Cytochrome P450 oxidoreductase gene and its differentially terminated cDNAs from the white rot fungus Phanerochaete chrysosporium.
The white rot fungus Phanerochaete chrysosporium metabolizes a range of xenobiotics via P450 mono-oxygenation, particularly under peroxidase-suppressing culture conditions. Here we report the cloning and analysis of the gene from this fungus for the cytochrome P450 oxidoreductase (CPR) and its differentially terminated cDNAs. Using a PCR-based approach with degenerate primers, a 285-bp genomic fragment was isolated from the two widely studied strains BKM-F 1767 and ME 446, and was identified as a CPR gene segment based on sequence comparison with the database. A clone containing the full-length CPR gene was isolated from a BKM-F 1767 genomic library using the PCR-generated segment as a probe, and the 3937-bp insert was sequenced by gene walking. Based on the detection of conserved CPR motifs, a coding region of 2381 bp was identified with a 991-bp segment 5' to the putative ATG start codon. Two cDNAs with differentially terminated transcripts were isolated and sequenced. Comparison of the gene and the cDNA sequences confirmed the presence of three introns (62 bp, 50 bp, and 58 bp). Sequence identity and a phylogenetic comparison of the deduced protein (736 aa) with other CPRs in the database suggested that P. chrysosporium CPR is the largest CPR known and is more closely related to animal (36-38%) and yeast (37-38%) CPRs than to plant CPRs (33-35%). The availability of this gene will facilitate further studies on understanding the potent xenobiotic mono-oxygenation systems in this model white rot fungus.
Yadav
J S
JS
Department of Environmental Health, University of Cincinnati, College of Medicine, OH 45267-0056, USA. yadavjs@email.uc.edu
Loper
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics*; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D015723:Gene Library; D005800:Genes, Fungal / Q000235:genetics*; D006801:Humans; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020075:Phanerochaete / Q000201:enzymology* / Q000235:genetics*; D010802:Phylogeny; D020218:Response Elements / Q000235:genetics; D016415:Sequence Alignment; D016385:TATA Box / Q000235:genetics
D018076:DNA, Complementary; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D005486:Flavin Mononucleotide; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase