BACULOVIRUS-MEDIATED EXPRESSION AND PURIFICATION OF HUMAN FMO3 - CATALYTIC, IMMUNOCHEMICAL, AND STRUCTURAL CHARACTERIZATION

The baculovirus expression vector system was used to overexpress human FMO3 in insect cells for catalytic, structural, and immunochemical st udies. Membranes prepared from infected Trichoplusia ni cell suspensio ns catalyzed NADPH-dependent metabolism of methyl p-tolyl sulfide at r ates 20 times faster than those obtained with detergent-solubilized hu man liver microsomes. Sulfoxidation of the methyl and ethyl p-tolyl su lfides 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)-sulfoxid e. This chiral fingerprint recapitulated the metabolite profile obtain ed when detergent-treated human liver microsomes served as the enzyme source. Catalytically active human FMO3 was purified to apparent homog eneity by cholate solubilization and sequential column chromatography on Octyl-Sepharose, DEAE-Sepharose,and hydroxyapatite. Purified FMO3 e xhibited the same electrophoretic mobility as native microsomal enzyme , and immunoquantitation showed that this isoform represents similar t o 0.5% of human liver microsomal protein. Therefore, FMO3 is quantitat ively a major human liver monooxygenase. LC/electrospray-mass spectrom etry analysis of purified FMO3 identified >70% of the tryptic peptides , including fragments containing motifs far N-linked glycosylation and O-linked glycosylation. Although insect cells have the capacity for g lycan modification, MS analysis of the tryptic peptides demonstrated t hat these sites were not modified in the purified, recombinant enzyme. Edman degradation of the recombinant product revealed that posttransl ational modification of human FMO3 by insect cells was limited to clea vage at the N-terminal methionine, a process seen in vivo with animal orthologs of FMO3. These studies demonstrate the suitability of this e ukaryotic system for heterologous expression of human FMOs and future detailed analysis of their substrate specificities.