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.

Exploiting Electrode Nanoconfinement to Investigate the Catalytic Properties of Isocitrate Dehydrogenase (IDH1) and a Cancer-Associated Variant

“… 15 A typical FNR loading at pH 8 corresponds to 0.7 mM in a 1-μm-deep layer, ignoring the volume due to ITO material. 22 As controlled via an electrochemical workstation, FNR acts as the transducer, interconverting electrons and NADP(H) with high efficiency and reversibility in the nanoconfined environment. 16 The catalytic rate in either direction, reduction or oxidation, is displayed directly on a computer screen as current.…”

“…Further experiments were carried out to test the bidirectionality of catalysis by IDH1 and R132H. For IDH1-catalyzed 2OG/isocitrate interconversion ( Figure 2 A), the CO 2 that is required was produced catalytically in situ from bicarbonate present at 0.1 M in the buffer using carbonic anhydrase (CA), a highly active Zn-enzyme, that was coentrapped in the electrode nanopores; 22 notably a 50:1 2OG/isocitrate ratio was needed to equalize the oxidation and reduction currents. A formal reduction potential for the 2OG/isocitrate interconversion ( E 0′ 2OG/isocit ) of −0.40 V vs SHE at pH 7.7 at the local CO 2 level was calculated from the Nernst equation using the clearly defined zero-current potential ( E eq ) obtained with the 50/1 ratio.…”

Some fundamental insights into biological redox catalysis from the electrochemical characteristics of enzymes attached directly to electrodes

“…An even more significant finding emerged when it was shown that binding a NADP(H)-dependant dehydrogenase as a second enzyme in the ITO nanopore network resulted in tight electrochemical coupling to that enzyme's reaction [36] , [37] , [38] , [39] . The situation is represented in Fig.…”

“…5 E is of a ketone/alcohol interconversion that is easily driven in either direction [ 37 , 38 ]. Consequently it is now becoming possible to carry out electrochemical studies of enzymes (extending beyond the second enzyme) that do not involve electron transfer [39] . The FNR acts as a ‘ECE’ transducer to interconvert electrons and hydride (NADPH), and the nanoconfinement of FNR with the second enzyme leads to very rapid recycling of the nicotinamide cofactor.…”

From Enzyme Stability to Enzymatic Bioelectrode Stabilization Processes

“…They can be metal centers, including copper for oxygen reduction [6], iron and nickel for hydrogen evolution and uptake or CO 2 reduction [7][8][9], manganese for water oxidation in photosynthesis [10], etc. Some others are dependent on nicotinamide adenine dinucleotide phosphate (NADPH) to realize enzymatic transformations [11]. Such enzymes have been envisioned as biocatalysts in biosensors [12][13][14], biosynthesis reactors [15,16], or biofuel cells [17,18].…”