DNA sequence encoding flavin-containing monooxygenase

Details DNA sequence encoding flavin-containing monooxygenase DNA sequence encoding flavin-containing monooxygenase

2000-05-30

2004-07-13

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S189000, C435S320100, C435S419000, C435S252300, C435S254110, C536S023200, C536S023500

Reexamination Certificate

active

06762052

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the drug screening, diagnostic, and synthesis uses of the first recombinant derived adult human liver flavin-containing monooxygenase (form 3), also referred to as adult human liver FMO (form 3) or HLFMO 3.
BACKGROUND AND INTRODUCTION TO THE INVENTION
The mammalian flavin-containing monooxygenase (FMO, EC 1.14.13.8, Dimethylaniline N-oxidase) is a widely distributed enzyme that catalyzes the NADPH-dependent oxygenation of a wide variety of nucleophilic nitrogen-, sulfur-, and phosphorous-containing drugs, chemicals, and xenobiotics (Cashman,
Chem. Res. Toxicol
. 8:165-181 (1995); Ziegler,
Enzymatic Basis of Detoxication
1:201-225 (1980); and Ziegler,
Drug Metab. Rev
. 6:1-32 (1988)). To dates, many of the investigations examining hepatic FMO have been performed with animal tissues, possibly because of the thermal instability of adult human liver FMO preparations. In contrast to adult human liver cytochromes P-450, almost nothing is known about the structure of adult human liver FMO. Adult human liver FMO has been designated FMO3 (Lawton et al.,
Arch. Biochem. Biophys
. 308: 254-257 (1994)). A few studies with adult human liver microsomes have demonstrated FMO-like enzyme activity (Gold et al.,
Xenobiotica
3:179-189 (1973) Lemoine et al.,
Arch. Biochem. Biophys
. 276:336-342 (1990); McManus et al.,
Drug Metab. Dispos
. 15:256-261 (1987)) and immunoreactivity with the antibody against pig liver FMO1 (Lemoine et al.,
Arch. Biochem. Biophys
. 276:336-342 (1990); Dannan et al.,
Mol. Pharmacol
. 22:787-794 (1982)). Dimethylaniline N-oxygenation was observed in adult (Gold et al.,
Xenobiotica
3:179-189 (1973)) and fetal (Rane,
Clin. Pharmacol. Ther
. 15:32-38 (1973)) human liver microsome preparations. In contrast to dimethylaniline N-oxygenation, which was observed in both kidney and liver tissues, imipramine N-oxygenation was only observed in microsome preparations from human kidney, but not from human liver (Lemoine et al.,
Arch. Biochem. Biophys
. 276:336-342 (1990)). The conclusion from these studies was that FMO was present in human liver tissue, albeit with low specific activity and possibly as multiple enzyme forms. This has been verified with the cloning of five forms of FMO cDNA from human liver cDNA libraries (Phillips et al.,
Chem. Biol. Interact
. 96:17-32 (1995)).
In animals, FMO has been reported to be present as at least one pulmonary form (Williams et al.,
Biochem. Biophys. Res. Commun
. 124:116-122 (1984); Tynes et al.,
Biochem. Biophys. Res. Commun
. 126:1069-1075 (1985)) and as two or more hepatic forms (e.g., forms 1 and 3) (Yamada et al.,
Arch. Biochem. Biophys
. 280:305-312 (1990); Ozols,
J. Biol. Chem
. 265:10289-10299 (1990)). It is more recently recognized that FMOs are present in multiple tissues and “hepatic” and “pulmonary” forms are misnomers. In rabbit liver, form 1 and 3 are only 55% identical to one another, but the amino acid sequence identity between hog liver FMO1 and rabbit liver FMO form 1 is approximately 87% (Ozols,
Arch. Biochem. Biophys
. 290:103-115 (1991)). Although studies are limited, forms 1 and 3 FMO apparently differ in many important properties including substrate specificity (Yamada et al.,
Arch. Biochem. Biophys
. 280:305-312 (1990)), enzyme stability (Ozols,
Arch. Biochem. Biophys
. 290:103-115 (1991)) and other physical properties.
For example, hepatic form 1 FMO activity is stimulated by primary aliphatic alkylamines and form 1 FMO catalyzes the N-oxygenation of secondary and tertiary amines (Ziegler, Enzymatic Basis of Detoxication 1, 201-225 (1980)). In contrast, form 3 FMO apparently N-oxygenates primary aliphatic alkylamines as well as secondary and tertiary amines (Yamada et al.,
Arch. Biochem. Biophys
. 280:305-312. (1990)). Aliphatic primary amines are sequentially N-oxygenated by FMO3 to hydroxylamine and oximes. The pharmacological activity of these metabolities are largely unknown but if FMO3 catalyzes efficient oxime formation from endogenous amines, this could be important in cellular homeostasis. Abnormal amine metabolism by FMO3 could be important in numerous disease states that are associated with abnormal amine metabolism. Some aliphatic tertiary amines such as chlorpromazine are preferentially N-oxygenated by form 3 FMO (Yamada et al.,
Arch. Biochem. Biophys
. 280:305-312 (1990)) but a detailed description of animal FMO3 activity has not been described.
FMO has been purified to homogeneity from a number of sources (Ziegler,
Drug Metab. Rev
. 19:1-32 (1988)) and it is the pig liver enzyme (FMO form I) which has been the subject of the most extensive studies. Using probes directed against the pig liver FMO and using a fetal human liver cDNA library, a cDNA encoding a FMO has been cloned (Dolphin et al.,
J. Biol. Chem
. 266:12379-12385 (1991)). Thus, fetal human liver flavin-containing monooxygenase (FMO) shares approximately 86% identity with pig liver FMO and 87% identity with rabbit liver FMO form I deduced from the cDNA data (ibid.). Fetal human liver FMO has been designated form 1. Substrate specificity differences are apparent for hepatic form 1 and 3 FMOs from in vitro animal liver enzyme studies (Yamada et al.,
Arch. Biochem. Biophys
. 280:305-312 (1990)), but almost nothing is known about the human liver enzymes.
A number of studies have shown that adult human liver microsomes are capable of tertiary amine N-oxygenation (Gold & Ziegler,
Xenobiotica
3:179-189 (1973); McManus et al.,
Drug Metab. Dispos
. 15:256-261 (1987); Lemoine et al.,
Arch. Biochem. Biophys
. 276:336-342 (1990); Rane,
Clin. Pharmacol. Ther
. 15:32-38 (1973)) and thiobenzamide S-oxygenation (McManus et al.,
Drug Metab. Dispos
. 15:256-261 (1987)).
Adult human liver FMO-dependent N- and S-oxygenation activity is quite thermally labile and activity is maximal at pH 8.4 (Gold and Ziegler, supra; McManus et al., supra; and Lemoine et al., supra, although considerable intersample variation has been observed. Most physical properties of animal FMOs are shared by human liver FMO forms although differences in substrate specificity have not been extensively examined. For example, human liver microsomes did not N-oxygenate imipramine even though imipramine was an excellent substrate for pig liver FMO form I (Lemoine et al., supra). Immunoquantitation of human liver FMO has relied on antibodies directed against animal FMOs. Thus, polyclonal antibodies prepared against pig liver FMO recognized a 60,000 Da human liver protein, although the immunoblot was characterized as very faint. Antisera raised against rat liver FMO recognized an adult human kidney protein, but did not recognize anything in the adult human liver (Lemoine et al., supra (1990)). This is another indication that multiple forms of FMO are present in the adult human liver and kidney.
For over 25 years, the literature has described a few people who, instead of N-oxygenating trimethylamine (TMA) to the polar, readily excreted trimethylamine N-oxide (TMANO), excreted large amounts of unmetabolized TMA in the urine and secreted the volatile and malodorous TMA in their breath, sweat and skin (Humbert, et al.,
Lancet
i:770-771 (1970); Higgins et al.,
Biochem. Med
. 6:392-396 (1972); Danks et al.,
N. Engl. J. Med
. 25:962 (1976)). TMA smells like the essence of rotting fish and people who suffer from this apparent metabolic disorder have what is referred to as the “fish-odor syndrome.” In humans, trimethylaminuria is an autosomal recessive disorder involving deficient N-oxygenation of TMA (Al-Waiz et al.,
Br. J. Clin. Pharmacol
. 25:664p-665p (1993); Ayesh, et al.,
Br. Med. J
., 655-657 (1993); Ayesh and Smith,
Pharmacol. Ther
. 45:387-401 (1990)). Normally, over 95% of a dose of TMA from dietary sources or otherwise is converted to TMANO that is excreted in the urine. The ability to N-oxygenate TMA is apparently distributed polymorphically (at least in some Caucasian populations evaluated thus far) and people with “fish-odor syndrome” are apparently homozygous for an allele that determines an indi

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