Manjunath S. Shet

APPLICATION OF ELECTROCHEMISTRY FOR P450-CATALYZED REACTIONS

Publisher Summary The heme proteins called P450 catalyze the metabolism of variety of different chemicals. Many of these are oxidation reactions where P450s function as mixed function oxidases. The oxygen chemistry associated with these reactions permits the introduction of an atom of molecular oxygen in a regio- and stereo-specific manner. P450s are excellent candidates as catalysts for the synthesis of high-value speciality chemicals that are difficult to synthesize by conventional chemical oxidation techniques. In addition to molecular oxygen and the chemical substrate to be metabolized, P450s require a source of electrons (NADPH) and an electron transport protein(s) for the transfer of two electrons (in sequence) from NADPH to the hemeprotein. The use of NADPH as a source of electrons serves as an economic limitation for designing reaction systems that require large-scale incubations and may operate for extended time periods. Electrochemical method for a number of P450-catalyzed reactions is used. The results summarized in the chapter show the applicability of the method for a variety of reactions catalyzed by different P450s. The use of bulk electrolysis to drive a P450-catalyzed reaction, in place of NADPH, permits the construction of reactor systems using P450s as catalysts.

[2] Construction of plasmids and expression in Escherichia coli of enzymatically active fusion proteins containing the heme-domain of a P450 linked to NADPH-P450 reductase

Publisher Summary This chapter discusses the construction of plasmids and expression in Escherichia coli of enzymatically active fusion proteins containing the heme-domain of a P450 linked to NADPH-P450 reductase. The chapter also describes the design of constructs. An important consideration in the creation of any construct by mutagenesis is to minimize the region of DNA that is subjected to mutagenesis and to confirm by sequencing that only the desired mutations have been introduced into this mutagenized region. The use of (low error rate) thermostable DNA polymerases with proofreading activities has been heralded for use in mutagenesis. The chapter discusses development of P450 catalysts that might be of commercial value for chemical synthetic processes. Therefore, it was important to engineer and express fusion proteins that might be immobilized to a matrix permitting the regenerative flow through of reactant chemicals. In addition, it was important to include additional domains in the fusion protein that might influence a P450 reaction, for example the domain for cytochrome b 5 or the presence of a second flavoprotein-domain.

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

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.