Catalytic Kinetics Considerations and Molecular Tools for the Design of Multienzymatic Cascade Nanoreactors

Chauhan, Kanchan; Zarate-Romero, A.; Sengar, Prakhar; Medrano, C.; Vázquez-Duhalt, Rafael

doi:10.1002/cctc.202100604

Cited by 20 publications

(20 citation statements)

References 131 publications

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“…Overall, we observe a highly idiosyncratic response to SP fusion and encapsulation among MK orthologues. It should be noted that for encapsulated enzymes, classical Michaelis–Menten kinetics may not provide the best model due to the possible impacts of the protein cage and confinement on the catalytic properties of encapsulated enzymes . Nevertheless, in the absence of a model that corrects for these factors, the use of a common reaction between orthologous enzymes encapsulated within the same protein cage is informative.…”

Section: Resultsmentioning

confidence: 99%

“…It should be noted that for encapsulated enzymes, classical Michaelis−Menten kinetics may not provide the best model due to the possible impacts of the protein cage and confinement on the catalytic properties of encapsulated enzymes. 57 Nevertheless, in the absence of a model that corrects for these factors, the use of a common reaction between orthologous enzymes encapsulated within the same protein cage is informative. These findings point to a need to empirically determine the optimal encapsulation strategy and loading density for the biocatalytic nanoreactor assembly.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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Rapid Assembly and Prototyping of Biocatalytic Virus-like Particle Nanoreactors

et al. 2022

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Protein cages are attractive as molecular scaffolds for the fundamental study of enzymes and metabolons and for the creation of biocatalytic nanoreactors for in vitro and in vivo use. Virus-like particles (VLPs) such as those derived from the P22 bacteriophage capsid protein make versatile self-assembling protein cages and can be used to encapsulate a broad range of protein cargos. In vivo encapsulation of enzymes within VLPs requires fusion to the coat protein or a scaffold protein. However, the expression level, stability, and activity of cargo proteins can vary upon fusion. Moreover, it has been shown that molecular crowding of enzymes inside VLPs can affect their catalytic properties. Consequently, testing of numerous parameters is required for production of the most efficient nanoreactor for a given cargo enzyme. Here, we present a set of acceptor vectors that provide a quick and efficient way to build, test, and optimize cargo loading inside P22 VLPs. We prototyped the system using a yellow fluorescent protein and then applied it to mevalonate kinases (MKs), a key enzyme class in the industrially important terpene (isoprenoid) synthesis pathway. Different MKs required considerably different approaches to deliver maximal encapsulation as well as optimal kinetic parameters, demonstrating the value of being able to rapidly access a variety of encapsulation strategies. The vector system described here provides an approach to optimize cargo enzyme behavior in bespoke P22 nanoreactors. This will facilitate industrial applications as well as basic research on nanoreactor-cargo behavior.

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Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

Rapid Assembly and Prototyping of Biocatalytic Virus-like Particle Nanoreactors

et al. 2022

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“…[30,31] These compounds showed antioxidant and antiviral activity dependent from the number of carbon atoms in the side-chain. [32] Enzymatic cascade is an advanced tool for multiple synthetic transformations in one-pot condition, [33,34] promoting the fast exchange of substrate between adjacent enzymes. [35,36] Supported multienzyme cascades are reported, and their efficiency, selectivity, stability and reusability deeply discussed.…”

Section: Introductionmentioning

confidence: 99%

Lignin Nanoparticles Support Lipase‐Tyrosinase Enzymatic Cascade in the Synthesis of Lipophilic Hydroxytyrosol Ester Derivatives

et al. 2022

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Lipophilic esters of hydroxytyrosol have been synthesized in a one-pot procedure from tyrosol and long side-chain carboxylic acids by a lignin nanoparticles supported enzymatic cascade of lipase M and tyrosinase. The reaction involved the initial esterification of the aliphatic carboxylic group with the hydroxyl group of tyrosol followed by ortho-hydroxylation of the aromatic moiety. Lipase M and tyrosinase were immobilized on lignin nanoparticles by layer-by-layer procedure using chitosan as positive charged polyelectrolyte and Concanavalin A as molecular spacer to control the optimal distance between the active sites of the two enzymes. The occurrence of substrate channeling effect was hypothesized on the basis of FRET analysis. Lipophilic hydroxytyrosol esters were synthesized in high yield in 2-methyltetrahydrofuran and in the presence of a very low amount of buffer to generate the hydration sphere of the enzyme.

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“…Artificially confined enzyme cascades are of considerable interest for biocatalysis technology and fundamental insight, and they have been extensively reviewed. − In this section, we highlight some recent approaches to advance the design of scaffolds and interpretation of data.…”

Section: Nanoconfining and Energizing Enzyme Cascades For Technologymentioning

confidence: 99%

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

et al. 2022

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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.

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Catalytic Kinetics Considerations and Molecular Tools for the Design of Multienzymatic Cascade Nanoreactors

Abstract: ations in cascade reactions on nanoassemblies are of great importance. Thus, in this work, important parameters of enzyme kinetics in cascade reaction and the molecular tools for the design and optimization of bi-and multi-enzymatic nanobioreactors reactions are discussed.

Cited by 20 publications

References 131 publications

Rapid Assembly and Prototyping of Biocatalytic Virus-like Particle Nanoreactors

Rapid Assembly and Prototyping of Biocatalytic Virus-like Particle Nanoreactors

Lignin Nanoparticles Support Lipase‐Tyrosinase Enzymatic Cascade in the Synthesis of Lipophilic Hydroxytyrosol Ester Derivatives

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

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