P450 in biotechnology:: zinc driven ω-hydroxylation of p-nitrophenoxydodecanoic acid using P450BM-3 F87A as a catalyst

Cytochrome P450 enzymes require the delivery of two electrons to the heme protein for their enzymatic function. NADPH or NADH are usually used as reduction equivalents. In the absence of a substrate, NADPH may inactivate P450 enzymes. Furthermore, it is expensive, making it unsuitable for the preparative synthesis of fine chemicals. Approaches for replacing NADPH with an electrochemically generated reduction by using platinum-electrodes and different mediators are known. In the present study, NADPH was substituted by the mediator cobalt(III)sepulchrate and zinc dust that serves as an electron source. The mutated fatty acid hydroxylase P450 BM-3 F87A from Bacillus megaterium was chosen as a catalyst, since it shows a three-fold higher sensitivity and a nearly five-fold higher activity for p-nitrophenoxydodecanoic acid (12-pNCA) than the wild-type enzyme. The formation of p-nitrophenolate can easily be monitored using a photometer at 410 nm. The turnover rate of the zinc/cobalt(III)sepulchrate system reaches 20% of the NADPH activity. Compared to the electrochemical approaches the activity is at least 77% higher (turnover 125 eq min(-1)). The presented alternative cofactor system can be used instead of NADPH or expensive electrochemical devices (platinum electrodes) for fine chemical synthesis. (C) 2000 Elsevier Science B.V. All rights reserved.