PMID: 8969926Nov 1, 1996Paper

The use of electrochemistry for the synthesis of 17 alpha-hydroxyprogesterone by a fusion protein containing P450c17

Endocrine Research
R W EstabrookC W Fisher

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 incubatio...Continue Reading

References

Nov 15, 1992·Proceedings of the National Academy of Sciences of the United States of America·C W FisherR W Estabrook
Jul 1, 1991·Proceedings of the National Academy of Sciences of the United States of America·H J BarnesM R Waterman
Aug 15, 1995·Proceedings of the National Academy of Sciences of the United States of America·K M FaulknerR W Estabrook
Dec 15, 1993·Proceedings of the National Academy of Sciences of the United States of America·M S ShetR W Estabrook
Mar 1, 1996·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·M J CoonL L Bestervelt

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Citations

Nov 10, 1998·Clinical and Experimental Pharmacology & Physiology·E M Gillam
Jun 26, 2003·DNA and Cell Biology·Pavel A NazarovLyudmila A Novikova
Dec 12, 2001·Protein Engineering·S G BellL L Wong
Jan 3, 2001·Proceedings of the National Academy of Sciences of the United States of America·O DöhrC R Wolf
May 4, 2018·Biomedit︠s︡inskai︠a︡ khimii︠a︡·A V KuzikovV V Shumyantseva
Mar 8, 2006·Chembiochem : a European Journal of Chemical Biology·Jovana Nazor, Ulrich Schwaneberg
Dec 11, 2007·Chemical Research in Toxicology·Elizabeth M J Gillam
Oct 25, 2001·Archives of Biochemistry and Biophysics·K NakamuraF P Guengerich
Jul 17, 1998·Current Opinion in Chemical Biology·L L Wong
Apr 19, 2005·Journal of Inorganic Biochemistry·Victoria V ShumyantsevaAlexander I Archakov

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