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Department of Medicinal Chemistry (R.L.H., A.P.H., A.J.M.S.,
A.E.R.), University of Washington; and
Laboratory of Cellular and
Molecular Pharmacology (R.M.P.), National Institute of Environmental
Health Sciences
The baculovirus expression vector system was used to overexpress
human FMO3 in insect cells for catalytic, structural, and immunochemical studies. Membranes prepared from infected
Trichoplusia ni cell suspensions catalyzed NADPH-dependent
metabolism of methyl p-tolyl sulfide at rates 20 times
faster than those obtained with detergent-solubilized human liver
microsomes. Sulfoxidation of the methyl and ethyl p-tolyl
sulfides by recombinant human FMO3 proceeded with little stereochemical
preference, whereas sulfoxidation of the n-propyl and
n-butyl homologs demonstrated increasing selectivity for
formation of the (R)-sulfoxide. This chiral fingerprint
recapitulated the metabolite profile obtained when detergent-treated
human liver microsomes served as the enzyme source. Catalytically
active human FMO3 was purified to apparent homogeneity by cholate
solubilization and sequential column chromatography on Octyl-Sepharose,
DEAE-Sepharose, and hydroxyapatite. Purified FMO3 exhibited the same
electrophoretic mobility as native microsomal enzyme, and
immunoquantitation showed that this isoform represents ~0.5% of
human liver microsomal protein. Therefore, FMO3 is quantitatively a
major human liver monooxygenase. LC/electrospray-mass spectrometry
analysis of purified FMO3 identified >70% of the tryptic peptides,
including fragments containing motifs for N-linked
glycosylation and O-linked glycosylation. Although insect
cells have the capacity for glycan modification, MS analysis of the
tryptic peptides demonstrated that these sites were not modified in the
purified, recombinant enzyme. Edman degradation of the recombinant
product revealed that posttranslational modification of human FMO3 by
insect cells was limited to cleavage at the N-terminal methionine, a process seen in vivo with animal orthologs of
FMO3. These studies demonstrate the suitability of this eukaryotic
system for heterologous expression of human FMOs and future detailed analysis of their substrate specificities.
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