Human Flavin-Containing Monooxygenase Form 3: cDNA Expression

Trimethylaminuria is an autosomal recessive human disorder affecting a small part of the population as an inherited polymorphism. Individuals diagnose...
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AUGUST 1997 VOLUME 10, NUMBER 8 © Copyright 1997 by the American Chemical Society

Communications Human Flavin-Containing Monooxygenase Form 3: cDNA Expression of the Enzymes Containing Amino Acid Substitutions Observed in Individuals with Trimethylaminuria John R. Cashman,*,† Yi-An Bi,† Jing Lin,† Rima Youil,‡ Melanie Knight,‡ Susan Forrest,‡ and Eileen Treacy§ Seattle Biomedical Research Institute, 4 Nickerson Street, Suite 200, Seattle, Washington 98109, McGill University-Montreal Children’s Hospital Research Institute, 2300 rue Tupper Street, A-717, Montreal, Quebec H3H 1P3, Canada, and Murdoch Institute Royal Children’s Hospital, Flemington Road, Parkville 3052, Melbourne, Australia Received April 8, 1997X

Trimethylaminuria is an autosomal recessive human disorder affecting a small part of the population as an inherited polymorphism. Individuals diagnosed with trimethylaminuria excrete relatively large amounts of trimethylamine in their urine, sweat, and breath, and this results in a fishy odor characteristic of trimethylamine. Activity of the human flavin-containing monooxygenase (FMO) has been proposed to be deficient in trimethylaminuria patients causing a decrease in the metabolism of trimethylamine that results in a fishy body odor. Cohorts of Australian, American, and British individuals suffering from trimethylaminuria have been identified. The human FMO3 cDNA was amplified from lymphocytes of affected patients. We report preliminary evidence of substitutions detected by screening of the cDNA and genomic DNA. The variant human FMO3 cDNA was constructed from wild type human FMO3 cDNA by site-directed mutagenesis as maltose-binding protein fusions. Five distinct human FMO3 mutants were expressed as fusion proteins in Escherichia coli and compared with wild type human FMO3 maltose-binding proteins (FMO3-MBP) for the N-oxygenation of 10-[(N,Ndimethylamino)pentyl]-2-(trifluoromethyl)phenothiazine, tyramine, and trimethylamine. Human Lys158 FMO3-MBP and, to a greater extent, human Glu158 FMO3-MBP efficiently N-oxygenated the three amine substrates. Human Lys158 Ile66 FMO3-MBP, Glu158 Ile66 FMO3-MBP, Lys158 Leu153 FMO3-MBP, and Glu158 Leu153 FMO3-MBP were all constructed as mutants identified as possible FMO3 variants responsible for trimethylaminuria and were found to be inactive as N-oxygenases. The results suggest that mutations at codons 66 and 153 of FMO3 can cause trimethylaminuria in humans. We observed a common polymorphism of Lys to Glu at codon 158 of FMO3 that segregated with almost equal allele frequencies in a number of control Australian and North American samples studied. The Lys158 to Glu158 human FMO3 polymorphism does not decrease trimethylamine N-oxygenation for the cDNAexpressed enzyme and thus does not appear to be causative of trimethyaminuria. The data show that the functional activity of human FMO3 can be significantly altered by amino acid changes that have been observed in individuals with clinically diagnosed trimethylaminuria. S0893-228x(97)00053-2 CCC: $14.00

© 1997 American Chemical Society

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Introduction The hepatic human flavin-containing monooxygenase (FMO)1 (E.C. 1.14.13.8) catalyzes the NADPH-dependent N-oxygenation of primary, secondary, and tertiary xenobiotic and endogenous amines (1, 2). Human FMO3 N-oxygenase activity has been proposed to be deficient in individuals suffering from trimethylaminuria (1, 3, 4). Trimethylaminuria is an autosomal recessive human condition whereby affected subjects excrete relatively large amounts of unmetabolized trimethylamine (TMA) instead of the normal nontoxic and deoderation product trimethylamine N-oxide (TMANO) (3, 5). Consequently, individuals with trimethylaminuria possess a characteristic fishy body odor that comes from the elevated unmetabolized TMA present in the urine, sweat, and breath of affected persons (3, 5). Generally, humans convert TMA to TMANO in greater than 65% yield (6) or have more than 12-18 µmol of urinary TMA/mmol of creatinine under normal dietary conditions (7). Although TMA is a constituent present from diverse sources (i.e., bacterial degradation of choline and creatinine or from lecithin or reduction of TMANO), in humans it is usually obtained from dietary sources including egg yolk, liver, legumes, soybeans, and peas (8). In humans, TMA is metabolized to TMANO almost exclusively (9). Intestinal bacteria can also metabolize TMA to di- and monomethylamine and ultimately to ammonia and carbon dioxide, but this constitutes a minor pathway (10). In animals, TMA N-oxygenation has been proposed to be catalyzed by hepatic FMO1 (11), but in human liver, functional FMO1 is not present to a detectable extent (12) and FMO3 is the enzyme most likely responsible for TMANO formation. Human FMO3 efficiently N-oxygenates primary, secondary, and tertiary amines (1, 2). Thus, phenethylamine or tyramine is sequentially Noxygenated by human FMO3 to the corresponding hydroxylamine, and the hydroxylamine is converted to give exclusively the trans oxime (13, 14). The tertiary amine 10-[(N,N-dimethylamino)pentyl]-2-(trifluoromethyl)phenothiazine (5-DPT) is regioselectively N-oxygenated to give the corresponding tertiary amine N-oxide (15, 16). 5-DPT N-oxygenation has found use as a functional marker of human FMO3 activity that can be correlated with human microsomal FMO3 immunoreactivity (17, 18). TMA has been shown to be N-oxygenated by FMO from animals (19) but has not been examined as a substrate for human FMO3. To test the hypothesis that human FMO3 is responsible for TMA N-oxygenation and that abnormalities of the gene that encoded defective human FMO3 resulted in trimethylaminuria, we identified a number of patients with trimethylaminuria with documented diminished TMA N-oxygenation. Screening of the first 760 base * To whom reprint requests should be addressed. Telephone: (206) 284-8846, ext. 310. Fax: (206) 284-0313. E-mail: [email protected]. † Seattle Biomedical Research Institute. ‡ Murdoch Institute Royal Children’s Hospital. § McGill University-Montreal Children’s Hospital Research Institute. X Abstract published in Advance ACS Abstracts, August 1, 1997. 1Abbreviations: FMO, flavin-containing monooxygenase; TMA, trimethylamine; TMANO, trimethylamine N-oxide; 5-DPT, 10-[(N,Ndimethylamino)pentyl]-2-(trifluoromethyl)phenothiazine; MBP, maltosebinding protein; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PEG 8000, poly(ethylene glycol) 8000; FMO3-MBP, flavin-containing monooxygenase maltose-binding protein; DETAPAC, diethylenetriaminepentaacetic acid; RPHPLC, reverse phase highperformance liquid chromatography.

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pairs of the 5′-end of human FMO3 cDNA was done with a number of samples from these individuals. For one change, a common polymorphism at codon 158 was identified in controls and affected subjects. This was observed in strict linkage disequilibrium in the homozygous state in all of the Australian and American cases of trimethylaminuria examined. In addition, a human FMO3 mutation (i.e., Met66 f Ile 66) was also observed in the genomic DNA of one individual. The wild type and variant human FMO3 cDNAs were expressed as fusion proteins in Escherichia coli and compared with the fusion protein of a variant human FMO3 identified from a British cohort (20). The results suggested that abnormal in vivo metabolism of TMA that caused trimethylaminuria can be related in some cases to mutations of human FMO3. Two mutations have been characterized as causative of the trimethylaminuria phenotype in humans. In a third case, a frequent polymorphism was observed that was highly associated with the trimethylaminuria phenotype but was not in itself causative. Other amino acid substitutions or molecular rearrangements yet to be identified involving human FMO3 are likely to be causative of trimethylaminuria in affected individuals.2

Experimental Procedures Materials. Trimethylamine hydrochloride (TMA), trimethylamine N-oxide (TMANO), tyramine, and all other chemicals used for this study were purchased from Aldrich Chemical Co. (Milwaukee, WI). 10-[(N,N-Dimethylamino)pentyl]-2-(trifluoromethyl)phenothiazine (5-DPT) and 4-hydroxyphenethyl oxime were synthesized as previously described (14, 21). Triton X-100, L-R-phosphatidylcholine, and all the components of the NADPHgenerating system were obtained from Sigma Chemical Co. (St. Louis, MO). All other reagents and solvents used in this study were purchased from Fisher Scientific Inc. (Santa Clara, CA). The E94 plasmid and the human Lys158 FMO3 and Glu158 FMO3 fusion constructs were from previously described work (21). The pGEM(-) vector, restriction endonucleases, DNA polymerase Klenow fragment, T4 DNA ligase, phosphatase, Taq polymerase, and E. coli JMIO9 were purchased from Promega (Madison, WI). Mutagenesis reagents and the in vitro mutagenesis kit for site-directed mutagenesis were from Bio-Rad (Hercules, CA). Oligonucleotides for mutagenesis and sequencing were synthesized by CyberSyn (Lenni, PA). pMAL-c2 and amylose resin were purchased from New England Biolabs (Beverly, MA). Oligonucleotide sequencing was done by the DNA sequencing facility at the Seattle Biomedical Research Institute. Subcloning and cDNA Expression of Human FMO3 and Variant Fusion Proteins. The strategy to clone, sequence, and express the full-length human FMO3 open reading frame relied on using PCR to construct the cDNA encoding wild type and variant proteins and place into the expression vector pMALc2. The construction allowed for the fusion of human FMO3 cDNA at the 3′-end of a sequence encoding the maltose-binding protein (MBP) as described previously (21). Single oligonucleotide changes converted the Lys158 FMO3-MBP wild type or Glu158 FMO3-MBP cDNA to the desired substituted codon. The oligonucleotide change was accomplished by using site-directed mutagenesis. The mutagenesis was carried out on a subclone of human FMO3 cDNA (i.e., NcoI to SacI fragment) in the vector pGEM(-). Because there is a SacI site in the pMAL-2c vector, the mutated Nhe/SacI fragment cannot be directly subcloned into HFMO3-MBP fusion plasmid. The site-directed mutagenized Nhe/SacI fragment was thus first transferred into E94, and then the Nhe/HindIII fragment was transferred to one of the 2E.

Treacy, personal communication.

Communications two HFMO3-MBP fusion plasmids. The oligonucleotide designed for the Ile66 site-directed mutagenesis (i.e., 5′-GG GAA ACA CAT ATT CTC TTT GG-3′) corresponded to codons 63-70 of human FMO3 and contained two mismatches at codon 66. The oligonucleotide designed for the Leu153 site-directed mutagenesis (i.e., 5′-TAG GTT GAG ATA CAC ATG ATG TCC-3′) corresponded to codons 148-155 of human FMO3 and contained a single mismatch at codon 153. Automated sequencing (Applied Biosystems Model 373A) showed that each full-length human FMO3 cDNA was correctly inserted into the MBP fusion expression vector that encoded each pMAL-HFMO3. Competent JM109 E. coli cells were transformed with pMALHFMO3 and grown in a similar manner as previously described (15, 16, 22). The bacteria were harvested by centrifugation and lysed, and the bacterial lysate was cleared by centrifugation. The supernatants derived from a 4 L culture of JM109 E. coli containing pMAL-HFMO3 or variants were passed through an amylose column and selectively eluted as described before (21). Fractions were collected and assayed for protein concentration and fractionated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For some experiments, sequential precipitation of amylose column affinity-purified protein with 6% and then 14% poly(ethylene glycol) 8000 (PEG 8000) provided fractions as much as 90% pure and highly enriched in human FMO3-MBP activity. Protein concentration was determined by the BCA protein assay from Pierce (Rockford, IL). Electrophoresis and Immunoblotting. Amylose column affinity-purified human FMO3-MBP proteins were fractionated by electrophoresis on 12% SDS-PAGE, transferred to nitrocellulose, and immunostained according to the procedure described previously (23) except 0.12 mg/mL MBP was added as a blocking reagent in the primary antibody solution. An antibody directed against the human FMO3 was obtained by immunizing rabbits with purified human FMO3-MBP (R and R Rabbitry, Stanwood, WA) and used at a 1:100 (v:v) dilution. The immunoblot assay system utilized [125I]protein A for radiometric detection. Enzyme Preparations and Incubations. Assay and analysis of human FMO3-MBP N-oxygenation of 5-DPT were done as previously described (21). A typical incubation mixture for tyramine or TMA contained cDNA-expressed enzyme (10-600 µg of protein), NADP+ (4 mM), glucose-6-phosphate (4.0 mM), glucose-6-phosphate dehydrogenase (1 IU), diethylenetriaminepentaacetic acid (DETAPAC) (1.2 mM), and potassium phosphate buffer (50 mM, pH 8.4). Reactions were initiated by the addition of 1.2 mM tyramine or 2.0 mM TMA and stopped at designated times by addition of 6 volumes of cold dichloromethane/2-propanol (2:1, v:v, for tyramine) or acetonitrile (for TMA). After addition of 20 mg of Na2CO3 the incubations were mixed thoroughly and centrifuged to separate the protein and organic fractions. For analysis of tyramine metabolites, the organic fraction was evaporated to dryness, taken up in CH3CN, and analyzed with a Rainin analytical C-18 column interfaced to a Hitachi L-6200A HPLC with UV detection set at 278 nm. The mobile phase consisted of an isocratic system set at 85% A and 15% B, where A was water and B was acetonitrile containing 0.1% HClO4 (60% solution), at a flow rate of 1.5 mL/min that efficiently separated tyramine, 4-hydroxyphenethylhydroxylamine, trans- and cis-4-hydroxyphenethylamine oximes, and 4-hydroxybenzaldehyde that had retention times of 3.4, 4.3, 6.5, 7.9, and 8.1 min, respectively (14). Metabolites were quantified by comparing the metabolite and substrate peak areas of the chromatogram after accounting for the molar extinction coefficients of each metabolite. For the analysis of TMA, the protein was precipitated by the addition of CH3CN and centrifuged. The supernatant was lyophilized to dryness and dissolved in methanol, mixed thoroughly, centrifuged, and placed directly on HPLC. The formation of TMANO was directly analyzed with a Rainin analytical C-18 column using TMANO as an external standard. TMANO was separated and quantified by a Hitachi L-6200 HPLC employing an evaporative-lightscattering detector (Sedex 55 detector, Richard Scientific, No-

Chem. Res. Toxicol., Vol. 10, No. 8, 1997 839 Table 1. N-Oxygenation of 5-DPT, Tyramine, and Trimethylamine by Human Flavin-Containing Monooxygenase 3 and Variantsa product formation [nmol/(min‚mg of protein)] human FMO3 enzymes

5-DPT N-oxide

Lys158 Glu158 Lys158 Ile66 Glu158 Ile66 Lys158 Leu153 Glu158 Leu153

4.2 ( 0.23 8.8 ( 0.14 NDc ND ND ND

trans-4-hydroxy phenethyl oximeb 1.0 ( 0.24 2.7 ( 0.38 NDd ND ND ND

TMANO 2.6 ( 0.29 4.1 ( 0.02 NDe ND ND ND

a Incubations were carried out as described in the Experimental Procedures. The values are the mean of 3-4 determinations ( SD. The data are typical of 3-4 similar experiments that were done with other preparations of the same enzymes. b Only the trans oxime isomer was observed; no cis oxime was detected. c ND, not detectable; the limit of detection for 5-DPT N-oxide was 5 pmol/ (min‚mg of protein). d ND, not detectable; the limit of detection for the trans oxime was 20 pmol/(min‚mg of protein). e ND, not detectable; the limit of detection for TMANO was 120 pmol/ (min‚mg of protein).

vato, CA). The reverse phase HPLC (RPHPLC) system efficiently separated TMANO (i.e., retention time 3.2 min) from the solvent front using a mobile phase of 62% A, 35% B, and 3% C, where A was CH3CN, B was water, and C was methanol containing 0.4% saturated ammonium hydroxide.

Results Expression of Human FMO3-MBP cDNA in E. coli. Genomic DNA from 11 probands diagnosed with trimethyaminuria as well as 44 normal controls were isolated from lymphoblasts and amplified by PCR. The urinary TMA levels were previously reported to be in the affected range (i.e., between 49 and 235 µmol of TMA/mmol of creatinine) for the trimethylaminuria subjects and in the normal range for the controls examined (5, 7, 20). TMA levels or TMA/TMANO ratios were determined for all 11 of the trimethylaminuria patients examined showing that deficient TMA N-oxygenation was apparent (data not shown). Sequencing of the cDNA or genomic DNA of human FMO3 from affected individuals showed a number of changes that resulted in amino acid substitutions. The PCR products designed to give the full-length open reading frame cDNA encoding human FMO3-MBP enzymes identified from trimethylaminuria patients and listed in Table 1 were inserted into a pMAL-c2 expression vector. Complete DNA sequence analysis of both strands of the pMAL-HFMO3 cDNA confirmed that the entire coding strand was successfully extended and correctly inserted into the expression vector. The expression of pMAL-c2 in the E. coli host bacteria JM109 resulted in overproduction of full-length human FMO3-MBP enzymes. The IPTGinduced human FMO3-MBP enzymes from amylose affinity resin-purified bacterial lysates were detectable on SDS-PAGE. Using antibodies directed against Glu158 FMO3-MBP, a band at 100 kDa corresponding to each cDNA-expressed FMO3-MBP fusion protein was clearly detected (data not shown). Regio- and Stereoselective N-Oxygenation by Human FMO3-MBP and Variants. The regio- and stereoselective N-oxygenation of primary and tertiary amines with amylose resin affinity-purified human FMO3MBP was examined to investigate the involvement of protein structure on FMO3 function. 5-DPT and tyramine were excellent substrates for human Lys158 FMO3-MBP and Glu158 FMO3-MBP enzymes (Table 1). TMA was

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less efficiently N-oxygenated by these enzymes, but TMA was nevertheless a reasonable substrate to selectively study the role of human FMO3 mutations in endogenous substrate N-oxygenation. In all cases, product formation was directly determined by HPLC analysis of organic extracts. Preliminary studies showed that cDNA-expressed human Lys158 FMO3-MBP and Glu158 FMO3-MBP supplemented with NADPH catalyzed the N-oxygenation of 5-DPT, tyramine, and TMA. Formation of 5-DPT N-oxide was linearly dependent on affinity-purified human Lys158 FMO3-MBP or Glu158 FMO3-MBP (0-80 µg of protein) and on time (0-15 min) (data not shown). As reported previously, the Km for 5-DPT N-oxygenation was approximately 65 µM (16). As shown in Table 1, 5-DPT was efficiently N-oxygenated by human Lys158 FMO3-MBP or Glu158 FMO3-MBP but was not detectably N-oxygenated by the other proteins examined. Formation of trans4-hydroxyphenethyl oxime from tyramine was a linear function of affinity-purified human Lys158 FMO3-MBP or Glu158 FMO3-MBP (0-600 µg of protein) and of time (0-15 min) (data not shown). During the incubations with tyramine, the only product observed formed was the corresponding trans oxime. Although significant differences in the Km for formation of trans oxime from Lys158 FMO3-MBP and Glu158 FMO3-MBP (i.e., 952 and 216 µM, respectively) have been noted (14), saturating substrate was present in the incubations to assess primary amine N-oxygenase activity. The data of Table 1 show that tyramine was efficiently N-oxygenated by human Lys158 FMO3-MBP and Glu158 FMO3-MBP but was not detectably N-oxygenated by the other mutants listed. For TMA, production of TMANO was linearly dependent on protein concentration (0-1.0 and 0-0.65 mg of protein) and on time (0-25 and 0-20 min) for human Lys158 FMO3-MBP and Glu158 FMO3-MBP, respectively. The mean Km value for TMA N-oxygenation for both enzymes was 724 µM. The data of Table 1 show that TMANO formation was only observed for Lys158 FMO3-MBP and Glu158 FMO3-MBP and not for any of the other enzymes examined. N-Oxygenation of all three substrates by Lys158 FMO3-MBP and Glu158 FMO3-MBP was dependent on the pH of the reaction mixture, and the pH optimum was between 8.5 and 9.5. For human Lys158 FMO3-MBP and Glu158 FMO3-MBP, maximal N-oxygenation activity was dependent on 0.015% Triton X-100, but beyond 0.05% Triton X-100, a marked decrease in the N-oxygenation activity of the three substrates was observed (21). It is notable that the soluble FMO3-MBP does not suffer from the same problems associated with membrane-bound or detergent-solubilized human FMO3 and for that reason represents an efficient system to examine structure-function relations for mutagenized FMO enzymes, the analysis of which could otherwise be confounded by mutations that interfere with membrane association. The N-oxygenation of chemicals, endogenous substrates, and dietary constituents by human FMO3 enzymes in vitro was investigated to provide a correlation of amine N-oxidation in vivo for humans possessing those same FMO3 enzymes. Determination of N-oxygenation product regio- and steroselectivity results with human FMO3 enzymes could provide information about enzyme structure-function relationships. As shown in Table 1, 5-DPT, TMA, and tyramine were efficiently N-oxygenated by both human Lys158 FMO3-MBP and Glu158 FMO3MBP. Thus, Lys158 FMO3-MBP- and Glu158 FMO3-

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MBP-catalyzed formation of 5-DPT N-oxide, TMANO, and trans-4-hydroxyphenethyl oxime was quite efficient although Glu158 FMO3-MBP appeared to be a more efficient N-oxygenase.

Discussion Of the five forms of human FMO currently identified, hepatic FMO3 appears to be the most prominent catalytically active form responsible for lipophilic amine N-oxygenation. Generally, human FMO3-dependent amine N-oxygenation produces polar metabolites that are readily excreted. In the case of the tertiary amine TMA, N-oxide formation represents a detoxication and deodoration process. Human FMO3 has been proposed to be responsible for the N-oxygenation and deodoration of TMA, but to date, no reports describing the expression of mutants of human FMO3 causative of diminished TMA N-oxygenation have appeared in the literature. Analysis of the cDNA or genomic DNA of human FMO3 from individuals suffering from trimethylaminuria revealed a number of amino acid substitutions. In some cases, more than one change in human FMO3 cDNA was linked to abnormal TMA metabolism in vivo. One Australian patient suffering from trimethylaminuria had at least two changes in human FMO3 (i.e., Lys158 Met66 f Glu158 Ile66. However, the nucleotide transversion that changes lysine for glutamic acid at codon 158 is not by itself responsible for low human TMA N-oxygenation activity in vivo because cDNA expression of this protein gives an enzyme with increased TMA N-oxygenation activity in vitro compared with the wild type Lys158 FMO3 enzyme (Table 1). The human Glu158 FMO3MBP also N-oxygenates another tertiary amine, 5-DPT, and the primary amine tyramine more efficiently than wild type human Lys158 FMO3-MBP (Table 1). Because every Australian and American individual genotyped for human FMO3 mutations and diagnosed with trimethylaminuria examined thus far possessed the Lys158 f Glu158 substitution and because the Glu158 change by itself is not causative for the disease, another alteration present possibly near the 3′-end of the human FMO3 cDNA must be responsible for the loss of TMA Noxygenation activity. Further DNA sequencing must be done to verify this possibility. The 5′-ends of human FMO3 genomic DNA from 44 normal controls and 11 trimethylaminuria probands were sequenced, and only one trimethylaminuria patient carried a G f T nucleotide base change at codon 66. This change caused a Met66 f Ile66 substitution. To examine whether this mutation was causative for low human FMO3 activity and contributed to trimethylaminuria, human Lys158 Ile66 FMO3-MBP cDNA was expressed and the N-oxygenase activity was compared with cDNAexpressed human Glu158 Ile66 FMO3-MBP. For both mutants, no detectable 5-DPT, TMA, or tyramine Noxygenase activity was observed (Table 1). These results suggest that the human Met66 f Ile66 FMO3 mutation abrogates human FMO3 N-oxygenation activity. The results are consistent with a role of this mutation as causative for trimethylaminuria. Further studies need to be done to determine whether the human Met66 f Ile66 FMO3 mutation constitutes a secondary starting point for translation mechanisms or whether alteration of the amino acid (i.e., codon 66) causes abnormal protein folding and production of an inactive human FMO3. Recently, a proband with trimethylaminuria was reported in a British family suffering from trimethyl-

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aminuria (5, 20). Sequencing of the DNA showed that the proband carried a base change at codon 153 and resulted in a human Pro153 f Leu153 FMO3 amino acid change (20). This amino acid substitution was not present in the human FMO3 cDNA isolated from trimethylaminuria patients identified from Australia. To examine the N-oxygenase activity of the mutant that was encoded in the human Pro153 f Leu153 FMO3 cDNA substitution, both human Lys158 Leu153 FMO3-MBP and Glu158 Leu153 FMO3-MBP proteins were expressed. Neither mutant showed any detectable 5-DPT, TMA, or tyramine N-oxygenase activity. Thus, the human FMO3 Pro153 f Leu153 amino acid substitution may also be causative for trimethylaminuria. Because proline residues are often associated with fundamental structural motifs in enzymes, it is possible that the human Pro153 f Leu153 FMO3 amino acid change causes protein tertiary structure changes that result in abnormal protein folding and an inactive human FMO3 N-oxygenase. Both glutamic acid and lysine amino acids at codon 158 were examined to investigate whether this amino acid position influenced the mutation at position 66 or 153. In all cases examined, codon 158 did not influence the effect of codon 66 or 153 usage. We conclude that the substitutions at positions 66 and 153 alone are sufficient to cause structural changes in human FMO3 that abrogate the N-oxygenase activity. The ability to N-oxygenate TMA and other amines is distributed polymorphically, at least in the limited number of Caucasian populations studied thus far. The results shown herein support the hypothesis that trimethylaminuria likely represents compound heterozygotes for alleles resulting in deficient human FMO3 function. Trimethylaminuria is a complex phenotype, and detection of the fish-odor syndrome is dependent on dietary and environmental considerations as well as a genetic predetermination and pharmacogenetic polymorphism for diminished or absent activity of the human FMO3 enzyme/gene. Trimethylaminuria patients also suffer from additional metabolic and psychosocial abnormalities (24, 25) including low self-esteem, anxiety, clinical depression, and addiction to drugs (26). Because many of the clinical manifestations of trimethylaminuria (27) can be viewed as perturbations of normal central and sympathetic neural stimulation, it is possible that decreased human FMO3 enzyme function contributes to abnormal biogenic amine metabolism and may contribute to the neurochemical symptomology observed in trimethylaminuria patients.

Acknowledgment. The authors acknowledge helpful discussions with Dr. Alan Brunelle (Seattle, WA) and Dr. Colin Dolphin and Professor Ian Phillips (Queen Mary and Westfield College, London, England). This work was financially supported by the National Institutes of Health (Grant GM 36426).

References (1) Cashman, J. R. (1995) Structural and catalytic properties of the mammalian flavin-containing monooxygenase. Chem. Res. Toxicol. 8, 165-181. (2) Lin, J., Berkman, C. E., and Cashman, J. R. (1996) N-Oxygenation of primary amines and hydroxylamines and retroreduction of hydroxylamines by adult human liver microsomes and adult human flavin-containing monooxygenase 3. Chem. Res. Toxicol. 9, 1183-1193. (3) Al-Waiz, M., Ayesh, R., Mitchell, S. C., Idle, J. R., and Smith, R. L. (1987) Trimethylaminuria (fish odour syndrome): And inborn error of oxidative metabolism. Lancet. 1, 634-635.

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