Abstract
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.
Highlights
Cytochrome P450 enzymes (CYPs) constitute a large superfamily of heme-containing monooxygenases that are widely distributed in all kingdoms of life (Nelson 2009)
Kamei et al (2006) reported the congener-specific metabolism of 3,3’,4,4’-tetrachlorobiphenyl, 2,3,3’,4,4’-pentachlorobiphenyl, 2,3’,4,4’,5-pentachlorobiphenyl, 3,3’,4,4’,5-pentachlorobiphenyl and 2,3’,4,4’,5,5’-hexachlorobiphenyl in 11 Co-PCBs by P. brevispora and the detection of methoxylated metabolites in the culture containing each congener, suggesting that these metabolites are probably produced via hydroxylation of CoPCBs catalyzed by CYPs
To investigate the involvement of CYPs with the metabolism of dioxins, we first searched for CYP cDNA in P. brevispora
Summary
Cytochrome P450 enzymes (CYPs) constitute a large superfamily of heme-containing monooxygenases that are widely distributed in all kingdoms of life (Nelson 2009). Several studies pointed out that white-rot fungi are capable of degrading certain xenobiotics under culturing conditions that did not induce the production of lignin peroxidase, manganese-dependent peroxidase or laccase (Bumpus and Brock 1988; Mileski et al 1988; Yadav and Reddy 1993; Yadav et al 1995). Besides such lignin-degrading enzymes, alternative oxygenases, CYPs, are apparently involved in catalyzing degradation of several xenobiotics. Since whole genome sequencing of P. chrysosporium has been completed, the molecular diversity of CYPs and the presence of at least 150 CYP genes have been elucidated (Nelson 2009)
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