Sequencing, expression, and characterization of cDNA expressed flavin-containing monooxygenase 2 from mouse.
The cDNA clone of mouse flavin-containing monooxygenase 2 (FMO2) was obtained as an expressed sequence tag (EST) isolated from a female mouse kidney cDNA library from the I.M.A.G.E. consortium (I.M.A.G.E. CloneID 1432164). Complete sequencing of the EST derived a nucleotide sequence for mouse FMO2, which contains 112 bases of 5' flanking region, 1607 bases of coding region, and 309 bases of 3' flanking region. This FMO2 sequence encodes a protein of 535 amino acids including two putative pyrophosphate binding sequences (GxGxxG/A) beginning at positions 9 and 191. Additionally, this mouse FMO protein sequence shows 87 and 86% homology to rabbit and human FMO2 respectively. The mouse FMO2 sequence was subcloned into the expression vector pJL-2, a derivative of pKK233-2 and used to transform XL1-Blue Escherichia coli. FMO activity in particulate fractions isolated from isopropyl-beta-D-thiogalactopyanoside (IPTG) induced cells was heat stable (45 degrees C for 5 min) and demonstrated optimal activity at a relatively high pH of 10.5. The expressed FMO2 enzyme showed catalytic activity towards the FMO substrate methimazole and further analysis of E. coli fractions utilizing NADPH oxidation demonstrated that the mouse FMO2 enzyme also exhibits catalytic activity towards thiourea, trimethylamine, and the insecticide phorate.
Karoly
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Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695, USA.
Rose
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D020224:Expressed Sequence Tags; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018076:DNA, Complementary; D009249:NADP; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)