Heterologous expression and mechanistic investigation of a fungal cytochrome P450 (CYP5150A2): involvement of alternative redox partners.
A fungal cytochrome P450 monooxygenase (CYP5150A2) from the white-rot basidiomycete Phanerochaete chrysosporium was heterologously expressed in Escherichia coli and purified as an active form. The purified CYP5150A2 was capable of hydroxylating 4-propylbenzoic acid (PBA) with NADPH-dependent cytochrome P450 oxidoreductase (CPR) as the single redox partner; the reaction efficiency was improved by the addition of electron transfer protein cytochrome b5 (Cyt-b5). Furthermore, CYP5150A2 exhibited substantial activity with redox partners Cyt-b5 and NADH-dependent Cyt-b5 reductase (CB5R) even in the absence of CPR. These results indicated that a combination of CB5R and Cyt-b5 may be capable of donating both the first and the second electrons required for the monooxygenation reaction. Under reaction conditions in which the redox system was associated with the CB5R-dependent Cyt-b5 reduction system, the exogenous addition of CPR and NADPH had no effect on the PBA hydroxylation rate or on coupling efficiency, indicating that the transfer of the second electron from Cyt-b5 was the rate-limiting step in the monooxygenase system. In addition, the rate of PBA hydroxylation was significantly dependent on Cyt-b5 concentration, exhibiting Michaelis-Menten kinetics. This study provides indubitable evidence that the combination of CB5R and Cyt-b5 is an alternative redox partner facilitating the monooxygenase reaction catalyzed by CYP5150A2.
Kyushu University, Hakozaki, Higashi-ku, Fukuoka, Japan. ichinose@agr.kyushu-u.ac.jp
Hirofumi
H
Ichinose
Hiroyuki
H
Wariishi
D000595:Amino Acid Sequence; D055162:Biocatalysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase / Q000378:metabolism; D015786:Cytochromes b5 / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D020075:Phanerochaete / Q000201:enzymology* / Q000235:genetics*; D013057:Spectrum Analysis
D009243:NAD; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D042966:Cytochrome-B(5) Reductase