ABSTRACT
Enterobactin is a high-affinity iron chelator produced and secreted by Escherichia coli and Salmonella typhimurium to scavenge scarce extracellular Fe3+ as a micronutrient. EntC and EntB are the first two enzymes in the enterobactin biosynthetic pathway. Isochorismate, produced by EntC, is a substrate for EntB isochorismatase. By using a competing isochorismate-consuming enzyme (the E. coli SEPHCHC synthase MenD), we found in a coupled assay that residual EntB isochorismatase activity decreased as a function of increasing MenD concentration. In the presence of excess MenD, EntB isochorismatase activity was observed to decrease by 84%, indicative of partial EntC-EntB channeling (16%) of isochorismate. Furthermore, addition of glycerol to the assay resulted in an increase of residual EntB isochorismatase activity to approximately 25% while in the presence of excess MenD. These experimental outcomes supported the existence of a substrate channeling surface identified in a previously reported protein-docking model of the EntC-EntB complex. Two positively charged EntB residues (K21 and R196) that were predicted to electrostatically guide negatively charged isochorismate between the EntC and EntB active sites were mutagenized to determine their effects on substrate channeling. The EntB variants K21D and R196D exhibited a near complete loss of isochorismatase activity, likely due to electrostatic repulsion of the negatively charged isochorismate substrate. Variants K21A, R196A, and K21A/R196A retained partial EntB isochorismatase activity in the absence of EntC; in the presence of EntC, isochorismatase activity in all variants increased to near wild-type levels. The MenD competition assay of the variants revealed that while K21A channeled isochorismate as efficiently as wild-type EntB (~ 15%), the variants K21A/R196A and R196A exhibited an approximately 5-fold loss in observed channeling efficiency (~3%). Taken together, these results demonstrate that partial substrate channeling occurs between EntC and EntB via a leaky electrostatic tunnel formed upon dynamic EntC-EntB complex formation and that EntB R196 plays an essential role in isochorismate channeling.
Subject(s)
Enterobactin , Escherichia coli Proteins , Escherichia coli , Enterobactin/biosynthesis , Enterobactin/metabolism , Enterobactin/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli/enzymology , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Chorismic Acid/metabolism , Chorismic Acid/chemistry , HydrolasesABSTRACT
The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α2ß2 tetrameric enzyme in which the α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) active sites, and the ß-chain provides the 3-ketoacyl-CoA thiolase (KAT) active site. Linear, medium-chain and long-chain 2E-enoyl-CoA molecules are the preferred substrates of MtTFE. Previous crystallographic binding and modeling studies identified binding sites for the acyl-CoA substrates at the three active sites, as well as the NAD binding pocket at the HAD active site. These studies also identified three additional CoA binding sites on the surface of MtTFE that are different from the active sites. It has been proposed that one of these additional sites could be of functional relevance for the substrate channeling (by surface crawling) of reaction intermediates between the three active sites. Here, 226 fragments were screened in a crystallographic fragment-binding study of MtTFE crystals, resulting in the structures of 16 MtTFE-fragment complexes. Analysis of the 121 fragment-binding events shows that the ECH active site is the `binding hotspot' for the tested fragments, with 41 binding events. The mode of binding of the fragments bound at the active sites provides additional insight into how the long-chain acyl moiety of the substrates can be accommodated at their proposed binding pockets. In addition, the 20 fragment-binding events between the active sites identify potential transient binding sites of reaction intermediates relevant to the possible channeling of substrates between these active sites. These results provide a basis for further studies to understand the functional relevance of the latter binding sites and to identify substrates for which channeling is crucial.
Subject(s)
Acyl Coenzyme A , Bacterial Proteins , Catalytic Domain , Mycobacterium tuberculosis , Mycobacterium tuberculosis/enzymology , Crystallography, X-Ray , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Acyl Coenzyme A/metabolism , Acyl Coenzyme A/chemistry , Substrate Specificity , Binding Sites , Models, Molecular , Enoyl-CoA Hydratase/metabolism , Enoyl-CoA Hydratase/chemistry , Protein Binding , 3-Hydroxyacyl CoA Dehydrogenases/chemistry , 3-Hydroxyacyl CoA Dehydrogenases/metabolismABSTRACT
Docking domains (DDs) located at the C- and N-termini of polypeptides play a crucial role in directing the assembly of polyketide synthases (PKSs), which are multienzyme complexes. Here, we determined the crystal structure of a complex comprising the C-terminal DD (CDDMlnB) and N-terminal DD (NDDMlnC) of macrolactin trans-acyltransferase (AT) PKS that were fused to a functional enzyme, AmpC EC2 ß-lactamase. Interface analyses of the CDDMlnB/NDDMlnC complex revealed the molecular intricacies in the core section underpinning the precise DD assembly. Additionally, circular dichroism and steady-state kinetics demonstrated that the formation of the CDDMlnB/NDDMlnC complex had no influence on the structural and functional fidelity of the fusion partner, AmpC EC2. This inspired us to apply the CDDMlnB/NDDMlnC assembly to metabolon engineering. Indeed, DD assembly induced the formation of a complex between 4-coumarate-CoA ligase and chalcone synthase both involved in flavonoid biosynthesis, leading to a remarkable increase in naringenin production in vitro.
ABSTRACT
Hydrogen-bonded organic frameworks (HOF) represent an emerging category of organic structures with high crystallinity and metal-free, which are not commonly observed in alternative porous organic frameworks. These needle-like porous structure can help in stabilizing enzymes and allow transfer of molecules between enzymes participating in cascade reactions for enhanced substrate channelling. Herein, we systematically synthesized and investigated the stability of HOF at extreme conditions followed by one-pot encapsulation of single and bi-enzyme systems. Firstly, we observed HOF to be stable at pHâ 1 to 14 and at high temperatures (up to 115 °C). Secondly, the encapsulated glucose oxidase enzyme (GOX) showed 80 % and 90 % of its original activity at 70 °C and pHâ 11, respectively. Thirdly, transient time close to 0â seconds was observed for HOF encapsulated bi-enzyme cascade reaction system demonstrating a 4.25-fold improvement in catalytic activity when compared to free enzymes with enhanced substrate channelling. Our findings showcase a facile system synthesized under ambient conditions to encapsulate and stabilize enzymes at extreme conditions.
Subject(s)
Glucose Oxidase , Hydrogen Bonding , Metal-Organic Frameworks , Glucose Oxidase/chemistry , Glucose Oxidase/metabolism , Metal-Organic Frameworks/chemistry , Porosity , Hydrogen-Ion Concentration , Temperature , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , CatalysisABSTRACT
Co-assembling enzymes with nanoparticles (NPs) into nanoclusters allows them to access channeling, a highly efficient form of multienzyme catalysis. Using pyruvate kinase (PykA) and lactate dehydrogenase (LDH) to convert phosphoenolpyruvic acid to lactic acid with semiconductor quantum dots (QDs) confirms how enzyme cluster formation dictates the rate of coupled catalytic flux (kflux) across a series of differentially sized/shaped QDs and 2D nanoplatelets (NPLs). Enzyme kinetics and coupled flux were used to demonstrate that by mixing different NP systems into clusters, a >10× improvement in kflux is observed relative to free enzymes, which is also ≥2× greater than enhancement on individual NPs. Cluster formation was characterized with gel electrophoresis and transmission electron microscopy (TEM) imaging. The generalizability of this mixed-NP approach to improving flux is confirmed by application to a seven-enzyme system. This represents a powerful approach for accessing channeling with almost any choice of enzymes constituting a multienzyme cascade.
Subject(s)
L-Lactate Dehydrogenase , Lactic Acid , Nanoparticles , Phosphoenolpyruvate , Pyruvate Kinase , L-Lactate Dehydrogenase/metabolism , L-Lactate Dehydrogenase/chemistry , Lactic Acid/metabolism , Lactic Acid/chemistry , Pyruvate Kinase/metabolism , Pyruvate Kinase/chemistry , Nanoparticles/chemistry , Phosphoenolpyruvate/metabolism , Quantum Dots/chemistry , KineticsABSTRACT
Nanozymes mimic enzymes and that includes their selectivity. To achieve selectivity, significant inspiration for nanoparticle design can come from the geometric and molecular features that make enzymes selective catalysts. The two central features enzymes use are control over the arrangement of atoms in the active site and the placing of the active site down a nanoconfined substrate channel. The implementation of enzyme-inspired features has already been shown to both improve activity and selectivity of nanoparticles for a variety of catalytic and sensing applications. The tuning and control of active sites on metal nanoparticle surfaces ranges from simply changing the composition of the surface metal to sophisticated approaches such as the immobilization of single atoms on a metal substrate. Molecular frameworks provide a powerful platform for the implementation of isolated and discrete active sites while unique diffusional environments further improve selectivity. The implementation of nanoconfined substrate channels around these highly controlled active sites offers further ability to control selectivity through altering the solution environment and transport of reactants and products. Implementing these strategies together offers a unique opportunity to improve nanozyme selectivity in both sensing and catalysis.
Subject(s)
Metal Nanoparticles , Metal Nanoparticles/chemistry , Catalysis , Catalytic DomainABSTRACT
Spatial organization of biocatalytic activities is crucial to organisms to efficiently process complex metabolism. Inspired by this mechanism, artificial scaffold structures are designed to harbor functionally coupled biocatalysts, resulting in acellular materials that can complete multistep reactions at high efficiency and low cost. Substrate channeling is an approach for efficiency enhancement of multistep reactions, but fast diffusion of small molecule intermediates poses a major challenge to achieve channeling in vitro. Here, we explore how multistep biocatalysis is affected, and can be modulated, by cofactor-enzyme colocalization within a synthetic bioinspired material. In this material, a heterogeneous protein macromolecular framework (PMF) acts as a porous host matrix for colocalization of two coupled enzymes and their small molecule cofactor, nicotinamide adenine dinucleotide (NAD). After formation of the PMF from a higher order assembly of P22 virus-like particles (VLPs), the enzymes were partitioned into the PMF by covalent attachment and presentation on the VLP exterior. Using a collective property of the PMF (i.e., high density of negative charges in the PMF), NAD molecules were partitioned into the framework via electrostatic interactions after being conjugated to a polycationic species. This effectively controlled the localization and diffusion of NAD, resulting in substrate channeling between the enzymes. Changing ionic strength modulates the PMF-NAD interactions, tuning two properties that impact the multistep efficiency oppositely in response to ionic strength: cofactor partitioning (colocalization with the enzymes) and cofactor mobility (translocation between the enzymes). Within the range tested, we observed a maximum of 5-fold increase or 75% decrease in multistep efficiency as compared to free enzymes in solution, which suggest both the colocalization and the mobility are critical for the multistep efficiency. This work demonstrates utility of collective behaviors, exhibited by hierarchical bioassemblies, in the construction of functional materials for enzyme cascades, which possess properties such as tunable multistep biocatalysis.
Subject(s)
NAD , Porins , Biocatalysis , Porosity , Diffusion , Macromolecular SubstancesABSTRACT
Coordinating microbial consortia to realize complex synthetic pathways is an area of great interest in the rapidly growing field of biomanufacturing. This work presents a programmable method for assembling living cells based on the surface display of affinity groups, enabling whole-cell catalysis with optimized catalytic efficiency through the rational arrangement of cell assemblies and enzymes. In the context of d-phenyllactic acid (d-PLA) synthesis, four enzymes were rationally arranged considering substrate channeling and protein expression levels. The production efficiencies of d-PLA catalyzed by engineered microbial consortia were 1.31- and 2.55-fold higher than those of biofilm and whole-cell catalysts, respectively. Notably, substrate channeling was identified between the coimmobilized rate-limiting enzymes, resulting in a 3.67-fold improvement in catalytic efficiency compared with hybrid catalysts (free enzymes coupled with whole-cell catalysts). The highest yield of d-PLA catalyzed by microbial consortia was 102.85 ± 3.39 mM with 140 mM benzaldehyde as the substrate. This study proposes a novel approach to cell enzyme assembly for coordinating microbial consortia in multiple enzymatic biosynthesis processes.
Subject(s)
Escherichia coli , Polyesters , Escherichia coli/genetics , Escherichia coli/metabolism , Catalysis , Polyesters/metabolism , Microbial ConsortiaABSTRACT
Chorismate mutase (CM) and cyclohexadienyl dehydratase (CDT) catalyze two subsequent reactions in the intracellular biosynthesis of l-phenylalanine (Phe). Here, we report the discovery of novel and extremely rare bifunctional fusion enzymes, consisting of fused CM and CDT domains, which are exported from the cytoplasm. Such enzymes were found in only nine bacterial species belonging to non-pathogenic γ- or ß-Proteobacteria. In γ-proteobacterial fusion enzymes, the CM domain is N-terminal to the CDT domain, whereas the order is inverted in ß-Proteobacteria. The CM domains share 15% to 20% sequence identity with the AroQγ class CM holotype of Mycobacterium tuberculosis (∗MtCM), and the CDT domains 40% to 60% identity with the exported monofunctional enzyme of Pseudomonas aeruginosa (PheC). In vitro kinetics revealed a Km <7 µM, much lower than for ∗MtCM, whereas kinetic parameters are similar for CDT domains and PheC. There is no feedback inhibition of CM or CDT by the pathway's end product Phe, and no catalytic benefit of the domain fusion compared with engineered single-domain constructs. The fusion enzymes of Aequoribacter fuscus, Janthinobacterium sp. HH01, and Duganella sacchari were crystallized and their structures refined to 1.6, 1.7, and 2.4 Å resolution, respectively. Neither the crystal structures nor the size-exclusion chromatography show evidence for substrate channeling or higher oligomeric structure that could account for the cooperation of CM and CDT active sites. The genetic neighborhood with genes encoding transporter and substrate binding proteins suggests that these exported bifunctional fusion enzymes may participate in signaling systems rather than in the biosynthesis of Phe.
ABSTRACT
Ectoine and its derivative 5-hydroxyectoine are compatible solutes initially found in the hyperhalophilic bacterium Ectothiorhodospira halochloris, which inhabits the desert in Egypt. The habitat of ectoine producers implies the primary function of ectoine as a cytoprotectant against harsh conditions such as high salinity, drought, and high radiation. More extensive and in-depth studies have revealed the multiple functions of ectoine in its native producer bacterial cells and other types of cells and its biomolecular components (such as proteins and DNA) as a general protective agent. Its chemical properties as a bio-based amino acid derivative make it attractive for basic scientific research and related industries, such as the food/agricultural industry, cosmetic manufacturing, biologics, and therapeutic agent preparation. This article first discusses the functions and applications of ectoine and 5-hydroxyectoine. Subsequently, more emphasis was placed on advances in bio-based ectoine and/or 5-hydroxyectoine production. Strategies for developing more robust cell factories for highly efficient ectoine and/or 5-hydroxyectoine production are further discussed. We hope this review will provide a valuable reference for studies on the bio-based production of ectoine and 5-hydroxyectoine.
ABSTRACT
An enzyme cascade was established previously consisting of a recycling system with an l-amino acid oxidase (hcLAAO4) and a catalase (hCAT) for different α-keto acid co-substrates of (S)-selective amine transaminases (ATAs) in kinetic resolutions of racemic amines. Only 1â mol % of the co-substrate was required and l-amino acids instead of α-keto acids could be applied. However, soluble enzymes cannot be reused easily. Immobilization of hcLAAO4, hCAT and the (S)-selective ATA from Vibrio fluvialis (ATA-Vfl) was addressed here. Immobilization of the enzymes together rather than on separate beads showed higher reaction rates most likely due to fast co-substrate channeling between ATA-Vfl and hcLAAO4 due to their close proximity. Co-immobilization allowed further reduction of the co-substrate amount to 0.1â mol % most likely due to a more efficient H2 O2 -removal caused by the stabilized hCAT and its proximity to hcLAAO4. Finally, the co-immobilized enzyme cascade was reused in 3 cycles of preparative kinetic resolutions to produce (R)-1-PEA with high enantiomeric purity (97.3 %ee). Further recycling was inefficient due to the instability of ATA-Vfl, while hcLAAO4 and hCAT revealed high stability. An engineered ATA-Vfl-8M was used in the co-immobilized enzyme cascade to produce (R)-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethanamine, an apremilast-intermediate, with a 1,000 fold lower input of the co-substrate.
Subject(s)
Amines , Transaminases , Amines/chemistry , Transaminases/chemistry , L-Amino Acid Oxidase , Enzymes, Immobilized/chemistry , Catalase , Keto AcidsABSTRACT
Metabolons are temporary structural-functional complexes of sequential enzymes of a metabolic pathway that are distinct from stable multi-enzyme complexes. Here we provide a brief history of the study of enzyme-enzyme assemblies with a particular focus on those that mediate substrate channeling in plants. Large numbers of protein complexes have been proposed for both primary and secondary metabolic pathways in plants. However, to date only four substrate channels have been demonstrated. We provide an overview of current knowledge concerning these four metabolons and explain the methodologies that are currently being applied to unravel their functions. Although the assembly of metabolons has been documented to arise through diverse mechanisms, the physical interaction within the characterized plant metabolons all appear to be driven by interaction with structural elements of the cell. We therefore pose the question as to what methodologies could be brought to bear to enhance our knowledge of plant metabolons that assemble via different mechanisms? In addressing this question, we review recent findings in non-plant systems concerning liquid droplet phase separation and enzyme chemotaxis and propose strategies via which such metabolons could be identified in plants. We additionally discuss the possibilities that could be opened up by novel approaches based on: (i) subcellular-level mass spectral imaging, (ii) proteomics, and (iii) emergent methods in structural and computational biology.
Subject(s)
Metabolic Networks and Pathways , PlantsABSTRACT
In multienzymes cascade reaction, the inter-enzyme spacing is supposed to be a factor affecting the cascade activity. Here, a simple and efficient Y-shaped DNA scaffold is assembled using two partially complementary DNA single strands on magnetic microspheres, which is used to coimmobilize glucose oxidase (GOD) and horseradish peroxidase (HRP). As a result, on poly(vinyl acetate) magnetic microspheres (PVAC), GOD/HRP-DNA@PVAC multienzyme system is obtained, which can locate GOD and HRP accurately and control the inter-enzyme distance precisely. The distance between GOD and HRP is regulated by changing the length of DNA strand. It showed that the cascade activity is significantly distance-dependent. Moreover, the inter-enzyme spacing is not the closer the better, and too short distance would generate steric hindrance between enzymes. The cascade activity reached the maximum value of 967 U mg-1 at 13.6 nm, which is 3.5 times higher than that of free enzymes. This is ascribed to the formation of substrate channeling.
Subject(s)
Enzymes, Immobilized , Glucose Oxidase , Horseradish Peroxidase , Microspheres , DNAABSTRACT
Many biocatalytic processes inside cells employ substrate channeling to control the diffusion of intermediates for improved efficiency of enzymatic cascade reactions. This inspirational mechanism offers a strategy for increasing efficiency of multistep biocatalysis, especially where the intermediates are expensive cofactors that require continuous regeneration. However, it is challenging to achieve substrate channeling artificially in vitro due to fast diffusion of small molecules. By mimicking some naturally occurring metabolons, nanoreactors are developed using P22 virus-like particles (VLPs), which enhance the efficiency of nicotinamide adenine dinucleotide (NAD)-dependent multistep biocatalysis by substrate channeling. In this design, NAD-dependent enzyme partners are coencapsulated inside the VLPs, while the cofactor is covalently tethered to the capsid interior through swing arms. The crowded environment inside the VLPs induces colocalization of the enzymes and the immobilized NAD, which shuttles between the enzymes for in situ regeneration without diffusing into the bulk solution. The modularity of the nanoreactors allows to tune their composition and consequently their overall activity, and also remodel them for different reactions by altering enzyme partners. Given the plasticity and versatility, P22 VLPs possess great potential for developing functional materials capable of multistep biotransformations with advantageous properties, including enhanced efficiency and economical usage of enzyme cofactors.
Subject(s)
Biomimetics , NAD , NAD/metabolism , BiocatalysisABSTRACT
Yeast surface display is an appealing technique for constructing multienzyme cascades. This technique is commonly achieved using a scaffold for the ordered arrangement of various enzymes. However, this method is typically complicated because scaffold use may engender extra metabolic burden on the cell host. Here, we established a direct yeast surface codisplay strategy by employing two complementary anchor motifs, Agα1 and Pir1. These motifs allow for the codisplay of sequential uridine diphosphate-glycosyltransferase (UGT) and sucrose synthase (SUS) on the surface of Pichia pastoris (syn. Komagataella phaffii) for the glycosylation of natural products. We manipulated the displayed stoichiometry, amount, and assembly order of UGT and SUS by coupling them with anchor motifs. Furthermore, their effect on enzyme activity was thoroughly investigated. The surface-codisplayed strain UGT-Pir-SUS-Agα exhibited greater thermostability than the single-displayed strains and their free counterparts. Moreover, the strain UGT-Pir-SUS-Agα was successfully applied to glycyrrhetinic acid (GA) glycosylation to produce GA-3-O-Glc, with sucrose being the sugar donor in this process. This generated 7.5- to 20- and 5.3-fold higher GA-3-O-Glc concentration compared with the free counterparts (enzyme mass loading of 20-fold in excess) and mixed single-displayed strains of UGT-Agα and SUS-Pir, respectively. This increase was due to the improved biochemical properties and substrate channeling effect of strain UGT-Pir-SUS-Agα. This controllable direct surface codisplay strategy, based on complementary anchor motifs, is readily extendable to other enzyme cascades.
Subject(s)
Biological Products , Glycosylation , Biological Products/metabolism , Glycosyltransferases/metabolism , Pichia/genetics , Pichia/metabolismABSTRACT
The pursuit of single-assembled molecular cage reactors for complex tandem reactions is a long-standing target in biomimetic catalysis but still a grand challenge. Herein, nanozyme-like organic cages are reported by engineering air-stable radicals into the skeleton upon photoinduced electron transfer. The generation of radicals is accompanied by single-crystal structural transformation and exhibits superior stability over six months in air. Impressively, the radicals throughout the cage skeleton can mimic the peroxidase of natural enzymes to decompose H2 O2 into OH· and facilitate oxidation reactions. Furthermore, an integrated catalyst by encapsulating Au clusters (glucose oxidase mimics) into the cage has been developed, in which the dual active sites (Au cluster and radical) are spatially isolated and can work as cascade nanozymes to prominently promote the enzyme-like tandem reaction via a substrate channeling effect.
ABSTRACT
REPI is a pivotal point enzyme in plant benzylisoquinoline alkaloid metabolism as it promotes the evolution of the biosynthetic branch of morphinan alkaloids. Experimental studies of its activity led to the identification of two modules (DRS and DRR) that catalyze two sequential steps of the epimerization of (S)- to (R)-reticuline. Recently, special attention has been paid to its genetic characterization and evolutionary history, but no structural analyses of the REPI protein have been conducted to date. We present here a computational structural characterization of REPI with heme and NADP cofactors in the apo state and in three complexes with substrate (S)-reticuline in DRS and intermediate 1,2-dehydroreticuline in DRS and in DRR. Since no experimental structure exists for REPI, we used its AlphaFold model as a scaffold to build up these four systems, which were submitted to all-atom molecular dynamics (MD) simulations. A comparison of MD results for the four systems revealed key dynamic changes associated with cofactor and ligand binding and provided a dynamic picture of the evolution of their structures and interactions. We also explored the possible dynamic occurrence of tunnels and electrostatic highways potentially involved in alternative mechanisms for channeling the intermediate from DRS to DRR.
Subject(s)
Alkaloids , Papaver , Papaver/genetics , Papaver/chemistry , Papaver/metabolism , Molecular Dynamics Simulation , Alkaloids/chemistryABSTRACT
Fusion proteins, understood as those created by joining two or more genes that originally encoded independent proteins, have numerous applications in biotechnology, from analytical methods to metabolic engineering. The use of fusion enzymes in biocatalysis may be even more interesting due to the physical connection of enzymes catalyzing successive reactions into covalently linked complexes. The proximity of the active sites of two enzymes in multi-enzyme complexes can make a significant contribution to the catalytic efficiency of the reaction. However, the physical proximity of the active sites does not guarantee this result. Other aspects, such as the nature and length of the linker used for the fusion or the order in which the enzymes are fused, must be considered and optimized to achieve the expected increase in catalytic efficiency. In this review, we will relate the new advances in the design, creation, and use of fused enzymes with those achieved in biocatalysis over the past 20 years. Thus, we will discuss some examples of genetically fused enzymes and their application in carboncarbon bond formation and oxidative reactions, generation of chiral amines, synthesis of carbohydrates, biodegradation of plant biomass and plastics, and in the preparation of other high-value products.
Subject(s)
Multifunctional Enzymes , Protein Engineering , Amines/chemistry , Amines/metabolism , Biocatalysis , Carbohydrates , Carbon , Enzymes/chemistry , Multifunctional Enzymes/metabolism , Plastics/metabolismABSTRACT
Enzyme-enzyme assemblies commonly occur naturally, yet the factors that lead to their transient nature are not fully understood. Mitkas et al. have shown how clustered regularly interspaced short palindromic repeats (CRISPR) enzymes and RNA scaffolds allow synthetic enzyme complexes to be formed and disassembled as needed, providing powerful new tools for metabolic engineering.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , RNA , CRISPR-Cas Systems , Metabolic Engineering , RNA/metabolismABSTRACT
Proteins are not designed to be standalone entities and must coordinate their collective action for optimum performance. Nature has developed through evolution the ability to co-localize the functional partners of a cascade enzymatic reaction in order to ensure efficient exchange of intermediates. Inspired by these natural designs, synthetic scaffolds have been created to enhance the overall biological pathway performance. In this chapter, we describe several DNA- and protein-based scaffold approaches to assemble artificial enzyme cascades for a wide range of applications. We highlight the key benefits and drawbacks of these approaches to provide insights on how to choose the appropriate scaffold for different cascade systems.