THE USE OF ELECTROCHEMISTRY FOR THE SYNTHESIS OF 17-ALPHA-HYDROXYPROGESTERONE BY A FUSION PROTEIN CONTAINING P450C17

A method has been developed for the commercial application of the uniq ue oxygen chemistry catalyzed by various cytochrome P450s. This is ill ustrated here for the synthesis of hydroxylated steroids. This method requires the preparation of large amounts of enzymatically functional P450 proteins that can serve as catalysts and a technique for providin g electrons at an economically acceptable cost. To generate large amou nts of enzymatically active recombinant P450s we have engineered the c DNAs for various P450s, including bovine adrenal P450c17, by linking t hem to a modified cDNA for rat NADPH-P450 reductase and placing them i n the plasmid pCWori(+). Transformation of E. coli results in the high level expression of an enzymatically active protein that can be easil y purified by affinity chromatography. Incubation of the purified enzy me with steroid in a reaction vessel containing a platinum electrode a nd a Ag/AgCl electrode couple poised at -650 mV, together with the ele ctromotively active redox mediator, cobalt sepulchrate, results in the 17 alpha-hydroxylation of progesterone at rates as high as 25 nmoles of progesterone hydroxylated/min/nmole of P450. Thus, high concentrati ons of hydroxylated steroids can be produced with incubation condition s of hours duration without the use of costly NADPH. Similar experimen ts have been carried out for the generation of the 6 beta-hydroxylatio n product of testosterone (using a fusion protein containing human P45 0 3A4). It is apparent that this method is applicable to many other P4 50 catalyzed reactions for the synthesis of large amounts of hydroxyla ted steroid metabolites. The electrochemical system is also applicable to drug discovery studies for the characterization of drug metabolite s.