Expression and characterization of CYP51, the ancient sterol 14-demethylase activity for cytochromes P450 (CYP), in the white-rot fungus Phanerochaete chrysosporium.
Phanerochaete chrysosporium was the first fungal genome to exhibit more than one hundred cytochrome P450 (CYP) genes for a fungus within its genome (approximately 150). It can metabolize lignocellulose and a wide range of environmental xenobiotics including many carcinogens and pollutants where cytochromes P450 may be involved. In the present paper we describe the heterologous expression and characterization of an ancestral CYP form, sterol 14alpha-demethylase (CYP51-EC1.14.13.70), from this organism. CYP51 was cloned from a cDNA library and expressed in both Escherichia coli, where it exhibited high affinity for azole antifungals, and Saccharomyces cerevisiae. Proof of function was observed by complementation of a conditional knock-down mutant of yeast CYP51. The CYP51 gene was found to be 1956 bases long and contained 7 exons and 6 introns coding for a polypeptide 550 amino acids long (62 kDa). The CYP51 protein exhibited high affinity (k (d) 0.25-0.45 microM) for azole antifungal compounds.
Institute of Life Science, School of Medicine, Swansea University, Singleton Park, Swansea, SA2 8PP, UK.
Andrew
A
Warrilow
Cynthia
C
Ugochukwu
David
D
Lamb
Diane
D
Kelly
Steven
S
Kelly
D000595:Amino Acid Sequence; D001393:Azoles / Q000378:metabolism; D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D016681:Genome, Fungal; D008031:Lignin / Q000378:metabolism; D008969:Molecular Sequence Data; D020075:Phanerochaete / Q000201:enzymology* / Q000235:genetics; D010802:Phylogeny; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D058886:Sterol 14-Demethylase; D015262:Xenobiotics / Q000378:metabolism
D001393:Azoles; D005656:Fungal Proteins; D015262:Xenobiotics; C036909:lignocellulose; D008031:Lignin; D003577:Cytochrome P-450 Enzyme System; D058886:Sterol 14-Demethylase