Research Article: Identification of cytochrome P450 monooxygenase genes from the white-rot fungus Phlebia brevispora

Date Published: January 25, 2012

Publisher: Springer

Author(s): Ryoich Nakamura, Ryuichiro Kondo, Ming-hao Shen, Hideharu Ochiai, Shin Hisamatsu, Shigenori Sonoki.


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.

Partial Text

Cytochrome P450 enzymes (CYPs) constitute a large superfamily of heme-containing monooxygenases that are widely distributed in all kingdoms of life (Nelson 2009). CYPs are involved in the metabolism of a wide variety of endogenous and xenobiotic compounds by catalyzing regio- and stereospecific monooxygenation with an oxygen atom generated from molecular oxygen. Mammalian CYPs have been studied extensively because of their leading role in drug and xenobiotic metabolism and detoxification (Allis et al. 2002; Inouye et al. 2002; McGraw JE and Waller 2006; Shimada 2006; Vrba et al. 2004; Warner et al. 2009; Yamazaki 2000; Zhang et al. 2006). CYPs from bacteria, yeast and fungi have also been well studied in the biosynthesis of essential compounds like ergosterol, which is a constituent of fungal cell membranes, and in the detoxification and biodegradation of a broad spectrum of environmental chemical pollutants (Kelly et al. 1997; Kelly et al. 2003; Lamb et al. 2000; Seth-Smith et al. 2008; van den Brink et al. 1998).

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 Co-PCBs catalyzed by CYPs. To investigate the involvement of CYPs with the metabolism of dioxins, we first searched for CYP cDNA in P. brevispora. There is little information concerning CYP genes from P. brevispora; however, some useful information about nucleotide sequences of CYP cDNAs from P. chrysosporium is available. Hence, two sets of degenerate primers were constructed to search for CYP cDNAs from P. brevispora, as shown in Table 1, based on the nucleotide sequence of CYP cDNAs from P. chrysosporium presented by Kullman and Matsumura (1997), and the nucleotide sequences registered on the cytochrome P450 homepage organized by Nelson (2009). We describe three unique full-length cDNAs encoding CYP genes pb-1, pb-2 and pb-3 in P. brevispora. As a result of BLAST nucleotide sequence homology searching of these three CYP cDNAs, we found they were closely related to the members of the representative multigene family CYP63, CYP63A1-CYP63A4, found in P. chrysosporium (Doddapaneni et al. 2005; Doddapaneni and Yadav 2004; Nelson 2009). CYPs are classified and named based primarily on the level of aa sequence similarity. A family is generally defined as those CYPs having > 40% aa sequence similarity, and a subfamily is defined as those CYPs having > 55% aa sequence similarity. The deduced aa sequence alignments of pb-1-pb-3 showed that these three CYPs are members of the same family, and pb-1 and pb-3 are in the same subfamily. Furthermore, pb-1 and pb-3 appeared to belong to the subfamily of CYP63A1-CYP63A3, and pb-2 to CYP63A4.

The authors declare that they have no competing interests.