Atypical kinetics of cytochromes P450 catalysing 3'-hydroxylation of flavone from the white-rot fungus Phanerochaete chrysosporium.
We cloned full-length cDNAs of 130 cytochrome P450s (P450s) derived from Phanerochaete chrysosporium and successfully expressed 70 isoforms in Saccharomyces cerevisiae. To elucidate substrate specificity of P. chrysosporium P450s, we examined various substrates including steroid hormones, several drugs, flavonoids and polycyclic aromatic hydrocarbons using the recombinant S. cerevisiae cells. Of these P450s, two CYPs designated as PcCYP50c and PcCYP142c with 14% identity in their amino acid sequences catalyse 3'-hydroxylation of flavone and O-deethylation of 7-ethoxycoumarin. Kinetic data of both enzymes on both reactions fitted not to the Michaelis-Menten equation but to Hill's equation with a coefficient of 2, suggesting that two substrates bind to the active site. Molecular modelling of PcCYP50c and a docking study of flavone to its active site supported this hypothesis. The enzymatic properties of PcCYP50c and PcCYP142c resemble mammalian drug-metabolizing P450s, suggesting that their physiological roles are metabolism of xenobiotics. It is noted that these unique P. chrysosporium P450s have a potential for the production of useful flavonoids.
Kasai
Noriyuki
N
Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, Japan.
Ikushiro
Shinichi
S
Hirosue
Shinji
S
Arisawa
Akira
A
Ichinose
Hirofumi
H
Uchida
Yujirou
Y
Wariishi
Hiroyuki
H
Ohta
Miho
M
Sakaki
Toshiyuki
T
D000595:Amino Acid Sequence; D002384:Catalysis; D020134:Catalytic Domain; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D047309:Flavones; D005419:Flavonoids / Q000737:chemistry* / Q000378:metabolism; D006900:Hydroxylation; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D020075:Phanerochaete / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D047309:Flavones; D005419:Flavonoids; D003577:Cytochrome P-450 Enzyme System; C043562:flavone