-containing monooxygenase (FMO) enzymes catalyze oxidation at nucleophilic, heteroatom centers and are important for drug, xenobiotic, and endogenous substrate metabolism. In human liver, human FMO3 (hFMO3) is the most abundant FMO isoform and is known to contribute to the hepatic clearance of a variety of clinical drugs. The purpose of the current study was to express and compare the dog (beagle) FMO3 (dFMO3) to hFMO3. A full-length dFMO3 cDNA was obtained from liver by reverse transcription-polymerase chain reaction. Using a baculovirus expression system in Spodoptera frugiperda insect cells, dFMO3 was expressed to protein levels of 0.50 nmol/mg, as determined by liquid chromatography-fluorescence detection. nmol/min/nmol of enzyme. dFMO3 also catalyzed sulindac sulfide S-oxidation with 6.8 nmol/min/nmol of enzyme being the highest velocity observed. Finally, Western blot analysis indicated protein expression levels of dFMO3 in pooled dog liver and lung microsomes to be 27 and 9 pmol/mg, respectively. In summary, dFMO3 appears to be a functional enzyme expressed at appreciable levels in liver, but one with some kinetic properties that are substantially different from its human homolog hFMO3.The flavin-containing monooxygenases (FMOs) are a family of enzymes capable of catalyzing the oxidation of various drugs, xenobiotics, and endogenous substrates containing a soft nucleophile, usually nitrogen or sulfur (Cashman, 2000;Krueger and Williams, 2005). In humans, FMO-dependent drug metabolism can have important clinical implications (Cashman, 2000). Like cytochrome P450s (P450s), the FMOs are microsomal enzymes that require NADPH and O 2 , and FMOs have shown overlapping substrate specificity with P450s. FMOs also typically convert their xenobiotic substrates into more polar products that are less pharmacologically active and more easily excreted, thereby enhancing their elimination from the body (Cashman, 1995). The mammalian FMO gene family includes five different isoforms (FMO1 through FMO5) (Lawton et al., 1994). In humans, as well as in a variety of preclinical species, tissue distribution patterns of FMO isoforms have been described previously (Cashman and Zhang, 2006;Phillips and Shephard, 2008). FMO3 is the most abundantly expressed isoform in adult human liver, existing at levels similar to the major human liver P450 isoform, CYP3A4 . FMO3 has been observed to contribute to the metabolic clearance of a variety of drugs, e.g., cimetidine, nicotine, and tamoxifen, as well as the diet-derived substrate trimethylamine (Cashman et al., 1992(Cashman et al., , 1993Mani et al., 1993). It has been demonstrated that FMO3 is essential for the N-oxygenation and metabolic clearance of trimethylamine (Dolphin et al., 1997;Lang et al., 1998). This finding led to the discovery that human FMO3 (hFMO3) is also a highly polymorphic gene (Koukouritaki et al., 2005). In general, a total of 29 allelic variants of FMO3 have each been observed to be associated with the human condition known as trimethyla...

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Section: Expression and Characterization Of Dog Fmo3mentioning

confidence: 91%

Expression and Characterization of Functional Dog Flavin-Containing Monooxygenase 3

Lickteig

Riley²,

Melton³

et al. 2009

Drug Metab Dispos

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“…1). Indeed, a histidine in this same position is present in mouse FMO3 (Falls et al, 1997). Moreover, it was reported that mouse FMO3 as well as other FMO3s from different species are able to metabolize methimazole and TMA (Falls et al, 1997 …”

mentioning

confidence: 94%

TWO NEW POLYMORPHISMS OF THE FMO3 GENE IN CAUCASIAN AND AFRICAN-AMERICAN POPULATIONS: COMPARATIVE GENETIC AND FUNCTIONAL STUDIES

Lattard¹,

Zhang²,

Tran³

et al. 2003

Drug Metab Dispos

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“…The expression of recombinant proteins has greatly aided the characterization of FMOs isoforms. So, the cDNAs of FMO1, FMO2, FMO3, and FMO5 isoforms of various animal species have been expressed successfully in several expression systems (Lawton et al, 1991Itoh et al, 1997) and high levels of expression have been observed in Escherichia coli (Atta-Asafo-Adjei et al, 1993;Lawton and Philpot, 1993;Lomri et al, 1993;Overby et al, 1995;Falls et al, 1997).…”

mentioning

confidence: 99%

Cloning, Sequencing and Tissue Distribution of Rat Flavin-Containing Monooxygenase 4: Two Different Forms Are Produced by Tissue-Specific Alternative Splicing

Lattard¹,

Longin-Sauvageon²,

Benoît³

2003

Mol Pharmacol

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The nucleotide sequence of rat flavin-containing monooxygenase 4 (FMO4) mRNA was obtained by reverse transcriptionpolymerase chain reaction (RT-PCR) and 5Ј/3Ј terminal extension. Complete cDNA was amplified, cloned, and sequenced from the mRNA obtained from rat kidney and brain. Two different transcripts (short and long) stemming from the splicing of an internal region of 189 bases pair, corresponding to exon 4 were identified. This alternative splicing seems to be specific of the brain. The long cDNA encodes a protein of 560 amino acids with a predicted molecular mass of 63,395 Da. The short cDNA encodes a protein of 497 amino acids with a predicted molecular mass of 55,871 Da. Both of these encoded sequences contain the NADPH-and FAD-binding sites and a hydrophilic carboxyl terminus. These sequences are 80 and 79% identical to the sequences of human and rabbit FMO4. By Northern blotting and/or RT-PCR, the long-form FMO4 mRNA was detected in the rat kidney, intestine, and liver and the short form particularly in the brain. For the first time, the expression of FMO4 protein was demonstrated. By Western blotting using the two different forms of FMO4 antibodies, a long FMO4 protein was detected in the rat kidney, whereas in the rat brain, only the short form of FMO4 was observed.

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Molecular Cloning, Sequencing, and Expression inEscherichia coliof Mouse Flavin-Containing Monooxygenase 3 (FMO3): Comparison with the Human Isoform

Cited by 30 publications

References 45 publications

Expression and Characterization of Functional Dog Flavin-Containing Monooxygenase 3

Expression and Characterization of Functional Dog Flavin-Containing Monooxygenase 3

TWO NEW POLYMORPHISMS OF THE FMO3 GENE IN CAUCASIAN AND AFRICAN-AMERICAN POPULATIONS: COMPARATIVE GENETIC AND FUNCTIONAL STUDIES

Cloning, Sequencing and Tissue Distribution of Rat Flavin-Containing Monooxygenase 4: Two Different Forms Are Produced by Tissue-Specific Alternative Splicing

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