NADP(H)-dependent biocatalysis without adding NADP(H)

Abstract

Isocitrate dehydrogenase 1 (IDH1) naturally copurifies and crystallizes in a resting state with a molecule of its exchangeable cofactor, NADP + /NADPH, bound in each monomer of the homodimer. We report electrochemical studies with IDH1 that exploit this property to reveal the massive advantage of nanoconfinement to increase the efficiency of multistep enzyme-catalyzed cascade reactions. When coloaded with ferredoxin NADP + reductase in a nanoporous conducting indium tin oxide film, IDH1 carries out the complete electrochemical oxidation of 6 mM isocitrate (in 4mL) to 2-oxoglutarate (2OG), using only the NADP(H) that copurified with IDH1 and was carried into the electrode pores as cargo—the system remains active for days. The entrapped cofactor, now quantifiable by cyclic voltammetry, undergoes ~160,000 turnovers during the process. The results from a variety of electrocatalysis experiments imply that the local concentrations of the two nanoconfined enzymes lie around the millimolar range. The combination of crowding and entrapment results in a 10 2 to 10 3 -fold increase in the efficiency of NADP(H) redox cycling. The ability of the method to drive cascade catalysis in either direction (oxidation or reduction) and remove and replace substrates was exploited to study redox-state dependent differences in cofactor binding between wild-type IDH1 and the cancer-linked R132H variant that catalyzes the “gain of function” reduction of 2OG to 2-hydroxyglutarate instead of isocitrate oxidation. The combined results demonstrate the power of nanoconfinement for facilitating multistep enzyme catalysis (in this case energized and verified electrochemically) and reveal insights into the dynamic role of nicotinamide cofactors as redox (hydride) carriers.

Design principles for a nanoconfined enzyme cascade electrode via reaction–diffusion modelling

“…This current enhancement can be compared to a recent experimental work that demonstrated an enhancement factor of B25 at [NADP + ] B50 mM. 31 The results here show that the coupled current does increase with higher [NADP + ] (by 4100-1000 times compared to the uncoupled current, depending on the E2 activity), but at higher [NADP + ] (4100 mM) the contribution from the coupled current can become obscured by the bulk supply of [NADP + ], resulting in markedly smaller enhancement factors. In the context of a cofactor recycling system, there is a need to balance the requirement for higher cofactor turnover number (i.e.…”

From Protein Film Electrochemistry to Nanoconfined Enzyme Cascades and the Electrochemical Leaf

“…As shown in Figure A, human IDH1 copurifies with one molecule of NADP­(H) bound at each monomer active site. − , The tight binding of the cofactor to a resting inactive state of the enzyme (which is not reflected in steady-state K M determinations) allows each half of the IDH1 molecule to deliver up to a 1:1 stoichiometric quantity of NADP­(H) into the pores alongside FNR, without any exogenous NADP­(H) being required. Based on a proposed model, only after Mg 2+ and isocitrate bind to the enzyme to initiate a catalytic cycle is the cofactor released for rapid recycling by a nearby FNR molecule . The loading of IDH1 with its cofactor cargo resembles advanced drug delivery mechanisms where shuttle particles carry tightly bound cargo molecules to specific locations before they are released when instructed.…”

“…Further experiments were carried out to address the question of whether pausing catalytic turnover results in preferential dissociation of NADP + (under an oxidizing potential) or NADPH (under a reducing potential) (Figure E,F). Chronoamperometry experiments in which the potential was periodically switched between oxidizing and reducing values revealed that the 2OG-reducing IDH1 R132H/FNR system loses activity after the first oxidative pause, whereas the isocitrate-oxidizing wild-type IDH1/FNR system is much more stable following a reductive pause . A useful analogy is found with an “electromagnetic gripper”, a machine that picks up or drops a steel load depending on the electrical command given.…”

“…Taking the quantities obtained alongside assumptions about ITO layer depth and void volume (section ), the concentration ranges for the two coloaded enzymes were estimated to be 0.6–1.2 mM for FNR and 0.6–1.3 mM for IDH1. A comparison with the estimates for limiting enzyme concentrations given in Figure indicate that the enzymes must be very crowded; likewise, the higher concentration of FNR that is achieved in the absence of IDH1 also suggests competition for space …”

“…Only NADP­(H) that was tightly held (rebound) by IDH1 or IDH1 R132H would remain trapped in the nanopores, allowing the affinity of each enzyme for particular redox (hydrogenation) states of NADP­(H) to be investigated. (Adapted with permission from ref . Copyright 2022 National Academy of Science.…”