Molecular Lego: design of molecular assemblies of P450 enzymes for nanobiotechnology

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).…”

“…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 enzyme into conducting polymer films (Funk et al, 2005), modifying the enzyme with an electrical relay (Hahm and Lieber, 2003), functionalizing the electrode with a biomembrane-like surfactant (Ohldag et al, 2006), and supporting the enzyme in a clay nanoparticle modified electrode (Shumyantseva et al, 2004).…”

Immobilisation of P450 BM‐3 and an NADP+ Cofactor Recycling System: Towards a Technical Application of Heme‐Containing Monooxygenases in Fine Chemical Synthesis

“…[20] Therefore, an in vitro reaction process would be highly desirable if a cost efficient way of supplying electrons to the heme iron can be found. The use of electron mediators [25] , direct electron supply from electrodes [26,27] as well as enzymatic [15,17] and organometallic [28] approaches have been suggested as sources of the required reduction equivalents.…”

Strategies for the construction of insect P450 fusion enzymes

“…PUPPET is a platform in which three proliferating cell nuclear antigen (PCNA) fusion proteins are used to form a heterotrimer that recruits P450 into a complex with its electron donors, hence PUPPET refers to "proliferating cell nuclear antigen-utilized protein complex of P450 and its two electron transfer-related proteins" [61,62]. Finally, the "Molecular Lego" method involves the combination of P450 enzymes with the BMR domain from the natural protein BM3 described above, to make recombinant enzymes with novel catalytic activities [63]. Soluble, self-sufficient human P450-BMR fusion enzymes have been created by removing the hydrophobic N-terminal membrane anchor domain from the insoluble human enzyme before fusion to the BMR sequence ( Figure 5).…”