Molecular biology of human xenobiotic-metabolizing cytochromes P450: role of vaccinia virus cDNA expression in evaluating catalytic function

Abstract

Mammalian xenobiotic-metabolizing cytochromes P450s are membrane-bound enzymes that use O 2 and electrons from NADPH to oxidize their substrates. For most chemical substrates, stable metabolites are produced that are destined for further metabolism and elimination from the cell. These enzymes are also capable of metabolically-converting promutagens and procarcinogens to their active proximate metabolites that can kill and transform cells. The xenobiotic-metabolizing P450s reside with three distinct families of the large P450 superfamily. To study the catalytic activities of P450s, particularly human P450s that cannot be easily purified, a cDNA expression system was developed using vaccinia virus. P450 cDNAs incorporated into this lytic virus are efficiently expressed into catalytically-active enzymes that can be used to determine substrate specificities of specific human P450s forms. Activation of the hepatocarcinogen aflatoxin B 1 was determined using a sseries of vaccinia virus-expressed P450s establishing that it is metabolically-activated to a DnA-binding derivative by several human P450 forms, albeit to differing extents.