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Sequencing, expression, and characterization of cDNA expressed flavin‐containing monooxygenase 2 from mouse
Author(s) -
Karoly Edward D.,
Rose Randy L.
Publication year - 2001
Publication title -
journal of biochemical and molecular toxicology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.526
H-Index - 58
eISSN - 1099-0461
pISSN - 1095-6670
DOI - 10.1002/jbt.10009
Subject(s) - complementary dna , flavin containing monooxygenase , microbiology and biotechnology , chemistry , monooxygenase , nucleic acid sequence , biochemistry , sequence analysis , expression vector , biology , enzyme , gene , recombinant dna , cytochrome p450
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‐β‐D‐thiogalactopyanoside (IPTG) induced cells was heat stable (45°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. © 2001 John Wiley & Sons, Inc. J Biochem Mol Toxicol 15:300–308, 2001