TY - JOUR
T1 - Molecular characterization of cytochrome P450 catalyzing hydroxylation of benzoates from the white-rot fungus Phanerochaete chrysosporium
AU - Matsuzaki, Fumiko
AU - Wariishi, Hiroyuki
N1 - Funding Information:
This research was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (to H.W). The authors thank Professors M. Okamoto and T. Hanai for their help in real-time PCR quantitative analysis.
PY - 2005/9/9
Y1 - 2005/9/9
N2 - 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 448 nm. 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.
AB - 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 448 nm. 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.
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U2 - 10.1016/j.bbrc.2005.07.013
DO - 10.1016/j.bbrc.2005.07.013
M3 - Article
C2 - 16039998
AN - SCOPUS:23044455846
SN - 0006-291X
VL - 334
SP - 1184
EP - 1190
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 4
ER -