The power of electrified nanoconfinement for energising, controlling and observing long enzyme cascades

Abstract

Multistep enzyme-catalyzed cascade reactions are highly efficient in nature due to the confinement and concentration of the enzymes within nanocompartments. In this way, rates are exceptionally high, and loss of intermediates minimised. Similarly, extended enzyme cascades trapped and crowded within the nanoconfined environment of a porous conducting metal oxide electrode material form the basis of a powerful way to study and exploit myriad complex biocatalytic reactions and pathways. One of the confined enzymes, ferredoxin-NADP⁺ reductase, serves as a transducer, rapidly and reversibly recycling nicotinamide cofactors electrochemically for immediate delivery to the next enzyme along the chain, thereby making it possible to energize, control and observe extended cascade reactions driven in either direction depending on the electrode potential that is applied. Here we show as proof of concept the synthesis of aspartic acid from pyruvic acid or its reverse oxidative decarboxylation/deamination, involving five nanoconfined enzymes.

Fig. 1. Comparing nanoconfined enzyme cascades.

Comparisons between: a catalysis by a nanoconfined enzyme cascade immobilized on a scaffold that is suspended in solution; b catalysis by an enzyme cascade confined in zones of nanopores formed naturally by electrodeposition of indium tin oxide nanoparticles on a conducting support. The nanopore-confined cascades are energizable and directly observable via electron flow through the transducer ferredoxin-NADP⁺ reductase (E1).

Fig. 2. Cyclic voltammetry of an extended enzyme cascade trapped in electrode nanopores.

a Scheme of the nanoconfined cascade driving reductive amination/carboxylation of pyruvate to aspartate in either direction through bidirectional electrocatalytic recycling of NADP(H) by the transducer enzyme FNR. CA carbonic anhydrase; FNR ferredoxin-NADP⁺-reductase; ME malic enzyme; FumC fumarase; AspA aspartate-amino-lyase. b Cyclic voltammetry (25 °C, pH 7.5, 1 mV s⁻¹) of the 5-enzyme cascade (0.1 CA/1 FNR/5 ME/1 FumC/1 AspA) in buffer 0.05 M HEPES, 20 mM pyruvate, 0.1 M KHCO₃, 0.1 M NH₄Cl, 4 mM MgCl₂, 1 mM MnCl₂. Gray: blank, no cofactor present. Red: injection of NADP⁺ (to 20 µM). Blue: injection of aspartate (to 20 mM).

Fig. 3. Controlling and monitoring the rate and directionality of an enzyme cascade.

a The nanoconfined cascade in downstream notation. Enzymes E1 and E2 are, respectively, FNR and a NAD(P)(H) dehydrogenase and they represent the transducer-engine pair at which the rate of reaction flow along the cascade is recorded directly as current. Here the rest of the cascade is downstream with respect to the cofactor-dependent step (E1–E2 pair). Intermediates are passed to the next enzyme or escape (dotted red lines) into the bulk solution. b The nanoconfined cascade in upstream notation: the rest of the cascade is upstream of the cofactor-dependent step. c Rotation-rate dependence experiment for the system (CA/FNR/ME/FumC/AspA)@ITO/PGE (0.03 cm²) in a buffer containing NH₄Cl, KHCO₃, and pyruvate at pH 7.5, 25 °C, rotation rate 1000 rpm, under N₂ atmosphere. Arrows signify injection of NADP⁺ (to a final concentration of 20 µM) and switching rotation off (0 rpm) and on (1000 rpm). d Continuation of the experiment carried out in panel c: the cell was washed carefully, a fresh buffer was added and rotation (1000 rpm) was switched on. Arrows signify injection of NADP⁺ (to 20 µM) and aspartate (substrate of E4, to 20 mM) and successive switching off and on of rotation. e Cascade with E1, E2, etc, identified with the different enzymes making up the cascade unit: downstream direction runs left to right.

Fig. 4. Analysis of products and intermediates.

Relative percentage of products in the downstream direction (reductive amination/carboxylation) (a), and upstream direction (oxidative deamination/decarboxylation) (b). In both cases, the relative product distribution was maintained after recharging the system with new buffer and substrates. Error bars represent standard errors of the mean taken from three independent repeat experiments (data points plotted as circles for stage 1 and squares after recharging, N = 3).