Expression in Escherichia coli of the cloned flavin-containing monooxygenase from pig liver.
The amino acid sequence of pig liver flavin-containing monooxygenase (PgLFMO) was determined by nucleotide analysis of cDNA clones isolated using synthetic oligonucleotide probes. Most of the clones isolated were missing the 5'-noncoding region and the first seven codons including the putative initiation codon ATG. A full-length cDNA clone, PgLFMO1, was obtained by extending the 5'-end sequence to include an ATG initiation codon and the codons corresponding to the 5'-end using the polymerase chain reaction. Restriction sites for EcoRI and SalI were incorporated at the 5'- and 3'-ends of the cDNA, respectively, by polymerase chain reaction to provide a full-length cDNA clone, pTrcPgLFMO1. Escherichia coli strain NM522 was transformed with pTrcPgLFMO1 and the PgLFMO was expressed under the control of the Trc promoter. Recombinant PgLFMO isolated from the bacterial lysates was purified to homogeneity and found to have N- and S-oxygenation kinetic properties similar to those of the native enzyme. Stereoselective S-oxygenation of (+)- and (-)-4-bromophenyl-1,3-oxathiolane or 2-methyl-1,3-benzodithiole by the expressed PgLFMO showed some differences from that of the native enzyme, however. These data indicate that active PgLFMO was expressed in E. coli but that the enzyme action was distinct from the native enzyme.
Lomri
N
N
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446.
Thomas
J
J
Cashman
J R
JR
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D013552:Swine
D011994:Recombinant Proteins; D004247:DNA; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); D010100:Oxygen