title: The use of electrochemistry for the synthesis of 17 alpha-hydroxyprogesterone by a fusion protein containing P450c17.
abstract: A method has been developed for the commercial application of the unique oxygen chemistry catalyzed by various cytochrome P450s. This is illustrated 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 providing electrons at an economically acceptable cost. To generate large amounts of enzymatically active recombinant P450s we have engineered the cDNAs for various P450s, including bovine adrenal P450c17, by linking them to a modified cDNA for rat NADPH-P450 reductase and placing them in the plasmid pCWori+. Transformation of E. coli results in the high level expression of an enzymatically active protein that can be easily purified by affinity chromatography. Incubation of the purified enzyme with steroid in a reaction vessel containing a platinum electrode and a Ag/AgCl electrode couple poised at -650 mV, together with the electromotively 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 concentrations of hydroxylated steroids can be produced with incubation conditions of hours duration without the use of costly NADPH. Similar experiments have been carried out for the generation of the 6 beta-hydroxylation product of testosterone (using a fusion protein containing human P450 3A4). It is apparent that this method is applicable to many other P450 catalyzed reactions for the synthesis of large amounts of hydroxylated steroid metabolites. The electrochemical system is also applicable to drug discovery studies for the characterization of drug metabolites.
authors.affiliation: Department of Biochemistry, University of Texas, Southwestern Medical Center, Dallas 75235-9038, USA.
authors.forename: R W
authors.initials: RW
authors.lastname: Estabrook
authors.forename: M S
authors.initials: MS
authors.lastname: Shet
authors.forename: K
authors.initials: K
authors.lastname: Faulkner
authors.forename: C W
authors.initials: CW
authors.lastname: Fisher
mesh_terms: D019326:17-alpha-Hydroxyprogesterone / Q000138:chemical synthesis*; D000311:Adrenal Glands / Q000201:enzymology; D000818:Animals; D002417:Cattle; D003035:Cobalt; D004563:Electrochemistry; D004566:Electrodes; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006900:Hydroxylation; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D010984:Platinum; D015202:Protein Engineering; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012834:Silver; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D003035:Cobalt; D012834:Silver; D010984:Platinum; D009249:NADP; D019326:17-alpha-Hydroxyprogesterone; D013254:Steroid 17-alpha-Hydroxylase; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase