EXPRESSION AND CHARACTERIZATION OF A MODIFIED FLAVIN-CONTAINING MONOOXYGENASE-4 FROM HUMANS

The inability to obtain flavin-containing monooxygenase 4 (FMO4) in he terologous systems lass hampered efforts to characterize this isoform of the FMO gene family. Neither the human nor the rabbit ortholog of F MO4, each of which has been cloned and sequenced, has been expressed. Attempts to achieve expression of FMO4 have beets made with Escherichi a coli, baculovirus, yeast, and COS systems. The cDNAs encoding FMO4 h ave extended coding regions compared with those encoding other FMO iso forms, The derived amino acid sequences of FMO1, -2, -3, and -5 from a ll species examined contain about the same number of residues (531-535 residues), whereas the derived sequences of human and rabbit FMO4 con tain 558 and 555 residues, respectively. We have investigated whether the elongation of the FMO4 coding region is related to the inability t o achieve expression, The cDNA encoding human FMO4 has been modified b y a single base change that introduces a stop codon at the consensus p osition. This modification allows for expression in E. coli, Lack of e xpression of intact FMO4 is caused by a problem that occurs following transcription, a problem that is overcome completely by relocation of the stop codon 81 bases to 5' of its normal position, Truncated FMO4 i s expressed as an active enzyme with characteristics typical of an FMO isoform. Possible functional changes resulting from altering the 3'-e nd of an FMO were investigated with human FMO3. Elongation of the codi ng region of the FMO3 cDNA to the next available stop codon (FMO3) re sulted in the expression of an enzyme with properties very similar to those of unmodified FMO3. Elongation of FMO3 lowered the level of expr ession in E. coli but did not eliminate it. As with FMO4, the differen ce in expression levels between FMO3 and elongated FMO3 (FMO3) appear s to be related to translation rather than transcription, The function al characteristics of FMO3 and FMO3 are not significantly different.