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 ha s been cloned, sequenced, and designated as CYP2K1. The present report desc ribes 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 previ ously purified LMC2 P450. Homogenates of Sf9 cells expressing the CYP2K1 en zyme and LMC2 both catalyzed the hydroxylation of lauric acid and the epoxi dation 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, glutath ione (GSH) together with mouse liver cytosol or purified rat GSH-transferas e. When the AFB1 epoxide-GSH conjugate produced by BV-CYP2K1 and purified L MC2 was analyzed by HPLC using a chiral column, it had a retention time ide ntical to that produced by CYP3A4, a human P450 known to form exclusively t he AFB1 exo-epoxide. These results, therefore, confirm that the cDNA-expres sed CYP2K1 protein is catalytically and immunologically identical to purifi ed trout LMC2 and that these two enzymes produce primarily the highly carci nogenic stereoisomeric exo-epoxide form of AFB1.