Interactive biocatalysis achieved by driving enzyme cascades inside a porous conducting material

Abstract

An emerging concept and platform, the electrochemical Leaf (e-Leaf), offers a radical change in the way tandem (multi-step) catalysis by enzyme cascades is studied and exploited. The various enzymes are loaded into an electronically conducting porous material composed of metallic oxide nanoparticles, where they achieve high concentration and crowding - in the latter respect the environment resembles that found in living cells. By exploiting efficient electron tunneling between the nanoparticles and one of the enzymes, the e-Leaf enables the user to interact directly with complex networks, rendering simultaneous the abilities to energise, control and observe catalysis. Because dispersion of intermediates is physically suppressed, the output of the cascade - the rate of flow of chemical steps and information - is delivered in real time as electrical current. Myriad enzymes of all major classes now become effectively electroactive in a technology that offers scalability between micro-(analytical, multiplex) and macro-(synthesis) levels. This Perspective describes how the e-Leaf was discovered, the steps in its development so far, and the outlook for future research and applications.

Fig. 1. The cascade enzymes are loaded into a porous electrode material.

a SEM image of a layer of indium tin oxide nanoparticles (size distribution 10–50 nm) deposited on indium tin oxide glass. b A helpful metaphor: bricks thrown into a pile create random spaces having dimensions relating in size to the bricks. c Enzymes deposited onto the surface enter the pores and permeate throughout the porous layer. The image at the right shows an impression of magnified pores crowded with enzymes (ferredoxin-NADP⁺ reductase, mean diameter ~55 Å, and two other enzymes, mean diameters 108 Å and 160 Å); enzymes and pores are to scale.

Fig. 2. Stages in the discovery of the e-Leaf.

a Cyclic voltammogram showing the reduction and reoxidation of the FAD cofactor in FNR when it is embedded in a porous ITO electrode. The result shown refers to pH 8.0. The narrow widths of oxidation and reduction peaks signified a two-electron transfer process having significant cooperativity and very little dispersion^, despite the unusual environment. b Achieving the reversible catalytic electrochemistry of NADP⁺/NADPH (red cyclic voltammogram, overlaid with the ‘non-turnover’ FNR signal shown in black). c Activation of cascade electrocatalysis following pre-loading of FNR and then injecting different dehydrogenases (to low concentration) into the cell solution, which also contained the substrate for the enzyme being studied. ADH alcohol dehydrogenase, RedAm reductive aminase, ME malic enzyme (malate dehydrogenase), (S)-IRED (S)-imine reductase. d Cyclic voltammograms for the electrocatalysis of ketone/alcohol interconversion: at pH 9.0 the rate is almost the same in each direction. Figures were adapted with permission: (a and b), (c) and (d).

Fig. 3. Useful metaphoric representations of the e-Leaf.

a As an urban transport map in which the enzymes (stations) are arranged in terms of next-neighbour in the catalytic sequence noting (as is obvious for transport) there is a minimum requirement for two ‘stations’, in this case E1 (FNR) and E2 (an NAD(P)(H)-dependent dehydrogenase); N, R and P represent NAD(P)(H), reactant and product, respectively; D (the dashboard) comprises the electrochemical workstation and other hardware. b The dashboard of a car, emphasising the various controls and displays that have equivalents in the e-Leaf.

Fig. 4. The thermodynamic and spatial ranges of nicotinamide cofactor recycling.

a A free energy profile for electron/hydride transfer from an electrode to a reactant undergoing catalytic reduction at dehydrogenase E2. Transfers indicated in green are favourable, and those in red are unfavourable. b The >100-fold nanoconfinement advantage, showing the current measured at increasing concentrations of NADP(H) in solution. The black curve shows results with FNR (E1) and isocitrate dehydrogenase (E2) loaded but without isocitrate in the solution. The current depends on the FNR-catalysed oxidation of NADPH entering the ITO layer from the solution. The red trace shows results with the two-enzyme cascade reaction activated by adding isocitrate (10 mM): the current now depends on the rate of localised NADP(H) recycling. A rotating disc electrode, held at +0.2 V vs SHE, was used in each case. The data focus on conditions where NADPH < 10 mM to emphasise the regime that shows the largest enhancement. c Localised recycling between crowded enzymes: FNR, IDH1, and NADP(H) drawn to-scale with an FNR-IDH1 mean centre-to-centre distance of 9.4 Å, equivalent to a combined concentration of 2 mM (see text). Panel b was adapted with permission.

Fig. 5. Examples of extended enzyme cascades driven in the e-Leaf.

a, b Two examples of extended cascades, shown in conventional mode (left) to focus on the chemical conversions, and as cascade maps (right) to show the interrelationships between enzymes and the flow of information that is transduced by E1 into electrical current. The relative sizes of the enzyme circles emphasise the higher loading that is required to offset lower activity.

Fig. 6. The e-Leaf in drug mechanisms and development.

Applying the e-Leaf to study the selective inhibition of a single-site variant enzyme having undesired gain-of-function activity, in this case, human isocitrate dehydrogenase I (IDH1). a Cascade map depicting oxidation catalysis by E2 (native IDH1) and reduction catalysis by E2’ (IDH1 R132H). The sizes of the spheres represent inverse relative activity, IDH1 R132H being the least active catalyst and required to be loaded in higher quantities. b The ‘living’ cyclic voltammogram (scan rate 1 mV/s) showing the simultaneous monitoring of two enzymes in a single experiment and how the injection of a drug into the solution inhibits the conversion of 2-oxoglutarate to 2-hydroxyglutarate by E2’ but does not inhibit the wildtype activity of E2. c The kinetics of the inhibition process can be studied under pseudo-first-order conditions, even with very low levels of the drug. A full analysis of the kinetics has been published: the results reveal how the drug first binds without inhibiting, then locks into its inhibitory position in a slow subsequent step. Panels b and c were adapted with permission from reference.