Quantitation and kinetic properties of hepatic microsomal and recombinant flavin-containing monooxygenases 3 and 5 from humans

doi:10.1016/S0009-2797(97)00055-0

Chemico-Biological Interactions

Volume 106, Issue 1, 29 August 1997, Pages 29-45

https://doi.org/10.1016/S0009-2797(97)00055-0 Get rights and content

Abstract

Variable amounts of flavin-containing monooxygenase isoforms 3 and 5 (FMO3 and FMO5) are present in microsomal preparations from adult, male, human liver. Quantitation with monospecific antibodies and recombinant isoforms as standards showed levels of FMO3 and of FMO5 that ranged from 12.5 to 117 and 3.5 to 34 pmol/mg microsomal protein, respectively. The concentration of FMO3 was greater than that of FMO5 in all samples, but the ratio of FMO3 to FMO5 varied from 2:1 to 10:1. Human hepatic microsomal samples also showed variable activities for the S-oxidation of methimazole. This activity was associated totally with FMO3; no participation of FMO5 was apparent. This conclusion was supported by several lines of evidence: first, the catalytic efficiency of FMO3 with methimazole was found to be ∼5000 times greater than that of FMO5; second, the rate of metabolism showed a direct, quantitative relationship with FMO3 content; third, the plot of the relationship between metabolism and FMO3 content extrapolated close to the origin. A second reaction, the N-oxidation of ranitidine, exhibited a much higher K_m with recombinant FMO3 than did methimazole (2 mM vs. 35 μM). However, a direct relationship between this reaction and FMO3 content in human hepatic microsomal preparations was also apparent. This result shows that even with a high K_m substrate, FMO3-catalyzed metabolism can account for the majority of the product formation with some drugs. Our findings demonstrate that the contribution of FMO isoforms to human hepatic drug metabolism can be assessed quantitatively on the basis of the characteristics of the enzymes expressed in Escherichia coli

Introduction

The flavin-containing monooxygenase (FMO) gene family contains five members that are expressed in a species- and tissue-dependant manner [1]. These enzymes catalyze the oxidative metabolism of a variety of nitrogen-, sulfur-, and phosphorous-containing compounds, some of which are of toxicological importance 2, 3. The involvement of FMO-mediated reactions in human drug metabolism has been associated with a number of substrates including dimethylaniline 4, 5, 6, tertiary amines 7, 8including imipramine [6]and chlorpromazine [9], thiobenzamide 5, 6, and tamoxifen [10].

The most thoroughly studied of the FMO isoforms are FMO1, the major form expressed in liver of adult pigs [1], and FMO2, the major form in lung of adult rabbit [11]. However, neither of these isoforms appears to be expressed to any significant extent in the adult human liver [12]. On the basis of catalytic, immunochemical and expression data, it has been suggested that FMO3 makes the greatest contribution to FMO-mediated drug metabolism in adult human liver 12, 13, 14, 15. FMO3 has been cloned from human [13]and rabbit [16]and purified from rabbit [17], and FMO3 orthologs from macaque [14]and rat [18]have been identified. Results with purified and recombinant FMO3 14, 19, 20and with human samples 21, 22have provided evidence that the unique stereoselectivity of human liver microsomes for the metabolism of prochiral FMO substrates is a function of FMO3. However, FMO3 has not been quantitated in human samples nor has its specific activity (catalytic constant) been determined.

In addition to FMO3, FMO5 has also been detected in human liver [23]. While the activity of FMO5 appears to be limited by low substrate affinity, its concentration in human liver and its ability to metabolize known FMO drug substrates have not been investigated. Transcript for FMO4 has been detected in samples from human liver [24], but no evidence for protein expression has been reported. Therefore, it appears that the major FMO enzymes in human liver are FMO3 and FMO5. We have now examined the kinetic properties of FMO3 and FMO5 expressed in Escherichia coli and have been able to associate these properties with FMO activities in human liver microsomal samples. Quantitation of FMO3 and FMO5 with monospecific antibodies has allowed for a direct relationship to be established between FMO3 concentration and FMO-mediated activity. In addition, the results show little relationship between the expression of FMO3 and FMO5 and no relationship between levels of mRNA and levels of enzyme.

Section snippets

Cloning and sequencing of human FMO3 and FMO5

Human FMO5 was cloned from a human hepatic library and sequenced as described by Overby et al. [23]. Several cDNA clones encoding human FMO3 were isolated from the same library by hybridization with a 620 bp 5′-fragment (EcoRI) of the cDNA for rabbit FMO3 [16]random labelled [25]with ³²PdCTP (Boehringer Mannheim, Indianapolis, IN). Plaque lifts were prehybridized and hybridized in 6×SSC, salmon sperm 100 mg/ml, 4×Denhardt, 0.5% SDS and 50% formamide. Subsequent to overnight hybridization at

Purification of recombinant rabbit FMO3 and FMO5 and preparation of monospecific antibodies

E. coli containing recombinant rabbit FMO3 or FMO5 was recovered from 1–2 l of medium, and the membrane fraction was prepared [30]. Purification of FMO3 and FMO5 from detergent-solubilized preparations was done by fractionation with polyethelene glycol and chromatography on DEAE cellulose as described by Overby et al. [23]. Antibodies were raised in goats and their specificity tested with recombinant FMO1, 2, 3, and 5. Antibodies to FMO5 (anti-5) did not cross-react with other FMO isoforms and

Expression of FMO3 and FMO5 in E coli

Particulate fractions (100 000 g) from E. coli transformed with FMO3, FMO5, and pJL alone were electrophoresed on polyacrylamide gels in the presence of SDS and stained for protein with Coomassie Blue. The protein band with the highest intensity of staining in the FMO3 or FMO5 samples was not present in the pJL sample (Fig. 1). The extent of expression of FMO3 or FMO5 was estimated by determination of flavin concentrations and was based on an assumption of 1 mole of FAD bound per mole of FMO. E

Discussion

Our results demonstrate that livers of adult, male humans contain highly variable amounts of FMO3 and FMO5. In five samples the total FMO3 plus FMO5 content ranged 10-fold, from a high of 150 to a low of 16 pmol/mg microsomal protein. FMO3 content was consistently greater than FMO5 content but the ratio of the two varied from 2:1 to 10:1. The variability in absolute levels of FMO3 we observed is somewhat greater than reported for relative levels of FMO3 determined immunochemically with an

References (46)

A. Lemoine et al.
Evidence for the presence of distinct flavin-containing monooxygenases in human tissues
Arch. Biochem. Biophys.
(1990)
A.J.M. Sadeque et al.
Purification of macaque liver flavin-containing monooxygenase: A form of the enzyme related immunochemically to an isozyme expressed selectively in adult human liver
Biochim. Biophys. Acta
(1993)
V.B. Burnett et al.
Cloning, sequencing, and characterization of flavin-containing monooxygenases FMO3 and FMO4 from rabbit
J. Biol. Chem.
(1994)
R.J. Krause et al.
Characterization of the methionine S-oxidase activity of rat liver and kidney microsomes: Immunochemical and kinetic evidence for FMO3 being the major catalyst
Arch. Biochem. Biophys.
(1996)
A.E. Rettie et al.
Prochiral sulfoxidation as a probe for multiple forms of the microsomal flavin-containing monooxygenase: Studies with rabbit FMO1, FMO2, FMO3 and FMO5 expressed in Escherichia coli
Arch. Biochem. Biophys.
(1994)
L.H. Overby et al.
Characterization of flavin-containing monooxygenase five (FMO5) from human and guinea pig: Evidence that the unique catalytic properties of FMO5 are not confined to the rabbit ortholog
Arch. Biochem. Biophys.
(1995)
A.P. Feinberg et al.
A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity
Anal. Biochem.
(1983)
K. Itagaki et al.
Expression and characterization of a modified flavin-containing monooxygenase 4 from humans
J. Biol. Chem.
(1996)
M.P. Lawton et al.
Functional characterization of flavin-containing monooxygenase 1B1 expressed in Saccharomyces cerevisiae and Escherichia coli and analysis of proposed FAD- and membrane-binding domains
J. Biol. Chem.
(1993)
A. Dixit et al.
Spectrophotometric assay of the flavin-containing monooxygenase and changes in its activity in female mouse liver with nutritional and diurnal conditions
Arch. Biochem. Biophys.
(1984)

J.M. Bailey et al.

Methylmercury as a reversible denaturing agent for agarose gel electrophoresis

Anal. Biochem.

(1976)

U.K. Laemmli et al.

Maturation of the head of bacteriophage T4 I. DNA packaging events

J. Mol. Biol.

(1973)

E.J. Faeder et al.

A rapid micromethod for determination of FMN and FAD mixtures

Anal. Biochem.

(1973)

O.H. Lowry et al.

Protein measurement with Folin phenol reagent

J. Biol. Chem.

(1951)

B.A. Domin et al.

Quantitation of rabbit cytochrome P-450, form 2, in microsomal preparations bound directly to nitrocellulose paper using a modified peroxidase-immunostaining procedure

Anal. Biochem.

(1984)

E. Atta-Asafo-Adjei et al.

Cloning, sequencing, distribution and expression of a mammalian flavin-containing monooxygenase from a third gene subfamily

J. Biol. Chem.

(1993)

D.M. Ziegler

Flavin-containing monooxygenases: Catalytic mechanism and substrate specificity

Drug Metab. Rev.

(1988)

D.M. Ziegler

Recent studies on the structure and function of multisubstrate flavin-containing monooxygenases

Annu. Rev. Pharmacol. Toxicol.

(1993)

M. Al-Waiz et al.

A genetic polymorphism of the N-oxidation of trimethylamine in humans

Clin. Pharmcol. Ther.

(1987)

M.S. Gold et al.

Dimethylaniline N-oxidase and aminopyrene N-demethylase activities of human liver tissue

Xenobiotica

(1973)

M.E. McManus et al.

Flavin-containing monooxygenase activity in human liver microsomes

Drug Metab. Dispos.

(1986)

P. Hlavica et al.

Some aspects of the role of cytochrome P-450 isozymes in the N-oxidative transformation of secondary and tertiary amine compounds

J. Biochem. Toxicol.

(1995)

R. Kuntzman et al.

N-oxide formation: a new route for inactivation of the antihistaminic chlorcyclizine

J. Pharmacol. Exp. Therap.

(1967)

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Quantitation and kinetic properties of hepatic microsomal and recombinant flavin-containing monooxygenases 3 and 5 from humans

Abstract

Introduction

Section snippets

Cloning and sequencing of human FMO3 and FMO5

Purification of recombinant rabbit FMO3 and FMO5 and preparation of monospecific antibodies

Expression of FMO3 and FMO5 in E coli

Discussion

Arch. Biochem. Biophys.

Biochim. Biophys. Acta

J. Biol. Chem.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Anal. Biochem.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

Anal. Biochem.

J. Mol. Biol.

Anal. Biochem.

J. Biol. Chem.

Anal. Biochem.

J. Biol. Chem.

Flavin-containing monooxygenases: Catalytic mechanism and substrate specificity

Drug Metab. Rev.

Recent studies on the structure and function of multisubstrate flavin-containing monooxygenases

Annu. Rev. Pharmacol. Toxicol.

A genetic polymorphism of the N-oxidation of trimethylamine in humans

Clin. Pharmcol. Ther.

Dimethylaniline N-oxidase and aminopyrene N-demethylase activities of human liver tissue

Xenobiotica

Flavin-containing monooxygenase activity in human liver microsomes

Drug Metab. Dispos.

Some aspects of the role of cytochrome P-450 isozymes in the N-oxidative transformation of secondary and tertiary amine compounds

J. Biochem. Toxicol.

N-oxide formation: a new route for inactivation of the antihistaminic chlorcyclizine

J. Pharmacol. Exp. Therap.