Heterologous expression of CYP2K1 and identification of the expressed protein (BV-CYP2K1) as lauric acid (omega-1)-hydroxylase and aflatoxin B1 exo-epoxidase.
LMC2 is the most abundant constitutively expressed hepatic cytochrome P450 found in sexually immature rainbow trout (Onchorynchus mykiss) and is also the isozyme that activates the carcinogen aflatoxin B1 (AFB1). This P450 has been cloned, sequenced, and designated as CYP2K1. The present report describes the heterologous expression of enzymatically active CYP2K1 (BV-CYP2K1) in baculovirus Spodoptera frugiperda (Sf9) insect cells and its catalytic and immunoreactivity characterization in comparison with that of the previously purified LMC2 P450. Homogenates of Sf9 cells expressing the CYP2K1 enzyme and LMC2 both catalyzed the hydroxylation of lauric acid and the epoxidation of AFB1 in the presence of rat NADPH-cytochrome P450 reductase. Both LMC2 and BV-CYP2K1 catalyzed the oxidation of lauric acid primarily at the (omega-1) position plus small amounts at the (omega-2) position. Formation of AFB1 epoxide was shown indirectly by the appearance of an AFB1 epoxide-glutathione conjugate when P450 incubation mixtures contained AFB1, glutathione (GSH) together with mouse liver cytosol or purified rat GSH-transferase. When the AFB1 epoxide-GSH conjugate produced by BV-CYP2K1 and purified LMC2 was analyzed by HPLC using a chiral column, it had a retention time identical to that produced by CYP3A4, a human P450 known to form exclusively the AFB1 exo-epoxide. These results, therefore, confirm that the cDNA-expressed CYP2K1 protein is catalytically and immunologically identical to purified trout LMC2 and that these two enzymes produce primarily the highly carcinogenic stereoisomeric exo-epoxide form of AFB1.
Yang
Y H
YH
Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon 97331-7301, USA.
Miranda
C L
CL
Henderson
M C
MC
Wang-Buhler
J L
JL
Buhler
D R
DR
D016604:Aflatoxin B1 / Q000378:metabolism; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D017931:DNA Primers; D018076:DNA, Complementary; D029941:Fish Proteins; D006801:Humans; D007850:Lauric Acids / Q000378:metabolism; D051379:Mice; D017686:Oncorhynchus mykiss; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D013250:Steroid Hydroxylases / Q000378:metabolism*
D017931:DNA Primers; D018076:DNA, Complementary; D029941:Fish Proteins; D007850:Lauric Acids; D011994:Recombinant Proteins; C030358:lauric acid; D003577:Cytochrome P-450 Enzyme System; D016604:Aflatoxin B1; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C088323:CYP2K1 protein, Oncorhynchus mykiss; D000072467:Cytochrome P450 Family 2