Identification of active flavin-containing monooxygenase isoform 2 in human lung and characterization of expressed protein.
Full-length human (hFMO2.1) and monkey (mFMO2) flavin-containing monooxygenase proteins, which share 97% sequence identity, were produced by baculovirus-mediated expression in insect cells and assayed for S-oxygenation under conditions known to affect FMO activity. Both enzymes demonstrated maximal activity at pH 9.5; but hFMO2.1 retained significantly more activity than mFMO2 did at pH 9.0 and higher. hFMO2.1 also retained significantly more activity than mFMO2 did in the presence of magnesium and all detergents tested. Although hFMO2.1 had more residual activity after heating at 45 degrees C than mFMO2, under some conditions, both had less than 10% of control activity, whereas expressed rabbit FMO2 retained over 50% activity. Screening for NADPH-oxygenation by hFMO2.1, indicated that substituted thioureas with a small cross-sectional area (2.4-4.3 A) are good substrates, whereas 1,3-diphenylthiourea (11.2 A) was not oxygenated. We confirmed the presence of hFMO2.1 in lung tissue from a heterozygous individual (hFMO2*1/hFMO2*2A) by Western analysis and confirmed activity by S-oxygenation. These microsomes also demonstrated a heat-associated loss of activity similar to expressed hFMO2.1. The heat sensitivity of hFMO2.1 may partially explain why activity in post mortem human lung samples has previously been unreported. Individuals that have the FMO2*1 allele-encoding full-length hFMO2.1 may exhibit altered drug metabolism in the lung.
Krueger
Sharon K
SK
Department of Environmental and Molecular Toxicology, The Linus Pauling Institute, Oregon State University, 571 Weniger, Corvallis, OR 97331-6512, USA.
Martin
Sarah R
SR
Yueh
Mei-Fei
MF
Pereira
Clifford B
CB
Williams
David E
DE
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D003470:Culture Media; D004795:Enzyme Stability; D005260:Female; D005786:Gene Expression Regulation; D006361:Heating; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007313:Insecta / Q000166:cytology; D008168:Lung / Q000201:enzymology* / Q000648:ultrastructure; D008253:Macaca mulatta; D008713:Methimazole / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D013045:Species Specificity; D014162:Transfection
D003470:Culture Media; D008713:Methimazole; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)