A direct electrode-driven P450 cycle for biocatalysis

Abstract

The large potential of redox enzymes to carry out formation of high value organic compounds motivates the search for innovative strategies to regenerate the cofactors needed by their biocatalytic cycles. Here, we describe a bioreactor where the reducing power to the cycle is supplied directly to purified cytochrome CYP101 (P450cam; EC 1.14.15.1) through its natural redox partner (putidaredoxin) using an antimony-doped tin oxide working electrode. Required oxygen was produced at a Pt counter electrode by water electrolysis. A continuous catalytic cycle was sustained for more than 5 h and 2,600 enzyme turnovers. The maximum product formation rate was 36 nmol of 5-exo-hydroxycamphor͞nmol of CYP101 per min.

Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

“…Considering these problems, the use of isolated oxygenases in enzyme reactors could be advantageous. [6][7][8][9] Regarding the application of isolated P450 monooxygenases in fine chemical synthesis, enzymes from bacterial sources turned out as more suitable than those originating from plants, fungi or vertebrates: besides a much higher activity compared to eucaryotic enzymes, bacterial P450s exhibit in many cases higher stability. [10] P450 enzymes consisting of a heme domain fused to an FAD-and FMN-containing P450 reductase domain are -in contrast to the majority of P450s -"self-sufficient", i.e., they do not have to be supplied with additional redox partners (reductases) apart from NADPH.…”

Electrocatalytic Drug Metabolism by CYP2C9 Bonded to A Self-Assembled Monolayer-Modified Electrode

“…Various methods for enzyme immobilization have been explored to solve the problems that were caused by direct attachment (Reipa et al, 1997;Gilardi et al, 2002;Joseph et al, 2003;Johnson et al, 2005;Shumyantseva et al, 2005). These methods include immobilizing the enzyme in a polyion film (Masuda and Fukuda, 1995), entrapping the enzyme in a sol-gel film (Masuda et al, 2000), incorporating the ABBREVIATIONS: P450, cytochrome P450; CPR, cytochrome P450 reductase; SAM, self-assembled monolayer; MUA, 11-mercaptoundecanoic acid; OT, EDC,-N-ethyl carbodiimide hydrochloride; NHS, N-hydroxysulfosuccinimide; PBS, phosphate-buffered solution; XPS, X-ray photoelectron spectroscopy; C 1s, carbon 1s electron; CV, cyclic voltammetry; LC, liquid chromatography; S 2p, sulfur 2p electron; N 1s, nitrogen 1s electron; HPLC, high-performance liquid chromatography.…”

“…In practice, it is difficult to isolate the individual contributions of these factors because they are often interdependent. Thus, it is of interest to devise model platforms that can be used to independently control these parameters to monitor their respective effects on metabolism.P450 catalysis requires a constant supply of NADPH as the electron source and cytochrome P450 reductase (CPR) to deliver the electrons.In attempts to obviate the requirement of these redox partners/cofactors for catalysis, P450 enzymes have been immobilized on electrodes so that the electrode supplies electrons to drive the P450 catalytic cycle (Estabrook et al, 1996;Reipa et al, 1997;Gilardi et al, 2002) with effective electrical communication between the electrode and the enzyme being critical (Yang et al, 2008). Direct adsorption of enzymes on bare electrodes, such as gold, platinum, and graphite, results in diminished biocatalytic activity (Habermüller et al, 2000;Joseph et al, 2003;Shumyantseva et al, 2005).…”

Microbial P450 enzymes in biotechnology

“…Recent improvements include the modification of the electrode surface and/or the use of mediators which assist in the catalytic cycle. So, a bioelectrochemical reactor was constructed where a continuous catalytic cycle from the cathode to P450cam via its natural redox partner -putidaredoxin led to a nearly stoichiometric conversion of camphor (Reipa et al 1997). Artificial redox partners can also assist in the electron transport between electrode and monooxygenase.…”