Abstract

The baculovirus expression vector system was used to overexpress human FMO3 in insect cells for catalytic, structural, and immunochemical studies. Membranes prepared from infectedTrichoplusia 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 andn-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.