Three cytochrome P450 monooxygenase (CYP) genes, designated pb-1, pb-2 and pb-3, were isolated from the white-rot fungus, Phlebia brevispora, using reverse transcription PCR with degenerate primers constructed based on the consensus amino acid sequence of eukaryotic CYPs in the O2-binding, meander and heme-binding regions. Individual full-length CYP cDNAs were cloned and sequenced, and the relative nucleotide sequence similarity of pb-1 (1788 bp), pb-2 (1881 bp) and pb-3 (1791 bp) was more than 58%. Alignment of the deduced amino acid (aa) sequences of pb-1-pb-3 showed that these three CYPs belong to the same family with > 40% aa sequence similarity, and pb-1 and pb-3 are in the same subfamily, with > 55% aa sequence similarity. Furthermore, pb-1-pb-3 appeared to be a subfamily of CYP63A (CYP63A1-CYP63A4), found in Phanerochaete chrysosporium. The phylogenetic tree constructed by 500 bootstrap replications using the neighbor-joining method showed that the evolutionary distance between pb-1 and pb-3 was shorter than that between pb-2 and pb-1 (or pb-3). Exon-intron analysis of pb-1 and pb-3 showed that both genes have nearly the same number, size and order of exons and the types of introns, also indicating both genes appear to be evolutionarily close. It is interesting that the transcription level of pb-3 was evidently increased above the pb-1 transcription level by exposure to 12 coplanar PCB congeners and 2,3,7,8-tetrachlorodibenzo-p-dioxin, though the two genes were evolutionarily close.
Keywords: cytochrome P450 monooxygenase, Phlebia brevispora, gene cloning, real-time RT-PCR, dioxins, CYP63A