The Flavin-containing Monooxygenase 2 Gene (FMO2) of Humans, but Not of Other Primates, Encodes a Truncated, Nonfunctional Protein (original) (raw)
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Organization and evolution of the flavin-containing monooxygenase genes of human and mouse
Pharmacogenetics, 2004
Objectives To date, six flavin-containing monooxygenase (FMO) genes have been identified in humans, FMOs 1, 2, 3, 4 and 6, which are located within a cluster on chromosome 1, and FMO5, which is located outside the cluster. The objectives were to review and update current knowledge of the structure and expression profiles of these genes and of their mouse counterparts and to determine, via a bioinformatics approach, whether other FMO genes are present in the human and mouse genomes.
Drug Metabolism and Disposition, 2009
Catalytically active human flavin-containing monooxygenase isoform 2 (FMO2.1) is encoded by an allele detected only in individuals of African or Hispanic origin. Genotyping and haplotyping studies indicate that S195L and N413K occasionally occur secondary to the functional FMO2*1 allele encoding reference protein Gln472. Sulfoxygenation under a range of conditions reveals the role these alterations may play in individuals expressing active FMO2 and provides insight into FMO structure. Expressed S195L lost rather than gained activity as pH was increased or when cholate was present. The activity of S195L was mostly eliminated after heating at 45°C for 5 min in the absence of NADPH, but activity was preserved if NADPH was present. By contrast, Gln472 was less sensitive to heat, a response not affected by NADPH. A major consequence of the S195L mutation was a mean 12-fold increase in K m for NADPH compared with Gln472. Modeling an S213L substitution, the equivalent site, in the structural model of FMO from the Methylophaga bacterium leads to disruption of interactions with NADP ؉. N413K had the same pattern of activity as Gln472 in response to pH, cholate, and magnesium, but product formation was always elevated by comparison. N413K also lost more activity when heated than Gln472; however, NADPH attenuated this loss. The major effects of N413K were increases in velocity and k cat compared with Gln472. Although these allelic variants are expected to occur infrequently as mutations to the FMO2*1 allele, they contribute to our overall understanding of mammalian FMO structure and function.
Pharmacogenetics, 2004
Objectives To date, six flavin-containing monooxygenase (FMO) genes have been identified in humans, FMOs 1, 2, 3, 4 and 6, which are located within a cluster on chromosome 1, and FMO5, which is located outside the cluster. The objectives were to review and update current knowledge of the structure and expression profiles of these genes and of their mouse counterparts and to determine, via a bioinformatics approach, whether other FMO genes are present in the human and mouse genomes.
Drug Metabolism and Disposition, 2006
To characterize the contribution of amino acid 360 to the functional activity of the human flavin-containing monooxygenase form 3 (FMO3) and form 1 (FMO1) in the oxygenation of drugs and chemicals, we expressed four FMO3 variants (i.e., Ala 360-FMO3, His 360-FMO3, Gln 360-FMO3 and Pro 360-FMO3) and one FMO1 variant (i.e., Pro 360-FMO1) and compared them to wild-type enzymes (Leu 360-FMO3 and His 360-FMO1), respectively. The amino acid substitutions were introduced into wild-type FMO3 or FMO1 cDNA by site directed mutagenesis. The thermal stability of variants of Leu 360 FMO3 was also studied and the thermal stability was significantly different from that of wild-type FMO3. The influence of different substrates to modulate the catalytic activity of FMO3 variants was also examined. Selective functional substrate activity was determined with mercaptoimidazole, chlorpromazine and 10-[(N,Ndimethylaminopentyl)-2-(trifluoromethyl)] phenothiazene. Compared with wild-type FMO3, the Ala 360-FMO3, and His 360-FMO3 variants were less catalytically efficient for mercaptoimidazole S-oxygenation. N-Oxygenation of chlorpromazine was significantly less catalytically efficient for His 360-FMO3 compared with wild-type FMO3. Human Pro 360-FMO1 was significantly more catalytically efficient at S-oxygenating mercaptoimidazole and chlorpromazine compared with wild-type FMO1. The data support the mechanism that the Pro 360 loci affect thermal stability of FMO3. Because different amino acids at position 360 affect substrate oxygenation in a unique fashion from that of FMO3 stimulation, we conclude that the mechanism of stimulation of FMO3 is distinct from that of enzyme catalysis. A molecular model of human FMO3 was also constructed to help explain the results. The increase in catalytic efficiency observed for This article has not been copyedited and formatted. The final version may differ from this version.
2006
Departments of Pediatrics (S.B.K., M.T.P., R.N.H.), Pharmacology and Toxicology (R.N.H.), and Biostatistics (N.M.P., T.W.), Medical College of Wisconsin, and Children's Research Institute, Children's Hospital and Health System (S.B.K., M.T.P., R.N.H.), Milwaukee, Wisconsin; and Department of Environmental and Molecular Toxicology, The Linus Pauling Institute, Oregon State University, Corvallis, Oregon (M.C.H., L.K.S., S.K.K., J.E.V., D.E.W.) JPET Fast Forward. Published on October 18, 2006 as DOI:10.1124/jpet.106.112268
Molecular …, 2005
The flavin-containing monooxygenases (FMOs) are important for xenobiotic metabolism. FMO3, the predominant FMO enzyme in human adult liver, exhibits significant interindividual variation that is poorly understood. This study was designed to identify common FMO3 genetic variants and determine their potential for contributing to interindividual differences in FMO3 expression. FMO3 single nucleotide polymorphism (SNP) discovery was accomplished by re-sequencing DNA samples from the Coriell Polymorphism Discovery Resource. Population-specific SNP frequencies were determined by multiplexed, single-base extension using DNA from 201 Hispanic-American (Mexican descent), 201 African-American, and 200 non-Latino White (northern European descent) subjects. Haplotypes were inferred and population frequencies estimated using PHASE V2.1. Multisite-directed mutagenesis was used to introduce inferred upstream haplotypes into an FMO3/ luciferase construct for functional analysis in HepG2 cells. Sequence analysis revealed seven FMO3 upstream SNPs, eleven exon SNPs, and twenty-two intron SNPs. Five of the latter fell within consensus splice sites. A g.72G>T variant (E24D) is predicted to impact the structure of the Rossmann fold involved in FAD binding, while a g.11177C>A variant (N61K) is predicted to disrupt the secondary structure of a conserved membrane interaction domain. Seven common (>1%) promoter region haplotypes were inferred in one or more of the study populations that differed in estimated frequency among the groups. Haplotype 2 resulted in an 8-fold increase in promoter activity, while haplotype 8 and 15 exhibited a near complete loss of activity. In conclusion, FMO3 promoter haplotype variants modulate gene function and likely contribute to interindividual differences in FMO3 expression. The flavin-containing monooxygenases (FMOs) (EC 1.14.13.8) are a family of NADPHand oxygen-dependent microsomal enzymes involved in the oxidative metabolism of many nucleophilic nitrogen-, sulfur-and phosphorous-containing drugs and toxicants (Cashman, 2002). Multiple human FMO genes have been identified: a five gene cluster at 1q24.3 (FMO1-4 and FMO6p) that encodes four active enzymes (FMO1-4), a second cluster of five
Archives of Biochemistry and Biophysics, 2021
Human flavin-containing monooxygenase 3 (FMO3) is a membrane-bound, phase I drug metabolizing enzyme. It is highly polymorphic with some of its variants demonstrating differences in rates of turnover of its substrates: xenobiotics including drugs as well as dietary compounds. In order to measure its in vitro activity and compare any differences between the wild type enzyme and its polymorphic variants, we undertook a systematic study using different engineered proteins, heterologously expressed in bacteria, purified and catalytically characterized with 3 different substrates. These included the full-length as well as the more soluble C-terminal truncated versions of the common polymorphic variants (E158K, V257M and E308G) of FMO3 in addition to the fulllength and truncated wild-type proteins. In vitro activity assays were performed with benzydamine, tamoxifen and sulindac sulfide, whose products were measured by HPLC. Differences in catalytic properties between the wild-type FMO3 and its common polymorphic variants were similar to those observed with the truncated, more soluble versions of the enzymes. Interestingly, the truncated enzymes were better catalysts than the full-length proteins. The data obtained point to the feasibility of using the more soluble forms of this enzyme for in vitro drug assays as well as future biotechnological applications possibly in high throughput systems such as bioelectrochemical platforms and biosensors.
Expression and Characterization of a Modified Flavin-containing Monooxygenase 4 from Humans
Journal of Biological Chemistry, 1996
The inability to obtain flavin-containing monooxygenase 4 (FMO4) in heterologous systems has hampered efforts to characterize this isoform of the FMO gene family. Neither the human nor the rabbit ortholog of FMO4, each of which has been cloned and sequenced, has been expressed. Attempts to achieve expression of FMO4 have been made with Escherichia coli, baculovirus, yeast, and COS systems. The cDNAs encoding FMO4 have extended coding regions compared with those encoding other FMO isoforms. The derived amino acid sequences of FMO1,-2,-3, and-5 from all species examined contain about the same number of residues (531-535 residues), whereas the derived sequences of human and rabbit FMO4 contain 558 and 555 residues, respectively. We have investigated whether the elongation of the FMO4 coding region is related to the inability to achieve expression. The cDNA encoding human FMO4 has been modified by a single base change that introduces a stop codon at the consensus position. This modification allows for expression in E. coli. Lack of expression of intact FMO4 is caused by a problem that occurs following transcription, a problem that is overcome completely by relocation of the stop codon 81 bases to 5 of its normal position. Truncated FMO4 is expressed as an active enzyme with characteristics typical of an FMO isoform. Possible functional changes resulting from altering the 3-end of an FMO were investigated with human FMO3. Elongation of the coding region of the FMO3 cDNA to the next available stop codon (FMO3*) resulted in the expression of an enzyme with properties very similar to those of unmodified FMO3. Elongation of FMO3 lowered the level of expression in E. coli but did not eliminate it. As with FMO4, the difference in expression levels between FMO3 and elongated FMO3 (FMO3*) appears to be related to translation rather than transcription. The functional characteristics of FMO3 and FMO3* are not significantly different. The flavin-containing monooxygenase (FMO, 1 EC 1.14.13.8) gene family encodes a minimum of five isoforms that catalyze