CYP12A1, A MITOCHONDRIAL CYTOCHROME-P450 FROM THE HOUSE-FLY

Eukaryotic P450 proteins are membrane proteins found predominantly in the endoplasmic reticulum, In vertebrates, several biosynthetic P450s are found in mitochondria as well, We cloned three putative insect mit ochondrial P450s from larval house fly cDNA. These P450s are members o f a new P450 family, CYP12. The CYP12 proteins are most closely relate d to the mammalian mitochondrial P450 of the CYP11, CYP24, and CYP27 f amilies. The most abundant cDNA, CYP12A1, was expressed in Escherichia coli and purified. NADPH-dependent reduction of CYP12A1 was rapid and efficient with the bovine mitochondrial proteins adrenodoxin reductas e and adrenodoxin as electron transfer partners. In contrast, house fl y microsomal NADPH cytochrome P450 reductase reduced CYP12A1 only poor ly. In a reconstituted system with the bovine mitochondrial electron d onors, CYP12A1 metabolized a variety of insecticides and other xenobio tics; but did not metabolize ecdysteroids, juvenoids, or fatty acids. Subcellular localization of CYP12A1 by immunogold histochemistry estab lished the mitochondrial nature of this protein. CYP12A1 mRNA levels a re constitutively higher in an insecticide-resistant strain than in a susceptible strain, and this trait maps to chromosome II: in the house fly, where the constitutive overexpression of the pesticide-metaboliz ing microsomal CYP6A1 also maps, Multiple mitochondrial P450s have evo lved in insects and may play a role in the metabolism of xenobiotics i n addition to their possibly ancestral functions in steroidogenesis. ( C) 1998 Academic Press.