Molecular characterization of cytochrome P450 catalyzing hydroxylation of benzoates from the white-rot fungus Phanerochaete chrysosporium.
We cloned full-length cDNA (PcCYP1f) encoding one of the cytochrome P450s in the lignin-degrading basidiomycete Phanerochaete chrysosporium, which showed high homology to P450s in the CYP53 family. PcCYP1f was expressed as an active microsomal protein using the methylotrophic yeast Pichia pastoris expression system. Using the microsomal fraction containing PcCYP1f, a typical P450 CO-difference spectrum was obtained with absorption maximum at 448nm. Recombinant PcCYP1f catalyzed the hydroxylation of benzoic acid into 4-hydroxybenzoic acid in the presence of NADPH and P. chrysosporium cytochrome P450 oxidoreductase. In contrast to other CYP53 P450s, this enzyme was shown to catalyze the hydroxylation of 3-hydroxybenzoate into 3,4-dihydroxybenzoate. Furthermore, 2- and 3-methylbenzoate were also shown to be substrates of PcCYP1f. This is the first report showing the expression of a functionally active Phanerochaete P450. Finally, real-time quantitative PCR analysis revealed that PcCYP1f is induced at a transcriptional level by exogenous addition of benzoic acid.
Matsuzaki
Fumiko
F
Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan.
Wariishi
Hiroyuki
H
D000595:Amino Acid Sequence; D019817:Benzoic Acid / Q000737:chemistry* / Q000494:pharmacology*; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006900:Hydroxylation; D008969:Molecular Sequence Data; D020075:Phanerochaete / Q000187:drug effects / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D019817:Benzoic Acid; D003577:Cytochrome P-450 Enzyme System