Multienzyme Cascades

In some cases, multienzyme cascades are used where an artificial metabolic pathway has been created on an electrode to take a specific substrate and electrocataly tic ally convert it to produce energy [5,12-14]. With these systems, coulometry is useful for determining coulombic efficiency, as well as combining coulometry with nuclear magnetic resonance (NMR) analysis to determine reaction intermediates and products, and elucidate bottlenecks in the artificial metabolic pathway, or to determine the energy density of a fuel cell. Please refer to Chapter 5 for further information regarding multienzyme cascades. 

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Increasing evidence indicates that accumulation of aberrant or misfolded proteins, protofibril formation, ubiquitin-proteasome system dysfunction, and the direct or indirect consequences of abnormal protein aggregation and accumulation represent deleterious events linked to neurodegeneration (255,256). Ubiquitination is an essential cellular process affected by a multienzyme cascade involving Els (ubiquitin-activating enzymes), E2s (ubiquitin-conjugation enzymes or UBCs), and E3s (ubiquitin-protein Ugases) (12,257) (see Fig. 10.4). 

PROMISCUOUS ENZYMES VERSUS MULTIENZYME CASCADES VERSUS METABOLONS 55... 

In Nature, such multienzyme cascades, or metabolons, are often spatially oriented such that sequential enzymes in the cascade are in close proximity to each other [63]. This allows the substrate to be efficiently channeled from one enzyme to the next along the cascade, maximizing throughput. Several techniques have been developed to implement such an organization in a BFC [58,64], and these have yielded impressive results. A pyruvate/air BFC using a group of Krebs cycle enzymes isolated from mitochondria demonstrates a 49% increase in current density and 32% increase in power density when cross-linked into their native ordered conformation, versus the same enzymes free in solution [65]. 

Multienzyme Cascades Involving (o-TA-Catalyzed Amination of Ketones... 

Multienzyme cascade for the conversion of i-amino acids to a-hydroxy acids employing an L-amino acid deaminase (l-AAD) and isocaproate reductases (Hie). 

TABLE 2.13 Selected Results of the Regioselective Monoamination-Cyclization-Desymmetrization Multienzyme Cascade ... 

Multienzyme cascade synthesis combining ene-reductases with alcohol dehydrogenases, (a) Synthesis of optically pure y-butyrolactones (the building block is circled) and (b) optically pure primary or secondary tetralin orchroman derivatives, (c) Three-enzyme system for the generation of a valuable chiral building block. 

Transaminases are most powerful tools for the synthesis of chiral amines, amino acids, and amino alcohols, hi this chapter several approaches for tiie preparation of fine chemicals or building blocks for pharmaceuticals were discussed, like asymmetric synthesis or kinetic resolution. The main limitations of transaminase-catalyzed reactions are the need to shift the equihbrium to the product side and substrate and product inhibition. Some solutions to overcome such inhibition were presented here for example, multienzyme cascades or biphasic extraction of the product. Protein engineering by directed evolution or rational enzyme design is a promising option to find transaminases with different substrate specificities and enantiopreferences. This is becoming more and more important for the pharmaceutical industry. Furthermore, it is a way to alter enzyme properties known so far, like thermostability and solvent and pH stability. Protein engineering has been assisted by the recently solved structures of certain transaminases. 

The oxidase is a cell membrane-multienzyme complex. It has a cell surface receptor linked to a G-protein that activates a phosphatidyl inositol cascade leading to assembly and activation of the oxidase complex. The receptor is activated by the following ... 

Multienzyme systems have been used in carbon paste electrodes, providing bio-catalytic cascades that result in an analytical amperometric signal. For example, acetylcholine esterase (AChE) and choline oxidase (ChOx) have been co-immobilized in carbon pastes, either with monomeric TTF [145] or flexible ferrocene-containing polymers [146] as electron mediators. The primary reaction includes the hydrolysis of acetylcholine biocatalyzed by AChE, then the choline produced is oxidized by the electrically contacted ChOx giving an analytical amperometric signal corresponding to the acetylcholine concentration. 

An enzyme that is controlled by the binding of an allosteric effector or phosphorylation allows fine-tuning of a stimulus at each step of the pathway of activation. This provides an avenue for modulation by other chemical communication pathways. Many cell-signaling systems use similar multienzyme phosphorylation cascades (e.g., HMG-CoA reductase. Fig. 12-15). 

Since enzymes generally function under the same or similar conditions, several biocatalytic reactions can be carried out in a reaction cascade in a single flask. Thus, sequential reactions are feasible by using multienzyme systems in order to simplify reaction processes, in particular if the isolation of an unstable intermediate can be omitted. Furthermore, an unfavorable equilibrium can be shifted towards the desired product by linking consecutive enzymatic steps. This unique potential of enzymes is increasingly being recognized as documented by the development of multienzyme systems, also denoted as artificial metabolism [15]. 

A biosynthetic multienzyme reaction of particular interest involves carbon dioxide fixation with the production of methanol [373, 374]. FDH catalyzes the reduction of carbon dioxide to formate, and methanol dehydrogenase (M DH) catalyzes the reduction of formate to methanol. Both of these enzymes require NAD+/NADH-cofactor, and in the presence of the reduced dimethyl viologen mediator (MV +), they can drive a sequence of enzymatic reactions. The cascade of biocatalytic reactions results in the reduction of GO2 to formate catalyzed by FDH, followed by the reduction of formate to methanol catalyzed by MDH. A more complex system composed of immobilized cells of Parococcus deni-trijicans has been demonstrated for the reduction of nitrate and nitrite [375]. 

Microreactors are ideal for directing complex enzymatic synthesis, such as multienzyme catalysis and cascade reactions. There have been a grovdng number of studies [173-178] that represent the implementation of microreactions for multistep enzymatic catalysis and a list is presented in Table 10.5. Microfluidic biocatalytic... 

Moore et al. reported the first in vitro assembly of a complete type 11PKS enzymatic pathway and demonstrated the multienzyme total synthesis of the bacteriostatic agent enterocin [53], They employed nine recombinant and three commercial proteins in a single reaction vessel along with the necessary cofactors (S-adenosyl-L-methionine (SAM), ATP, Mg +, and NADPH) to produce enterocin (51) from its primary metabolite precmsors (benzoic acid and malonyl-CoA) (Scheme 15.16). This remarkable enzyme cascade generates 10 C-C bonds, 5 C-O bonds, and 7 chiral centers in impressive 25% yield. 

Cascade architecture A graphical representation of all reactions in the mulhen-zyme process is the basis for describing the final model structure. It includes the primary reachons, secondary reactions, and competing reactions. For a single enzyme, reachon mechanisms are well developed, and they are then included into the full model to describe the multienzyme process by combining the effect of the individual enzymes. In this way, the different possible reaction schemes are generated to give the cascade structure. 

Biocatalysis for drug discovery and development with an industrial perspective, and biocatytic cascade reactions with e integration of biocatalysts with one or more additional reaction steps, and multistep biocatalytic reaction sequences and multienzyme-catalyzed conversions, are presented. 

In multienzyme reactions similar to other cascades the product of one reaction serves as the substrate for the next. 

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