Abstract
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 Km 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 kcat 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.
Footnotes
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This work was supported in part by the National Institutes of Health National Heart, Lung, and Blood Institute [PHS Grant HL038650]; the National Institutes of Health National Institute of Environmental Health Sciences [Grant P30-ES00210]; and the Cell Imaging and Analysis Facility Core and the Proteins Core of the Environmental Health Sciences Center, Oregon State University.
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Part of this work was previously presented as follows: Krueger SK, Henderson MC, Siddens LK, VanDyke JE, Furnes B, Schlenk D, and Williams DE (2007) Assessment of methyl p-tolyl sulfoxidation by human FMO2.1, p.S195L, and p.N413K flavin-containing monooxygenase variants. 2007 Experimental Biology meeting; 2007 Apr 28–May 2; Washington, DC. Federation of American Societies for Experimental Biology, Bethesda, MD.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.109.027201.
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ABBREVIATIONS: FMO, flavin-containing monooxygenase; PHT, phorate; ANTU, α-naphthylthiourea; SNP, single nucleotide polymorphism; PDB, Protein Data Bank; BVMO, Baeyer-Villiger monooxygenase; ETU, ethylenethiourea; HPLC, high-performance liquid chromatography; MTS, methyl-p-tolyl sulfide; MTSO, methyl-p-tolyl sulfoxide.
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The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
- Accepted April 30, 2009.
- Received February 18, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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