Isolation and sequence analysis of cytochrome P450 12B1: the first mitochondrial insect P450 with homology to 1 alpha,25 dihydroxy-D3 24-hydroxylase.

The biosynthesis of steroid hormones is an integral component of insect growth, development and reproduction. Although there is an abundance of biochemical data implicating both microsomal and mitochondrial cytochrome P450s in steroid metabolism, molecular genetic information on mitochondrial P450s is almost entirely limited to vertebrate sequences. In the current study, a degenerate polymerase chain reaction (PCR) primer was targeted to the highly conserved region of P450 genes that encodes the heme-binding decapeptide. Using a 5' rapid amplification of cDNA ends (RACE) approach, seven novel cytochrome P450 genes were isolated from Drosophila acanthoptera, including one sequence (CYP12B1) with high regional homology to vertebrate mitochondrial P450s. Sequence analysis of the conceptual translation of the full length gene, obtained by 5'RACE, revealed an amphipathic NH2-terminus rich in basic and hydrophilic amino acids, a characteristic feature of mitochondrial P450s that distinguishes them from their distantly related microsomal relatives. Phylogenetically, CYP12B1 appears to be most closely related to the mammalian mitochondrial P450s of the CYP24 family that play a critical role in calcium homeostasis through the metabolism of vitamin D3. The potential biological role of CYP12B1 is discussed in the light of what is currently known about arthropod calcium binding proteins and their regulation.