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.
NAD(P)H quinone oxidoreductase 1 (NQO1) catalyses the two electron reduction of quinones and a wide range of other organic compounds. Its physiological role is believed to be partly the reduction of ...free radical load in cells and the detoxification of xenobiotics. It also has non-enzymatic functions stabilising a number of cellular regulators including p53. Functionally, NQO1 is a homodimer with two active sites formed from residues from both polypeptide chains. Catalysis proceeds via a substituted enzyme mechanism involving a tightly bound FAD cofactor. Dicoumarol and some structurally related compounds act as competitive inhibitors of NQO1. There is some evidence for negative cooperativity in quinine oxidoreductases which is most likely to be mediated at least in part by alterations to the mobility of the protein. Human NQO1 is implicated in cancer. It is often over-expressed in cancer cells and as such is considered as a possible drug target. Interestingly, a common polymorphic form of human NQO1, p.P187S, is associated with an increased risk of several forms of cancer. This variant has much lower activity than the wild-type, primarily due to its substantially reduced affinity for FAD which results from lower stability. This lower stability results from inappropriate mobility of key parts of the protein. Thus, NQO1 relies on correct mobility for normal function, but inappropriate mobility results in dysfunction and may cause disease.
Enzymes' inherent chirality confers their exquisite enantiomeric specificity and makes their use as green alternatives to chiral metal complexes or chiral organocatalysts invaluable to the fine ...chemical industry. The most prevalent way to alter enzyme activity in terms of regioselectivity and stereoselectivity for both industry and fundamental research is to engineer the enzyme. In a recent article by Keinänen et al., published in Bioscience Reports 2018, 'Controlling the regioselectivity and stereoselectivity of FAD-dependent polyamine oxidases with the use of amine-attached guide molecules as conformational modulators', an inverse approach was presented that focuses on the manipulation of the enzyme substrate rather than the enzyme. This approach not only uncovered dormant enantioselectivity in related enzymes but allowed for its control by the use of guide molecules simply added to the reaction solution or covalently linked to an achiral scaffold molecule.
Protein film electrochemistry (PFE) has given unrivalled insight into the properties of redox proteins and many electron-transferring enzymes, allowing investigations of otherwise ill-defined or ...intractable topics such as unstable Fe–S centers and the catalytic bias of enzymes. Many enzymes have been established to be reversible electrocatalysts when attached to an electrode, and further investigations have revealed how unusual dependences of catalytic rates on electrode potential have stark similarities with electronics. A special case, the reversible electrochemistry of a photosynthetic enzyme, ferredoxin-NADP+ reductase (FNR), loaded at very high concentrations in the 3D nanopores of a conducting metal oxide layer, is leading to a new technology that brings PFE to myriad enzymes of other classes, the activities of which become controlled by the primary electron exchange. This extension is possible because FNR-based recycling of NADP(H) can be coupled to a dehydrogenase, and thence to other enzymes linked in tandem by the tight channelling of cofactors and intermediates within the nanopores of the material. The earlier interpretations of catalytic wave-shapes and various analogies with electronics are thus extended to initiate a field perhaps aptly named “cascade-tronics”, in which the flow of reactions along an enzyme cascade is monitored and controlled through an electrochemical analyzer. Unlike in photosynthesis where FNR transduces electron transfer and hydride transfer through the unidirectional recycling of NADPH, the “electrochemical leaf” (e-Leaf) can be used to drive reactions in both oxidizing and reducing directions. The e-Leaf offers a natural way to study how enzymes are affected by nanoconfinement and crowding, mimicking the physical conditions under which enzyme cascades operate in living cells. The reactions of the trapped enzymes, often at very high local concentration, are thus studied electrochemically, exploiting the potential domain to control rates and direction and the current–rate analogy to derive kinetic data. Localized NADP(H) recycling is very efficient, resulting in very high cofactor turnover numbers and new opportunities for controlling and exploiting biocatalysis.
In living cells, redox chains rely on nanoconfinement using tiny enclosures, such as the mitochondrial matrix or chloroplast stroma, to concentrate enzymes and limit distances that nicotinamide ...cofactors and other metabolites must diffuse. In a chemical analogue exploiting this principle, nicotinamide adenine dinucleotide phosphate (NADPH) and NADP+ are cycled rapidly between ferredoxin–NADP+ reductase and a second enzyme—the pairs being juxtaposed within the 5–100 nm scale pores of an indium tin oxide electrode. The resulting electrode material, denoted (FNR+E2)@ITO/support, can drive and exploit a potentially large number of enzyme‐catalysed reactions.
In close quarters: Nicotinamide adenine dinucleotide phosphate (NADPH) and NADP+ are cycled rapidly between ferredoxin–NADP+ reductase (FNR) and a dehydrogenase enzyme (E2) within the 5–100 nm pores of an indium tin oxide electrode. The electrode mimics the nanoconfinement strategies of natural systems, such as the chloroplast stroma or mitochondria in living cells.
Reduction of CO2 and its direct entry into organic chemistry is achieved efficiently and in a highly visible way using a metal oxide electrode in which two enzyme catalysts, one for electrochemically ...regenerating reduced nicotinamide adenine dinucleotide phosphate and the other for assimilating CO2 and converting pyruvate (C3) to malate (C4), are entrapped within its nanopores. The resulting reversible electrocatalysis is exploited to construct a solar CO2 reduction/water-splitting device producing O2 and C4 with high faradaic efficiency.
In a discovery of the transfer of chloroplast biosynthesis activity to an inorganic material, ferredoxin-NADP
reductase (FNR), the pivotal redox flavoenzyme of photosynthetic CO
assimilation, binds ...tightly within the pores of indium tin oxide (ITO) to produce an electrode for direct studies of the redox chemistry of the FAD active site, and fast, reversible and diffusion-controlled interconversion of NADP
and NADPH in solution. The dynamic electrochemical properties of FNR and NADP(H) are thus revealed in a special way that enables facile coupling of selective, enzyme-catalysed organic synthesis to a controllable power source, as demonstrated by efficient synthesis of l-glutamate from 2-oxoglutarate and NH
.
The final steps of [FeFe]-hydrogenase maturation Lampret, Oliver; Esselborn, Julian; Haas, Rieke ...
Proceedings of the National Academy of Sciences - PNAS,
08/2019, Letnik:
116, Številka:
32
Journal Article
Recenzirano
Odprti dostop
The active site (H-cluster) of FeFe-hydrogenases is a blueprint for the design of a biologically inspired H₂-producing catalyst. The maturation process describes the preassembly and uptake of the ...unique 2FeH cluster into apo-hydrogenase, which is to date not fully understood. In this study, we targeted individual amino acids by site-directed mutagenesis in the FeFe-hydrogenase CpI of Clostridium pasteurianum to reveal the final steps of H-cluster maturation occurring within apo-hydrogenase. We identified putative key positions for cofactor uptake and the subsequent structural reorganization that stabilizes the 2FeH cofactor in its functional coordination sphere. Our results suggest that functional integration of the negatively charged 2FeH precursor requires the positive charges and individual structural features of the 2 basic residues of arginine 449 and lysine 358, which mark the entrance and terminus of the maturation channel, respectively. The results obtained for 5 glycine-to-histidine exchange variants within a flexible loop region provide compelling evidence that the glycine residues function as hinge positions in the refolding process, which closes the secondary ligand sphere of the 2FeH cofactor and the maturation channel. The conserved structural motifs investigated here shed light on the interplay between the secondary ligand sphere and catalytic cofactor.