DNA sequence encoding flavin-containing monooxygenase Number:6,762,052 from the United States Patent and Trademark Office (PTO) owispatent

Title: DNA sequence encoding flavin-containing monooxygenase

Abstract: Novel flavin-containing monooxygenase in substantially pure form is provided. Also disclosed are the cDNA and the reduced amino acid sequences, and fragments and derivatives thereof. The enzymes are useful in metabolism studies, in screening of compounds for biological or pharmacological activity, as well as serving as a bio-indicator of disease states or susceptibility to disease states.

Patent Number: 6,762,052 Issued on 07/13/2004 to Cashman,   et al.

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Inventors:	Cashman; John R. (Seattle, WA), Lomri; Noureddine (San Francisco, CA)
Appl. No.:	09/583,310
Filed:	May 30, 2000

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Current U.S. Class:	435/325 ; 435/189; 435/252.3; 435/254.11; 435/320.1; 435/419; 536/23.2; 536/23.5
Current International Class:	C12N 9/02 (20060101)
Field of Search:	435/189,320.1,325,419,252.3,254.11 536/23.2,23.5

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Other References

Sambrook et al. "Molecular Cloning, 2nd Edition", 1989, Cold Spring Harbor Laboratory Press, 17.10-17.27.* . Dolphin, "H. Sapiens mRNA for flavin-containing monooxygenase 3 (FMO3)," EMBL, Accession No. Z47552 (1995). . Lomri, et al., Proc. Natl. Acad. Sci. USA 92:9910 (1995). . Naeve, et al., "Accuracy of Automated DNA Sequencing: A Multi-Laboratory Comparison of Sequencing Results," BioTechniques 19:448-453 (1995)..

Primary Examiner: Prouty; Rebecca E.
Assistant Examiner: Steadman; David J.
Attorney, Agent or Firm: Townsend and Townsend and Crew LLP

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Government Interests

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GOVERNMENT SUPPORT

The U.S. government may have certain rights in the invention pursuant to grant GM 36426 received from the U.S. National Institutes of Health.

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Parent Case Text

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RELATED APPLICATIONS

This application is a continuation of and claims the benefit of U.S. patent application Ser. No. 08/617,671, filed Mar. 27, 1996, now abandoned, which is a continuation-in-part of U.S. patent application Ser. No. 08/023,843, filed Feb. 26, 1993, now abandoned, the disclosures of which are incorporated herein by reference in their entirety.

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Claims

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What is claimed is:

1. An isolated DNA sequence encoding an adult human liver flavin-containing monooxygenase (FMOS), wherein the DNA sequence is as depicted as SEQ ID NO: 5.

2. A procaryotic or eucaryotic host cell containing a DNA sequences according to claim 1 with a heterologous regulatory control sequence in an expression vector therefor.

3. An isolated DNA sequence encoding an adult human liver flavin-containing monooxygenase (FMOS), wherein the DNA sequence is as depicted as SEQ ID NO: 7.

4. A procaryotic or eucaryotic host cell containing a DNA sequence according to claim 3 with a heterologous regulatory control sequence in an expression vector therefor.

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Description

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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 individuals ability to carry out the N-oxygenation reaction (Ayesh et al., Br. J. Clin. Pharmacol. 25:664p-665p (1993). The molecular defect in trimethylaminuria has not yet been defined although it has been attributed to a deficiency in human FMO1 (Dolphin et al., J. Biol. Chem. 266:12379-12385 (1991); Dolphin et al., Biochem J. 287:261-267 (1992). It is now known, however, in contrast to what has been previously described (Dannan and Guengerich, Mol. Pharmacol. 22:787-794 (1982)), that human FMO1 is not expressed to a measurable extent in adult human liver and it is adult humans that have been associated with the disease. It is not likely that other monooxygenases form TMANO from TMA, based on existing studies (Gut and Conney, Drug Metab. Drug Interacts. 9:201-208 (1991) and the fact that TMA is a very good substrate for FMO from rat liver (Horori and Benoit, Biochem. Biophys. Res. Commun. 212:820-826 (1995). As described herein, cDNA-expressed human FMO3 is a good catalyst for the formation of TMANO from TMA in vitro. It is likely that human FMO3 is largely responsible for the N-oxygenation of TMA.

The deficiency of human FMO3 as an explanation for trimethylaminuria is probably more prevalent than previously realized (Treacy et al., J. Inher. Dis. 18:306-312 (1995) and the fish-odor syndrome is a major social handicap to patients who are usually anxious to obtain treatment. In addition to the psychosocial consequences of trimethylaminuria (i.e., anxiety, clinical depression, paranoia, suicidal personality and addiction to cigarettes, alcohol and drugs) on drug and endogenous amine metabolism as a result of altered or deficient human FMO3 activity (Chen and Aiello Am. J. Med. Genet. 45:335-339 (1993)), the possibly more important consequences of trimethylaminuria is that it may foretell about other more serious human diseases. For example if human FMO3 is involved in biogenic or other endogenous amine metabolism, a deficiency of human FMO3 may have profound consequences for a wide spectrum of diseases related to abnormal amine metabolism including cardiovascular disease and associated disorders, hypertension, and central nervous system diseases including but not limited to depression, stress, epilepsy, Huntington's, Parkinson's and Alzheimers disease and infectious diseases.

In adult human liver microsomes, in addition to FMO there are numerous other enzymes present including esterases and other monooxygenases. In some cases the other esterases and monooxygenases compete with the adult human liver FMO for substrate activity. For example, an esterase that converts a methyl ester to a carboxylic acid could make that compound unusable as a substrate for the FMO. Other monooxygenases present in the adult human liver could also compete with FMO for substrate activity. Microsomes also generate hydrogen peroxide and alkyl peroxide. Peroxide generated by microsomes could oxidize substrates of FMO such as sulfur, nitrogen, and phosphorous-containing chemicals. Therefore, the presence of such peroxides and other monooxygenases make it difficult to determine the true enzymatic activity and substrate specificity for adult human liver FMO. For this FMO to have any practical use in the research and industrial areas, it must be obtained in a form that is free of human liver microsomal monooxygenases and peroxides generated from these preparations.

SUMMARY OF THE INVENTION

The present invention provides adult human liver flavin-containing monooxygenase (form 3) in substantially pure form. The invention also includes mutants, variants, and fusion products of adult human liver flavin-containing monooxygenase (form 3). The invention also concerns a DNA sequence, and fragments and derivatives thereof, encoding adult human liver flavin-containing monooxygenase (form 3), and host cells involved in the expression of the adult human liver flavin-containing monooxygenase (form 3).

Another aspect of the invention includes methods for in vitro screening of compounds for biological or pharmacological activity. These methods include incubating a monooxygenase with the compound to detect the amount of oxygen consumed, the amount of NADPH consumed, or products formed.

A further aspect of the invention involves methods for detecting cancer in liver cells. This involves generating antibodies that react with the fetal human liver flavin-containing monooxygenase (form 1) and not FMO (form 3). Another method for detecting cancer in liver cells is the use of oligonucleotide probes that bind to the mRNA of the fetal human liver flavin-containing monooxygenase (form 1) and not FMO (form 3) gene. An additional method for detecting liver cancer uses the technique of PCR to amplify the cDNA of the fetal human liver flavin-containing monooxygenase (form 1) gene for hybridization with a probe.

Another aspect of the invention includes a method of selectively oxidizing a nucleophilic compound by incubating a monooxygenase with the nucleophilic compound.

An additional aspect of the invention involves a method of producing a nucleophilic compound with a center of chirality by incubating a monooxygenase with a substrate. This can be coupled in an asymmetric chemi-enzymatic synthesis of a chiral chemical or drug by reacting the resulting oxidized substrate with a strong base and an electiophilic compound.

Another aspect of the invention includes a method of assembling a native or active protein or peptide by incubating a monooxygenase capable of forming disulfides with an unfolded protein or peptide.

A further aspect of the invention involves a method of expressing a monooxygenase in a cDNA expression system to act as a catalyst for the renaturation of proteins or peptides by facilitating disulfide bond formation and protein folding.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the restriction endonuclease map and sequencing strategy of the HLFMO 3 cDNA insert;

FIG. 2 represents a Southern blot analysis of human genomic DNA using the cDNA fragment of HLFMO 3 as a probe,

FIG. 3 represents the schematic for the construction of the expression vector pTrcHLFMO 3;

FIG. 4 shows the SDS-PAGE analysis of the products from the expression vector pTrcHLFMO 3;

FIG. 5 represents the Western blot analysis of the products from the expression vector pTrcHLFMO 3;

FIG. 6A shows pH requirements for HFMO3-MBP activity and

FIG. 6B shows detergent requirements for HFMO3-MBP activity.

DETAILED DESCRIPTION OF THE SPECIFIC EMBODIMENTS

"Expression vectors" refer to vectors which are capable of replicating and transcribing DNA sequences contained therein, where such sequences are linked to other regulatory sequences capable of affecting their expression.

These expression vectors must be propagated in the host organisms or systems either as autonomous episomes or as an integral part of the chromosomal DNA. One form of expression vector which is suitable for use in the invention is the bacteriophage, a virus which replicates in bacteria. The .lambda.-gt11 phage is particularly desirable for this purpose. .lambda.-gt11 is a general recombinant DNA expression vector capable of producing polypeptides specified by the inserted DNA. To minimize degradation, upon induction with a synthetic analog of lactose (IPTG), foreign proteins or portions there of are synthesized as fused proteins with the prokaryotic protein .beta.-galactosidase. The use of host cells defective in protein degradation pathways may also increase the longevity of novel proteins produced from the induced .lambda.-gt11 clones. Proper expression of foreign DNA in .lambda.-gt11 clones will depend upon the proper orientation and reading frame of the inserted DNA with respect to the .beta.-galactosidase gene. Another form of expression vector used in recombinant DNA techniques is the prokaryotic plasmid: an unintegrated (extrachromosomal), double-stranded DNA circle. A third class of expression vectors are the eukaryotic vectors: vectors capable of driving expression of the foreign DNA in a eukaryotic cell. These are generally derived from viral sources and may be either extrachromosomal or integrated. The invention includes any other form of expression vector which serves an equivalent function and which is or subsequently becomes known in the art. Recombinant vectors and methodology disclosed herein are suitable for use in a wide range of prokaryotic and eukaryotic host cells. These host cells include microbial strains, such as E. coli INVlalphaF', Saccharomyces cerevisiae, and cell lines derived from multicellular eukaryotic organisms.

From two adult human cDNA libraries, 2.times.10.sup.6 phage plaques were screened with a mixture of three 36-mer synthetic oligonucleotide probes derived from the cDNA sequences of pig liver FMO (Gasser et al., Biochemistry 29:119-124 (1990)).

A total of 5 clones were isolated and purified to homogeneity and mapped by restriction endonuclease digestion. The largest clone which contained approximately 2200 bp (FIG. 1), was subcloned into Bluescript vectors (KS.sup.+- and SK.sup.+-) and both strands were entirely sequenced by the dideoxy method (Sanger et al., Proc. Natl. Acad. Sci. USA 74:5453-5467 (1977)).

Restriction endonuclease map and sequencing strategy of the HLFMO 3 cDNA insert is depicted in FIG. 1. Only the restriction sites used for subcloning are shown. The 1599 nucleotide open reading frame (ORF) is indicated in the box. The 5' and 3' ends of the HLFMO 3 cDNA are shown. The sequencing strategy is indicated by horizontal arrows. The cDNA fragments were subcloned in Bluescript (KS.sup.+-, SK.sup.+-) and ordered deletions were produced using Exonuclease III/mung bean nuclease. The deleted and the restriction endonuclease-generated fragments were sequenced by the dideoxy termination method (Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977)). The letter B indicates the cDNA fragment used as a probe for hybridization analyses (FIG. 2).

The complete nucleotide sequence of the HLFMO 3 cDNA was determined. It contained a 5'-untranslated region of 136 bp, followed by an open reading frame (ORF) of 1599 bp, encoding a protein of Mr 159,179, a termination codon and a 3'-untranslated region of 384 bp. The sequence ACCATGG (SEQ ID No. 9; (bp 134-140)) contained the initiating codon ATG and corresponded to the sequence found to be optimal for initiation of transcription by eukaryotic ribosomes (Kozak, Cell 44, 283-292 (1986)).

Two consensus polyadenylation signals were also found: ATTAA (SEQ ID No. 10) (bp 1805)) and AATAA (SEQ ID No. 11); (bp 2083)), which were situated 15 nucleotides upstream from the poly(A) tail (Breathnach et al., Ann. Rev. Biochem. 50:349-383 (1981)).

The amino acid sequence of adult human liver FMO (form 3) deduced from the cDNA clone is shown in SEQ ID No. 8. Comparison of HLFMO 3 with human liver FMO form 1 (HLFMO 1) (Dolphin et al., J. Biol. Chem. 266:12379-12385 (1991)), pig liver FMO form 1 (PLFMO) (Gasser et al., Biochemistry 29:119-124 (1990)), rabbit liver FMO form 1 (RLFMO I) (Lawton et al., J. Biol. Chem. 265:5855-5861 (1990)) and rabbit lung FMO (RLuFMO) (Lawton et al., J. Biol. Chem. 265:5855-5861 (1990)) showed only a modest degree of primary sequence identity (e.g., 53-57%). HLFMO 3 contained a putative FAD binding domain at amino acid residues 9-14 (e.g., GAGVSG (SEQ ID No. 13)) and a putative NADP.sup.+ binding domain at residues 191-196 (e.g., GLGNSG SEQ ID No. 14)). These cofactor-binding regions were highly conserved among all of the mammalian FMO enzymes known as well as the FMO bacterial equivalent, cyclohexanone monooxygenase (Chen et al., J. Bacteriol. 179:781-789 (1988)). In contrast to other mammalian hepatic FMO forms, HLFMO 3 has only a single putative consensus N-glycosylation site (Asn-Xxx-Ser/Thr) at residues 61-63. It was notable that HLFMO 3 did not contain the putative N-glycosylation sites at residues 120-123 and 315-317 that were present in form 1 FMOs.

Adult human liver mRNA was analyzed using the cDNA clone shown in FIG. 1. This cDNA was radiolabeled and the .sup.32 P probe was hybridized to poly(A).sup.+ RNA. The radiolabeled probe detected one mRNA species (2300 bp) in human liver. Genomic DNA extracted from adult human liver was treated with restriction EcoRI (E), PstI (P), BamHI (B) and XhoI (X) (FIG. 2). The samples were fractionated on a 0.7% agarose gel, transferred to a nylon membrane and probed with HLFMO 3 EcoRI B fragment (FIG. 1). As shown in FIG. 2, this probe hybridized to a single band with each sample. The apparent sizes of these bands ranged from 800 bases (EcoRI) to 4000 bases (PstI, BamHI and XhoI).

Currently, evidence for five forms of FMO exist that have deduced amino acid sequences ranging between 52% and 57% identity across species lines (Lawton et al., Arch. Biochem. Biophys., 308:254-257 (1994); Hines et al., Toxicol. Appl. Pharmacol., 125:1-6 (1994)). Thus far, approximately twelve full length sequences of FMO have been reported in the literature of in GENBANK. In addition to the five distinct FMO's (i.e., FMO1, FMO2, FMO3, FMO4 and FMO5), several other published sequences represent orthologs from other species as well as allelic variants.

The screening of animal and human cDNA libraries with other cDNAs or synthetic oligonucleotides encoding FMO have provided cDNA inserts of 2.2-2.6 kb in length or smaller. Thus far, the FMO cDNAs reported in the literature encode for enzymes of approximately 533-535 amino acids but examples of FMOs with nineteen (Atta-Asafo-Adjei et al., J. Biol. Chem., 268:9681-9689 (1993)) or twenty-five (Dolphin et al., Biochem. J. 287: 261-267 (1992)) additional C-terminal amino acids have been observed. In addition, minor FMO structural variants have been observed in rabbit liver but the role of the fill variants in rabbit physiology in not known. It is possible that mutations in conserved regions of the five FMOs could lead to enzymes with significantly decreased catalytic or physical properties. To be considered a member of the family of mammalian FMOs, approximately 40% or more amino acid sequences are required to be identical. Related non-mammalian flavoenzymes do not belong to the family of flavoprotein monooxygenases because, for example in the case of cyclohexanone monooxygenase, it is only 25% identical to amino acid sequences of mammalian FMOS. For a particular, FMO to belong to a specific subfamily, the requirement is for the ortholog to have 80% or greater amino acid sequence identity. However, even under optimal conditions, low stringency screening of an adult human liver cDNA library with pig FMO1 did not identify cDNA clones encoding human FMO3. In fact, the screen identified several weakly hybridizing clones, all of which turned out to encode human FMO4 (Phillips et al., Chem.-Biol. Interacts. 96:17-32 (1995)). This result is surprising in view of the fact that the human FMO3 is now recognized as the major form of FMO in adult human liver. Clearly, the high stringency procedure employed previously and the quality of the adult human liver library led to the difference in the success of isolation of the adult human FMO3 clones (Lomri et al., Proc. Natl. Acad Sci. USA 89:1685-1689 (1992)). The hybridization conditions described in the isolation of human FMO3 clones will selectively hybridize with orthologs only about 95% or greater identity. An analysis of the hybridization melting temperatures of the oligonucleotides used (i.e., oligos PLFMO1 and PLFMO1, see below) employing the following equation: T.sub.m =[.DELTA.H/.DELTA.S+Rln(C.sub.t)]-273.15 showed that the Tm for adult human liver FMO3 was significantly higher that the Tm for the other human liver FMOs now known. Hybridization is detected or it is not (Benton and Davis, Science 916:180-182 (1987)). Under the conditions of the salts, temperature-and detergent only the adult human FMO3 was selectively hybridized under the stringent conditions employed.

Some FMO regions are substantially homologous even between families. For example, residues near the N-terminus and between residue 450 and the C-terminus of FMO contain relatively highly conserved regions indicating important amino acid structural and/or functional domains. Creating variants or exchanging one region of FMO with another to maintain over 95% identity can introduce improved catalytic or deisirable physical properties into the newly engineered FMO. On the other hand, some regions of FMO are important to enzyme function such that removal or substitution with unacceptable amino acids may significantly decrease catalytic activity or some other physical property. This is borne out by the highly conserved FAD- and NADP.sup.+ -binding domains (i.e., GXGXXG) near deduced amino acid positions 9-14 and between 186-196, respectively. Intact NADP.sup.+ and FAD cofactor binding domains are essential for FMO function because site-directed mutagenesis of the region 9-14, to GXGXXV, resulted in expression of an inactive protein to which no FAD was bound (Lawton and Philpot, J.Biol. Chem. 268:5728-5734 (1993)). Conservative amino acid changes on the N-terminal side of the FAD-binding domain of FMO resulted in an FMO that retained activity. Fusing a 35 amino acid portion of .beta.-galactosidase to the N-terminus of pig FMO1 significantly enhanced the level of expression in E. coli while not noticably affecting the specific activity of the enzyme. Other FMO fusion proteins are also very active. For example, the maltose binding protein fusion protein to FMO3 possesses excellent catalytic and physical properties. The N-terminal peptides of FMOs examined to date do not have any clearly discernible signal peptide sequences and, coupled with the presence of an FAD-binding region, does not suggest this region as a functional membrane insertion domain. To date, most of the membrane insertion correlations have focused on highly lipophilic amino acid sequences.

To define the interaction of FMO in the membrane, it was reasoned that the core glycosylation enzyme system required for N-glycosylation was localized to the iuminal side of the endoplasmic reticulum membrane. As most FMOs have a putative consensus N-glycosylation site, determination of the site of N-glycosylation would indicate the region of FMO that was translocated across the endoplasmic membrane. With the exception of FMO4, all FMOs sequenced to date have at least a single putative consensus N-glycosylation site (i.e., Asn-Xaa-Ser/Thr). Based on indirect evidence, it has been reported that pig FMO1 is glycosylated (Korsmeyer et al., Flavins and Flavoproteins (Curti, Ronchi and Zanetti, eds.) Walter de Gruyter and Co., Berlin, pp 243-246 (1991)) and subsequent HPLC-mass spectrometry studies showed that a saccharide was attached to Asn 120. The highly conserved nature of the putative consensus N-glycosylation sites indicates that the region has an important structural and/or functional role in enzyme action. Analysis of the distance between the N-glycosylation site and transmembrane hydrophobic segments indicates that the N-glycosylation site of FMO is separated from the membrane by at least 13 amino acid residues. Membrane association likely occurs between amino acid residues Cys 30 and Leu 90, because the NADP.sup.+ - and FAD-binding domains must be on the same side of the membrane to affect catalysis. A variant of FMO in this region that replaces the membrane association sequence with compatible amino acids can be used to provide a more water-soluble and tractable protein that possesses more desirable catalytic and physical properties. All mammalian FMOs possess very strong membrane association properties and in the highly purified state are extremely intractable proteins with poor solubility characteristics (Guan et al., Biochemistry 30:9892-9900 (1991)). For example, even with as many as 200 C-terminal amino acids deleted from rabbit FMO2, membrane association was still observed. Comparison of the amino acids of the 5 forms of FMOs (deduced from cDNA data) by hydropathy profiles shows that many regions of FMO are highly hydrophobic as well as highly conserved. This is true even for regions of the FMO isoform sequence that are only modestly identical (i.e., 25-30% amino acid identity). FMO membrane association is not a passive event dictated exclusively by hydrophobic C-terminal amino acid residues. Rather, the information for active FMO membrane association is believed to be encoded in an internal sequence proximal to the N-terminus that signals membrane association. Modification of the membrane insertion sequence or the substrate binding channel by discrete or random mutagenesis or by discovering naturally occurring variants that heave different amino acids in these regions can also provide FMO enzymes with improved catalytic or physical properties.

The cDNA sequence does not provide information about possible N-terminal modifications and in each case examined (i.e., FMO1, FMO2 and FMO3) the initiation amino acid methionine is not present and the following amino acid is N-acetylated. The results have primarily come from mass spectral studies of the purified proteins. Residues such as alanine or glycine near the N-terminus apparently promote the removal of methionine during FMO protein maturation (Flinta et al., Eur. J. Biochem. 154:193-196 (1986)).

In certain instances, one may employ changes in the sequence of recombinant FMO3 and allelic variants to substantially increase or even decrease the biological activity of FMO3, depending on the intended use of the preparation. The biological activity may be determined as demonstrated herein. Homologous sequences, allelic variations, and natural mutants; induced point, deletion, and insertion mutants; alternatively expressed variants; proteins encoded by DNA which hybridize to nucleic acids which encode naturally occurring FMO3 are included herein.

The disclosed sequences of FMO3 are used to identify and isolate FMO polynucleotide molecules from suitable hosts such as canine, ovine, bovine, caprine, lagomorph, avian or the like. Complementary DNA molecules encoding FMO3 may be obtained by constructing a cDNA library mRNA from, for example, liver. DNA molecules encoding FMO3 may be isolated from such a library using the disclosed sequences in standard hybridization techniques (e.g., Sambrook et al. ibid., and Bothwell, Yancopoulos and Alt, eds, Methods for Cloning and Analysis of Sukarvotic Genes, Jones and Bartlett Publishers, Boston, Mass. 1990) or by amplification of sequences using polymerase chain reaction (PCR) amplification (e.g, Loh et al. Science 243: 217-222, 1989; Frohman et al., Proc. Acad. Sci. USA 85: 8998-9002, 1968; and Erlich (ed.), PCR Technology: Principles and Applications for DNA Amplification, Stockton Press, 1989; and U.S. Pat. No. 4,683,195, which are incorporated by reference herein in their entirety). In a similar manner, genomic DNA encoding FMO3 is obtained using probes designed from the sequences disclosed herein. Suitable probes for use in identifying FMO 3 sequences are obtained from FMO3-specific sequences, such as those, e.g., that are highly conserved regions. Suitable PCR primers are between 7-50 nucleotides in length, more preferably between 15, sometimes 18-20 and 25 nucleotides in length. Alternatively, FMO3 polynucleotide molecules may be isolated using standard hybridization using probes of at least about 7 nucleotides in length and up to and including the full coding sequence.

The choice of hybridization conditions will generally be guided by the purpose of the hybridization, the type of hybridization (DNA-DNA or DNA-RNA), and the, level of relatedness between the sequences. Methods for hybridization are well established in the literature; See, for example: Sambrook, ibid.; Hames and Higgins, eds, Nucleic Acid Hybridization A Practical Approach, IRL Press, Washington D.C., 1985; Berger and Kimmel, eds, Methods in Enzymology, Vol. 52, Guide to Molecular Cloning Techniques, Academic Press Inc., New York, N.Y., 1987; and Bothwell, Yancopoulos and Alt, eds, Methods for Cloning and Analysis of Eukaryotic Genes, Jones and Bartlett Publishers, Boston, Mass. 1990; which are incorporated by reference herein in their entirety. The stability of nucleic acid duplexes will decrease with an increased number and location of mismatched bases; thus, the stringency of hybridization may be used to maximize or minimize the stability of such duplexes. Hybridization stringency can be altered by: adjusting the temperature of hybridization; adjusting the percentage of helix-destabilizing agents, such as formamide, in the hybridization mix; and adjusting the temperature and salt concentration of the wash solutions. In general, the stringency of hybridization is adjusted during the post-hybridization washes by varying the salt concentration and/or the temperature. Stringency of hybridization may be reduced by reducing the percentage of formamide in the hybridization solution or by decreasing the temperature of the wash solution. High stringency, conditions may involve high temperature hybridization (e.g., 65-68.degree. C. in aqueous solution containing 4-6.times.SSC, or 42.degree. C. in 50% formamide) combined with washes at high temperature (e.g., 5-25.degree. C. below the T.sub.m) at a low salt concentration (e.g., 0.1.times.SSC). Reduced stringency conditions may involve lower hybridization temperatures (e.g., 35-42.degree. C. in 20-50% formamide) with washes at intermediate temperature (e.g., 40-60.degree. C.) and in a higher salt concentration (e.g., 2-6.times.SSC). Moderate stringency conditions may involve hybridization at a temperature between 50.degree. C. and 55.degree. C. and washes in 0.1.times.SSC, 0.1% SDS at between 50.degree. C. and 55.degree. C.

The invention provides isolated and purified polynucleotide molecules encoding FMO3 capable of hybridizing under stringent conditions to an oligonucleotide of 15 or more contiguous nucleotides of SEQ ID NO: 7 or SEQ ID NO: 9 and their complementary strands. The isolated FMO3 polynucleotide molecules preferably encode FMO3 proteins or fragments thereof that have enzymatic activity.

The present invention provides methods for producing recombinant FMO3 by inserting a DNA molecule encoding FMO3 into a suitable expression vector, which is in turn used to transfect or transform a suitable host cell.

Suitable expression vectors for use in carrying out the present invention will generally comprise a promoter capable of directing the transcription of a polynucleotide molecule of interest in a host cell. Representative expression vectors may include both plasmid and/or viral vector sequences. Suitable vectors include retroviral vectors, vaccinia viral in vectors, CMV viral vectors, BLUESCRIPT, baculovirus vectors, and the like. Promoters capable of directing the transcription of a cloned gene or cDNA may be inducible or constitutive promoters and include viral and cellular promoters. For expression in mammalian host cells, suitable viral promoters include the immediate early cytomeglovirus promoter (Boshart et al., Cell 41: 521-530, 1985) and the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1: 854-864, 1981). Suitable cellular promoters for expression of proteins in mammalian host cells include but are not limited to the mouse metallothionien-1 promoter (Palmiter et al., U.S. Pat. No. 4,579,821), and tetracycline-responsive promoter (Gossen and Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-5551, 1992 and Pescini et al., Biochem. Biophys. Res. Comm. 202: 1664-1667, 1994). Also contained in the expression vectors is a transcription termination signal located downstream of the coding sequence of interest. Suitable transcription termination signals include the early or late polyadenylation signals from SV40 (Kaufman and Sharp, Mol. Cell. Biol. 2:1304-1319, 1982), the polyadenylation signal from the Adenovirus 5 e1B region and the human growth hormone gene terminator (DeNoto et al., Nucleic Acid. Res. 9: 3719-3730, 1981).

Mammalian cells may be transfected by a number of methods including calcium phosphate precipitation (Wigler et al., Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603, 1981; Graham and Van der Eb, Virology 52: 456, 1973); lipofection (Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7417, 1987), microinjection and electroporation (Neumann et al., EMBO J. 1: 8410845, 1982). Mammalian cells can be transduced with virus such as SV40, CMV and the like. In the case of viral vectors, cloned DNA molecules may be introduced by infection of susceptible cells with viral particles. Retroviral vectors may be preferred for use in expressing FMO3 in mammalian cells, particularly when FMO3 is used in methods of gene therapy (for review, see, Miller et al., Methods in Enzymology 217: 581-599, 1994; which is incorporated herein by reference in its entirety).

It may be preferable to use a selectable marker to identify cells that contain the cloned DNA. Selectable markers are generally introduced into the cells along with the cloned DNA molecules and include genes that confer resistance to drugs, such as neomycin, hygromycin and methotrexate. Selectable markers may also complement auxotrophies in the host cell. Yet other selectable markers provide detectable signals, such as beta-galactosidase to identify cells containing the cloned DNA molecules. Selectable markers may be amplifiable. Such amplifiable selectable markers may be used to amplify the number of sequences integrated into the host genome.

As would be evident to one of ordinary skill in the art, the polynucleotide molecules of the present invention may be expressed in Saccharomyces cerevisiae, filamentous fungi, and E. coli. Methods for expressing cloned genes in Saccharomyces cerevisiae are generally known in the art (see, "Gene Expression Technology," Methods in Enzymoloy, Vol. 185, Goeddel (ed.), Academic Press, San Diego, Calif., 1990 and "Guide to Yeast Genetics and Molecular Biology," Methods in Enzymology, Guthrie and Fink (eds.), Academic Press, San Diego, Calif., 1991; which are incorporated herein by reference). Filamentous fungi (e.g., strains of Asperpillus) may also be used to express the proteins of the present invention. Methods for expressing genes and cDNAs in cultured mammalian cells and in E. coli is discussed in detail in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; which is incorporated herein by reference). As would be evident to one skilled in the art, one could express the protein of the instant invention in other host cells such as avian, insect and plant cells using regulatory sequences, vectors and methods well established in the literature.

FMO3 proteins produced according to the present invention are purified using a number of established methods, such as affinity chromatography using anti-FMO3 antibodies coupled to a solid support and sequence-specific chromatography. Additional purification may be achieved using purification means such as liquid chromatography, gradient centrifugation and gel electrophoresis among others. Methods of protein purification are known in the art (see generally, Scopes, R., Protein Purification, Springer-Verlag, N.Y., 1982, which is incorporated herein by reference) and can be applied to the purification of recombinant FMO3 described herein.

Thus, as discussed above, the present invention provides FMO3 isolated from its natural cellular environment, substantially free of other cellular proteins. Purified FMO3 is also provided. Substantially pure FMO3 of at least about 50% is preferred, at least about 70-80% is more preferred, and 95-99% or more homogeneity most preferred. Once purified, partially or to homogeneity, as desired, the recombinant FMO3 or native FMO3 may be used to generate antibodies, in screening and diagnostic procedures, etc.

Expression of Adult Human Liver FMO 3 in E. Coli

The expression vector was constructed and amplified by PCR by ligating the appropriate components designed to create for the full-length adult human liver FMO (form 3) cDNA as described in Example 6. The PCR product which was designed to obtain the full-length open reading frame cDNA of HLFMO 3 was inserted into a pTrc99A expression vector to give the expression plasmid, pTrcHLFMO 3 (FIG. 3). Restriction enzyme and DNA sequence analyses (Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977)) of the pTrcHLFMO 3 DNA sequence confirmed that the 5'-end of the HLFMO 3 cDNA coding strand was successfully extended and correctly inserted into the pTrc99A vector (FIG. 3). The expression of pTrcHLFMO 3 in the E. coli host bacteria NM522 following incubation in the presence of the inducing agent IPTG produced active HLFMO 3. NM522 host bacteria transformed with the pTrc99A vector alone did not produce any detectable FMO activity when grown in the presence or absence of IPTG. As shown in FIG. 4, the IPTG-induced HLFMO 3 protein from lysates of the pTrcHLFMO 3 transformed bacterial cells was detectable on SDS-PAGE. The in solubilized proteins from transformed E. coli cells were analyzed by SDS PAGE in duplicate. One gel was used for the analysis by Coomassie-blue staining (FIG. 4) and the other gel was subjected to Western blot analysis using antibodies directed against the guinea pig form 3 FMO (FIG. 5). A band at 60 kDa corresponding to the expressed HFLMO 3 protein was clearly detected. It was estimated that less than it of the total solubilized expressed protein was present as HLFMO 3.

Substrates and Synthesis of N-Oxides and S-Oxides

Trifluoperazine, thiobenzamide and chlorpromazine were purchased from Aldrich Chemical Co. (Milwaukee, Wis.). The 10-(N,N-Dimethylaminoalkyl)-2-(trifluoromethyl)phenothiazines were obtained from Professor D. M. Ziegler (University of Texas, Austin, Tex.) (Nagata et al., Chem. Res. Toxicol. 3:372-376 (1990)). 2-Methyl-1,3-benzodithiole was obtained from Professor D. Boyd (The Queens University of Belfast, N. Ireland) (Boyd et al., J. Chem. Soc. Perkin Trans. 1:1105 (1992)). (+) and (-)-4-Bromophenyl-1,3-oxathiolane were provided by Professor J. Sandstrom (University of Lund, Sweden). All of the substrates were completely characterized by .sup.1 H NMR, MS, UV-vis and in some cases circular dichroism spectroscopic means. The synthesis of tertiary amine N-oxides of trifluoperazine and chlorpromazine was accomplished by the general method previously described (Cashman et al., Drug Metab. Dispos. 16:616-622 (1988); Sofer & Ziegler, Drug Metab. Dispos. 6:232-239 (1978)). The 10-(N,N-dimethylaminoalkyl)-2-(trifluromethyl)phenothiazine N-oxides were biosynthesized with pig liver microsomes as described before (Nagata et al., Chem. Res. Toxicol. 3:372-376 (1990)). All of the tertiary amine N-oxides were completely characterized by spectral means. Similarly, thiobenzamide S-oxide (Cashman & Hanzlik, J. Org. Chem. 47, 4645-4650 (1982)), 4-bromophenyl-1,3-oxathiolane S-oxide diastereomers (Andresen et al., 1993), and 2-methyl-1,3-benzodithiole S-oxide diastereomers (Boyd et al., J. Chem. Soc. Perkin Trans. 1:1105 (1992)) were synthesized and completely characterized by methods, previously described.

Substrate Oxygenation

The regio- and stereo-selective oxygenation of various chemicals and drugs by human liver microsomes and solubilized protein from E. coli cells transformed with pTrcHLFMO 3 were examined to selectively monitor FMO enzyme action as well as to examine possible involvement of cytochromes P-450 or non-enzymatic oxidation of the same substrate. Trifluoperazine, other tricyclic antidepressants and other phenothiazines provided excellent probes for monooxygenase action because these chemicals possess a nucleophilic tertiary amine center known to be selectively oxygenated by FMO (Sofer & Ziegler, Drug Metab. Dispos. 6: 232-239 (1978); Nagata et al., Chem. Res. Toxicol. 3:.372-376 (1990)) and an electrophilic sulfur atom known to be selectively oxidized by cytochromes P-450 or by non-enzymatic means (i.e., H.sub.2 O.sub.2 or ROOH)). Other substrates (i.e., thiobenzamide, 4-bromophenyl-1,3-oxathiolane and 2-methyl-1,3-benzothiole) were also examined for S-oxygenation activity. In all cases examined, product formation was directly determined by HPLC analysis of organic extracts.

Solubilized protein from E. coli cells transformed with pTrcHLFMO 3 was evaluated for N- and S-oxygenase activity with compound 5 (Nagata et al., Chem. Res. Toxicol. 3:372-376 (1990)). In parallel, E. coli cells transformed with non-recombinant plasmid pTrc 99A were solubilized with 1-Triton X-100 and centrifuged at 100,000.times.g to afford a supernatant and a 100,000.times.g pellet. Solubilization of the recombinant HLFMO 3 protein resulted in 84% N-oxygenase activity (i.e., 0.23 nmol/min/mg of protein) in the supernatant fraction. The pellet contained 16% of the N-oxygenase activity (i.e., 0.043 nmol/min/mg of protein). The non-recombinant solubilized protein did not possess any detectable FMO activity. Other tertiary amine and sulfur-containing substrates examined also gave similar supernatant: pellet FMO activity ratios. No detectable amount of non-enzymatic N-oxygenation was observed during the metabolic incubations examined. In addition, no detectable N-oxide reduction was observed.

Preliminary studies that showed crude homogenates or solubilized protein of transformed E. coli supplemented with NADPH catalyzed the S- or N-oxygenation of a variety of substrates. As a standard for comparison, the oxidation of substrates with human liver microsomes was also studied. Under the conditions of the experiments, no detectable amount of non-FMO mediated S- or N-oxygenation was observed. For compound 6, formation of the tertiary amine N-oxide was a linear function of bacterial lysate protein concentration (0-4.6 mg of protein) and of time (0-10 min). In the presence of human liver microsomes, tertiary amine N-oxide formation of compound 6 was a linear function of microsomal protein concentration (0-0.5 mg of protein) and of time (0-30 min) (data not shown). N-Oxygenation of compound 6 was dependent upon the pH of the reaction mixture, and microsomal protein and solubilized bacterial lysate of expressed HLFMO 3 gave virtually identical pH-rate profiles. The pH-optimum for tertiary amine N-oxygenation was approximately 10 and the pH-rate profile resembled a titration curve with the midpoint near a value of pH 9.5. Other sulfur-containing substrates (i.e., thiobenzamide and 4-bromophenyl-1,3-oxathiolane) showed a maximum S-oxygenase activity in the presence of microsomes and expressed HLFMO 3 of approximately pH 8.5. The true pH optimum is probably 8.5 and it is likely that deprotonation of the amines (pka=9.5) provided the nonprotonated substrate and thus influenced the apparent pH optimum of the enzyme. ##STR1##

As shown in Table 1, N-oxygenation of chlorpromazine and compounds 2-4 was detectable, but compounds 5 and 6, with longer side chains, were better substrates for human liver microsomes and solubilized protein from E. coli transformed with pTrcHLFMO 3. Dichloromethane extracts of metabolic incubations with microsomes and solubilized protein of selected substrates from transformed E. coli were subjected to mass spectral analyses. The liquid secondary ion mass spectra (+LSIMS) of the tertiary amine N-oxide metabolite of compounds isolated from pig liver microsomes was similar with the chemical ionization (CI) spectrum of the N-oxide metabolite isolated from human liver microsomes and solubilized protein from E. coli transformed with pTrcHLFMO 3 (Table 2A, B).

The S-oxygenation of sulfur-containing substrates for the HLFMO 3 was investigated to provide a sensitive and direct method to study HLFMO (3) stereoselectivity. Determination of HLFMO 3 stereoselectivity could reveal information about enzyme mechanism as well as the incidence of competing achiral non-enzymatic oxidation processes (Cashman et al., J. Amer. Chem. Soc. 111:4844-4852 (1989); Cashman & Olsen, Mol. Pharmacol. 38:573-585 (1990)). Comparison of the stereoselectivity results obtained with human liver microsomes and recombinant HLFMO 3 with the results obtained using other FMO enzymes could also provide correlative information about enzyme substrate binding site topology. As shown in Table 3, 4-bromophenyl-1,3-oxathiolane was exclusively converted to (-)-trans-4-bromophenyl-1,3-oxathiolane S-oxide by both human liver microsomes and solubilized protein from E. coli transformed with pTrcHLFMO 3.

In contrast, (+)-4-bromophenyl-1,3-oxathiolane was converted to a mixture of cis and trans-4-bromophenyl-1,3-oxathiolane S-oxides by both human liver microsomes and cDNA expressed HLFMO 3. In fact, (+)-4-bromophenyl-1,3-oxathiolane is stereoselectively S-oxygenated to the trans S-oxide. In good agreement with previous studies employing hepatic preparations from animals (Cashman et al., J. Amer. Chem. Soc. 111:4844-4852 (1989); Cashman & Williams, Mol. Pharmacol. 37:333-339 (1990); Cashman et al., Chem. Res. Toxicol. 3:344-349 (1990)), the FMO monooxygenase preferentially attacks the sulfur atom at the least sterically hindered lone pair. The results suggest that FMO may be solely responsible for 4-bromophenyl-1,3-oxathiolane S-oxide formation though this does not rule out the involvement of cytochromes P-450. The stereoselective S-oxygenation of 2-methyl-1,3-benzodithiole was also investigated.

As shown in Table 4 human liver microsomes form mainly the (+)-(1R,2R)trans-S-oxide diastereomer although both cis diastereomeric S-oxides were formed, albeit in lower amounts. In contrast, expressed HLFMO 3 formed mainly the (+)-(1R,2S)cis S-oxide diastereomer although significant amounts of the other cis and (+)(1R,2R)trans S-oxide diastereomers were formed. In the presence of expressed HLFMO 3 or human liver microsomes no detectable amount of (-)-(1S,2R)trans-2-methyl-1,3-benzodithiole S-oxide was also formed (Table 4). Because the major product formed by expressed and highly purified pig liver FMO (form 1) is the cis-(-)-(1S,2R) S-oxide, it is likely that the major trans S-oxide product formed in human liver microsomes is catalyzed by cytochromes P-450.

Kinetic constants for the N- and S-oxygenation of various substrates in the presence of human liver microsomes or solubilized protein from E. coli transformed with pTrcHLFMO 3 were calculated from the rate of N- or S-oxide formation at variable substrate concentrations by the HPLC procedures described in the Experimental Procedures. The Km and Vmax values obtained from double reciprocal plots of velocity versus substrate concentration were listed in Table 5. As shown by the kinetic constants listed in Table 5, the values obtained for human liver microsomes were comparable to those obtained in the presence of cDNA-expressed HLFMO 3. The kinetic constants (Table 5) show that thiobenzamide, trifluoperazine, 10-(N,N-dimethylaminopentyl)-2-trifluoromethyl)phenothiazine (compound 5), and (+) and (-)-4-bromophenyl-1,3-oxathiolane are excellent substrates for the expressed HLFMO 3 and adult human liver microsomes. The concentration of substrate required to half-saturate the enzyme is in the general range reported previously for similar compounds. The turnover at infinite substrate concentration is similar to that reported by others for microsomal transformation with these same substrates (McManus et al., Drug Metab. Dispos. 15:256-261 (1987); Lemoine et al., Arch. Biochem. Biophys. 276:336-342 (1990)). In agreement with earlier work for other FMO's, HLFMO 3 does not catalyze S-oxidation of the phenothiazine tricyclic sulfur atom (Nagata, Chem. Res. Toxicol. 3:372-376 (1990)). This result is consistent with the mechanism proposed for animal FMOs which requires a "soft," highly polarizable nucleophilic atom as oxygenatable substrates.

That no detectable amount of trifluoperazine S-oxide or other 10-(N,N-dimethylalkyl)-2-trifluoromethyl) phenothiazine S-oxide were detected in preparations of proteins from pTrcHLFMO 3 transformed or non-transformed E. coli suggests that non-enzymatic or non-HLFMO 3 enzymic oxidations do not contribute to the determination of the kinetic constants listed in Table 5.

Dichloromethane extracts of metabolic incubations of selected reactions catalyzed by human liver microsomes and solubilized protein from transformed E. coli were purified by preparative HPLC and were subjected to mass spectral analyses. The mass spectral data of S-oxide metabolites was listed in Table 2 and was virtually identical to the data of the authentic material. Taken together, the data for N- and S-containing compounds clearly showed that HLFMO 3 cDNA expressed in E. coli and adult human liver microsomes catalyzed selective oxygenation of various tertiary-amine and nucleophilic sulfur-containing substrates.

TABLE 1 N- and S-Oxygenation of Various 10-(N,N-Dimethylaminoalkyl)-2- (Trifluoromethyl) Phenothiazines by Human Liver Microsomes and cDNA-Expressed Human Liver Flavin-Containing Monoxygenase.sup.a Alkyl Side Human Liver Microsomes Expressed HFLMO Substrate Chain N-oxide S-oxide N-oxide S-oxide n pmol/min/mg of protein pmol/min/mg of protein Chlorpromazine 3 58.9 .+-. 7.4 29.3 .+-. 3.7 10.2 .+-. 1.2 ND Compound 2 2 27.8 .+-. 4.5 ND.sup.b 30.4 .+-. 6.8 ND Compound 3 3 149.9 .+-. 14.3 12.2 .+-. 3.3 53.2 .+-. 12.9 ND Compound 4 4 201.2 .+-. 18.8 31.0 .+-. 15.8 118.0 .+-. 7.8 ND Compound 5 5 252.1 .+-. 38.6 32.5 .+-. 33.7 200.0 .+-. 45.7 ND Compound 6.sup.c 6 -- -- -- -- .sup.a Incubations were performed as described in Example 8 with 0.1 mM substrate. The values are the mean of 5 determinations .+-. SD. .sup.b ND, not detectable, limit of detection 10 pmol/min/mg of protein. .sup.c In a separate experiment, Compound 6 gave similar values to the values listed above for Compound 5.

TABLE 2A Mass Spectral Properties of N- and S-Oxide Metabolites of cDNA-Expressed Human Liver Flavin-Containing Monooxygenase (form II) Metabolite MW CI.sup.a or LSIMS.sup.b EIMS m/z (relative intensity) m/z (relative intensity) 2-Methyl-1,3-benzodithiole 184 185.sup.a (100) 184 (38.1), 166 (4.8), S-oxide 153 (19.8), 140 (100), 134 (29.4), 96 (40.5) (-)-4-Bromophenyl-1,3- 260 253/261.sup.a (14.0/14.0), 262/260 (13.2/13.2), oxathiolane.sup.c 183 (100) 235/232 (15.9/15.9), S-oxide 206/204 (11.9/11.4), 185/183 (38.1/42.1, 57 (100) 10-(N,N-Dimethylaminopentyl- 396 381.sup.a (7.9), 292 (10.8), NP.sup.d 2-trifluoromethyl)- 258 (31.8), 57 (100) phenothiazine N-oxide Thiobenzamide 153 239.sup.a (100), 135 (4.3), 238 (28.6), 135 (100) S-oxide 121 (10.1) .sup.a CI, Chemical Ionization, carrier gas was ammonia resulting in protonated molecular ions. .sup.b LSIMS, Cs.sup.+ liquid secondary ion mass spectrometry using a thioglycerol matrix. .sup.c cis and trans diastereomers gave virtually identical spectra. .sup.d NP, not possible, the metabolite was unstable to the mass spectrometry experiment. .sup.e Under the conditions of the mass spectrometry experiment, the S-oxide dimerized to yield 3,5-diphenyl-1,2,4-thiadiazole (MW 238). This was confirmed by an independent synthesis and MS experiment of an authentic sample (Hanzlik and Cashman, J. Org. Ch m. 47 4645-4650 (1982)).

TABLE 2B Mass Spectral Properties of N- and S-Oxide Metabolites of Human Liver Microsomes Metabolite MW CI.sup.a or LSIMS.sup.b EIMS m/z (relative intensity) m/z (relative intensity) 2-Methyl-1,3-benzodithiole 184 185.sup.a (100) 184 (11.9), 169 (6.3), S-oxide 153 (19.8), 140 (71.4), 57 (100) (-)-4-Bromophenyl-1,3- 260 262/260.sup.a (55.7/55.7), 262/260 (6.7/6.7), oxathiolane.sup.c 234/232 (60.4/56.4) 234/232 (6.7/6.7), S-oxide 186/184 (9.4/9.4), 184/182 (15.5/15.5), 57 (100) 10-(N,N-Dimethylaminopentyl- 396 381.sup.a (37.3, 367 (28.6), NP 2-trifluoromethyl)- 292 (11.1), 258 (46.0), phenothiazine 123 (100) N-oxide Thiobenzamide 153 239.sup.a (23.0), 136 (29.8), NP S-oxide 122 (71.2) 74 (87.5), 59 (100) .sup.a CI, Chemical Ionization, carrier gas was ammonia resulting in protonated molecular ions. .sup.b LSIMS, Cs.sup.+ liquid secondary ion mass spectrometry using a thioglycerol matrix. .sup.c cis and trans diastereomers gave virtually identical spectra.

TABLE 2C Mass Spectral Properties of N- and S-Oxides from Synthetic Sources Metabolite MW CI.sup.a or LSIMS.sup.b EIMS m/z (relative intensity) m/z (relative intensity) 2-Methyl-1,3-benzodithiole 184 ND.sup.f 184 (16), 162 (11.5), S-oxide 151 (13.5), 153 (12), 140 (100) (-)-4-Bromophenyl-1,3- 260 ND 262/260 (30.4/29.5), oxathiolane 234/232 (43.6/40.6), S-oxide 165/183 (100/94.4) 1'10-(N,N-Dimethylaminopentyl- 396 382.sup.b (100), 359 (23.6), 380 (42.9), 366 (10.5), 2-trifluoromethyl)- 341 (57.3) 293 (4.4), 280 (9.1), phenothiazine 267 (10.9, 266 (16.2), N-oxide 248 (11.5), 100 (100) Thiobenzamide 153 ND 238 (34.6), 135 (100), S-oxide 103 (18.6) .sup.a CI, Chemical Ionization, carrier gas was ammonia resulting in protonated molecular ions. .sup.b LSIMS, Cs.sup.+ liquid secondary ion mass spectrometry using a thioglycerol matrix. .sup.f ND, not done.

TABLE 3 S-oxygenation of (+) and (-)-4-Bromophenyl-1,3-Oxathiolane by Adult Human Liver Microsomes and CDNA-Expressed Human Liver Flavin-Containing Monooxygenase.sup.a Human Liver Microsomes Expressed HLFMO Substrate cis S-oxide trans S-oxide cis S-oxide trans S-oxide % (nmol/min/mg of protein) % (nmol/min/mg of protein) (+)-4-Bromophenyl- 40.4 59.5 (7.8 .+-. 1.7) 38.5 (0.5 .+-. 0.3) 61.4 (0.8 .+-. 0.2) 1,3-oxathiolane (5.3 .+-. 0.9) (-)-4-Bromophenyl- ND.sup.b 100 (2.4 .+-. 0.9) ND 100 (0.7 .+-. 0.1) 1,3-oxathiolane .sup.a Incubations were carried out as described in Example 8. The values are the mean of determinations .+-. SD. .sup.b ND, not detectable, limit of detection 15 pmol/min/mg of protein.

TABLE 4 Stereoselective S-Oxygenation of 2-Methyl-1,3-Benzodithiole by Microsomes and cDNA-Expressed Flavin-Containing Monooxygenise from Human Liver.sup.a S-Oxide Product Formed Enzyme cis cis trans trans Preparation (+)-(1R,2S) (-)-(1S,2R) (+)-(1R,2R) (-)-(1S,2R) % Diastereomer (amount formed, pmol/min/mg of protein) Human Liver Microsomes 28.2 (176 .+-. 34) 12.3 (77 .+-. 27) 59.5 (372 .+-. 35) ND.sup.b Expressed FMO 48.6 (54 .+-. 22) 19.8 (22 .+-. 2) 31.5 (35 .+-. 27) ND .sup.a Incubations were carried out in the presence of a 0.5 mM NADPH generating system, 0.6 mM DETAPAC, 125 nmol substrate, potassium phosphate buffer (pH 8.4) and enzyme (0.75 mg microsomes or 1.4 mg expressed protein). Each value was determined by HPLC and was the mean of 5 determinations of .+-. SD. .sup.b Not detectable, ND, limit of detection was 10 pmol/min/mg of protein.

TABLE 5 Kinetic Constants for Oxygenation of Tertiary Amines and Sulfur-Containing Compounds by Adult Human Liver Microsomes and cDNA-Expressed Human Liver Flavin-Containing Monooxygenase.sup.a Human Liver Microsomes Expressed HLFMO K.sub.m V.sub.max K.sub.m V.sub.max Substrate .mu.M (nmol/min/mg of protein) .mu.M (nmol/min/mg of protein) Thiobenzamide 14.9 1.3 72.6 1.44 Trifluoperazine 92.7 0.253 139.0 0.204 Compound 5 6.8 0.098 64.6 0.023 (+)-4-Bromophenyl.sup.b - 50.0 2.87 142.3 0.180 1,3-oxathiolane (-)-4-Bromophenyl.sup.b - 166.0 5.2 310.8 2.94 1,3-oxathiolane .sup.a The kinetic constants were calculated from initial velocity measurements by the