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

Cytochrome P450 enzymes require the delivery of two electrons to the heme p rotein for their enzymatic function. NADPH or NADH are usually used as redu ction equivalents. In the absence of a substrate, NADPH may inactivate P450 enzymes. Furthermore, it is expensive, making it unsuitable for the prepar ative synthesis of fine chemicals. Approaches for replacing NADPH with an e lectrochemically generated reduction by using platinum-electrodes and diffe rent mediators are known. In the present study, NADPH was substituted by th e mediator cobalt(III)sepulchrate and zinc dust that serves as an electron source. The mutated fatty acid hydroxylase P450 BM-3 F87A from Bacillus meg aterium was chosen as a catalyst, since it shows a three-fold higher sensit ivity and a nearly five-fold higher activity for p-nitrophenoxydodecanoic a cid (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. C ompared to the electrochemical approaches the activity is at least 77% high er (turnover 125 eq min(-1)). The presented alternative cofactor system can be used instead of NADPH or expensive electrochemical devices (platinum el ectrodes) for fine chemical synthesis. (C) 2000 Elsevier Science B.V. All r ights reserved.