Supramolecular Enzyme Research Articles

Protein–Polymer Conjugates: Advancing Enzyme Catalysis in Synthetic Chemistry

AbstractEnzyme catalysis is prominent in chemical synthesis due to its specificity, selectivity, and efficiency. However, enzymes often face challenges, such as inactivation and inhibition in practical applications, and they are also limited by narrow reaction scopes in complex catalytic scenarios like cascade reactions. These persistent issues have driven the development of chemical modifications of enzymes, aiming to enhance enzyme catalysis with external chemical entities. Polymers are particularly notable among these entities for their functional moieties and protective effects on biomolecules, enhancing enzyme properties, and even creating new‐to‐nature catalysis by harnessing catalytic promiscuity. This concept aims to introduce the field of protein–polymer conjugates for diverse emerging applications across emulsion biocatalysis, artificial enzymes, and supramolecular enzyme catalysis.

Integrating Enzymes with Supramolecular Polymers for Recyclable Photobiocatalytic Catalysis

AbstractChemical modifications of enzymes excel in the realm of enzyme engineering due to its directness, robustness, and efficiency; however, challenges persist in devising versatile and effective strategies. In this study, we introduce a supramolecular modification methodology that amalgamates a supramolecular polymer with Candida antarctica lipase B (CalB) to create supramolecular enzymes (SupEnzyme). This approach features the straightforward preparation of a supramolecular amphiphilic polymer (β‐CD@SMA), which was subsequently conjugated to the enzyme, resulting in a SupEnzyme capable of self‐assembly into supramolecular nanoparticles. The resulting SupEnzyme nanoparticles can form micron‐scale supramolecular aggregates through supramolecular and electrostatic interactions with guest entities, thus enhancing catalyst recycling. Remarkably, these aggregates maintain 80 % activity after seven cycles, outperforming Novozym 435. Additionally, they can effectively initiate photobiocatalytic cascade reactions using guest photocatalysts. As a consequence, our SupEnzyme methodology exhibits noteworthy adaptability in enzyme modification, presenting a versatile platform for various polymer, enzyme, and biocompatible catalyst pairings, with potential applications in the fields of chemistry and biology.

Integrating Enzymes with Supramolecular Polymers for Recyclable Photobiocatalytic Catalysis.

Chemical modifications of enzymes excel in the realm of enzyme engineering due to its directness, robustness, and efficiency; however, challenges persist in devising versatile and effective strategies. In this study, we introduce a supramolecular modification methodology that amalgamates a supramolecular polymer with Candida antarctica lipase B (CalB) to create supramolecular enzymes (SupEnzyme). This approach features the straightforward preparation of a supramolecular amphiphilic polymer (β-CD@SMA), which was subsequently conjugated to the enzyme, resulting in a SupEnzyme capable of self-assembly into supramolecular nanoparticles. The resulting SupEnzyme nanoparticles can form micron-scale supramolecular aggregates through supramolecular and electrostatic interactions with guest entities, thus enhancing catalyst recycling. Remarkably, these aggregates maintain 80 % activity after seven cycles, outperforming Novozym 435. Additionally, they can effectively initiate photobiocatalytic cascade reactions using guest photocatalysts. As a consequence, our SupEnzyme methodology exhibits noteworthy adaptability in enzyme modification, presenting a versatile platform for various polymer, enzyme, and biocompatible catalyst pairings, with potential applications in the fields of chemistry and biology.

Point-of-care detection of hydroxyurea drug in serum using a supramolecular enzyme mimetic

The sole approved therapeutic drug for sickle cell disease management is provided by hydroxyurea, requiring its precise detection and monitoring in biological matrices such as human blood serum for personalized treatment. Herein, we introduce a direct colorimetric detection method for hydroxyurea in serum using a metallo-supramolecular assembly called Vesi-ImCu that mimics the peroxidase enzyme. The Vesi-ImCu enzyme mimetic comprises a catalytically active copper center coordinated to synthetic imidazole containing surfactant and iminodiacetic acid capping moieties. By leveraging the peroxidase-like activity of Vesi-ImCu, we have demonstrated that it catalyzes the efficient production of nitric oxide from hydroxyurea that reacts with the Griess reagent, producing an intense pink color. This approach enables the naked-eye detection of hydroxyurea, with notable efficacy even in complex biological matrices such as human blood serum. The Vesi-ImCu suprazyme-based approach offers several advantages, including high sensitivity (limit of detection of 75 µM), clinically relevant dynamic range of hydroxyurea detection, minimum batch-to-batch variation, and the ability to perform mobile phone-based quantification. To our knowledge, this is the first report on direct colorimetric quantification of HU in serum. With its visual detection ability, high-end equipment-free quantification, user-friendliness and portability, the present enzyme mimetic holds great promise for point-of-care detection applications.

SupEnzyme: Combining enzymes with supramolecules for recyclable catalysis

Enzymes are formidable homogeneous biocatalysts, yet their recyclability remains a substantial impediment in the chemical industry. To address this, we introduce a supramolecular engineering approach that chemically conjugates enzymes with supramolecular β-cyclodextrin (β-CD), furnishing them with the capability for on-demand recyclable catalysis. This method generates a supramolecular enzyme, termed SupEnzyme, in which enzymes are endowed with supramolecular plug-and-play functionalities. Consequently, it can function both as a water-soluble homogeneous catalyst and, when interacting with guest polymers, as a recyclable heterogeneous catalyst. The selective introduction of guests and competitive guests allows for dynamic switching between homogeneous and heterogeneous states, showcasing the adaptability of this approach for catalysis and recycling. Moreover, this concept is extended to the integration of two distinct enzymes for recyclable multienzymatic cascades. Hence, this supramolecular strategy is envisioned to lay the foundation for the next generation of chemical enzyme engineering.

π‐π Catalysis Made Asymmetric—Enantiomerization Catalysis Mediated by the Chiral π‐System of a Perylene Bisimide Cyclophane

AbstractEnzymes actuate catalysis through a combination of transition state stabilization and ground state destabilization, inducing enantioselectivity through chiral binding sites. Here, we present a supramolecular model system which employs these basic principles to catalyze the enantiomerization of [5]helicene. Catalysis is hereby mediated not through a network of functional groups but through π‐π catalysis exerted from the curved aromatic framework of a chiral perylene bisimide (PBI) cyclophane offering a binding pocket that is intricately complementary with the enantiomerization transition structure. Although transition state stabilization originates simply from dispersion and electrostatic interactions, enantiomerization kinetics are accelerated by a factor of ca. 700 at 295 K. Comparison with the meso‐congener of the catalytically active cyclophane shows that upon configurational inversion in only one PBI moiety the catalytic effect is lost, highlighting the importance of precise transition structure recognition in supramolecular enzyme mimics.

π-π Catalysis Made Asymmetric-Enantiomerization Catalysis Mediated by the Chiral π-System of a Perylene Bisimide Cyclophane.

Enzymes actuate catalysis through a combination of transition state stabilization and ground state destabilization, inducing enantioselectivity through chiral binding sites. Here, we present a supramolecular model system which employs these basic principles to catalyze the enantiomerization of [5]helicene. Catalysis is hereby mediated not through a network of functional groups but through π-π catalysis exerted from the curved aromatic framework of a chiral perylene bisimide (PBI) cyclophane offering a binding pocket that is intricately complementary with the enantiomerization transition structure. Although transition state stabilization originates simply from dispersion and electrostatic interactions, enantiomerization kinetics are accelerated by a factor of ca. 700 at 295 K. Comparison with the meso-congener of the catalytically active cyclophane shows that upon configurational inversion in only one PBI moiety the catalytic effect is lost, highlighting the importance of precise transition structure recognition in supramolecular enzyme mimics.

Markov State Study of Electrostatic Channeling within the Tricarboxylic Acid Cycle Supercomplex.

The high efficiency of cascade reactions in supramolecular enzyme nanoassemblies, known as metabolons, has attracted substantial attention in various fields ranging from fundamental biochemistry and molecular biology to recent applications in biofuel cells, biosensors, and chemical synthesis. One reason for the high efficiency of metabolons is the structures formed by sequential enzymes that allow the direct transport of intermediates between consecutive active sites. The supercomplex of malate dehydrogenase (MDH) and citrate synthase (CS) is an ideal example of the controlled transport of intermediates via electrostatic channeling. Here, using a combination of molecular dynamics (MD) simulations and a Markov state model (MSM), we examined the transport process of the intermediate oxaloacetate (OAA) from MDH to CS. The MSM enables the identification of the dominant transport pathways of OAA from MDH to CS. Analysis of all pathways using a hub score approach reveals a small set of residues that control OAA transport. This set includes an arginine residue previously identified experimentally. MSM analysis of a mutated complex, where the identified arginine is replaced by alanine, led to a 2-fold decrease in transfer efficiency, also consistent with experimental results. This work provides a molecular-level understanding of the electrostatic channeling mechanism and will enable the further design of catalytic nanostructures utilizing electrostatic channeling.

Assembly status transition offers an avenue for activity modulation of a supramolecular enzyme.

Nature has evolved many supramolecular proteins assembled in certain, sometimes even seemingly oversophisticated, morphological manners. The rationale behind such evolutionary efforts is often poorly understood. Here, we provide atomic-resolution insights into how the dynamic building of a structurally complex enzyme with higher order symmetry offers amenability to intricate regulation. We have established the functional coupling between enzymatic activity and protein morphological states of glutamine synthetase (GS), an old multi-subunit enzyme essential for cellular nitrogen metabolism. Cryo-EM structure determination of GS in both the catalytically active and inactive assembly states allows us to reveal an unanticipated self-assembly-induced disorder-order transition paradigm, in which the remote interactions between two subcomplex entities significantly rigidify the otherwise structurally fluctuating active sites, thereby regulating activity. We further show in vivo evidences that how the enzyme morphology transitions could be modulated by cellular factors on demand. Collectively, our data present an example of how assembly status transition offers an avenue for activity modulation, and sharpens our mechanistic understanding of the complex functional and regulatory properties of supramolecular enzymes.

Construction of supramolecular laccase enzymes and understanding of catalytic dye degradation using multispectral and molecular docking approaches

A non-covalent supramolecular enzyme system which was successfully constructed by non-covalent interaction of enzyme with substrates analogs can effectively recognize and degrade 13 kinds of dyes.

Biomimetic Membrane-Structured Nanovesicles Carrying a Supramolecular Enzyme to Cure Lung Cancer.

Platforms for enzyme delivery must simultaneously have plasma stability, high catalytic activity, and low/no immunogenicity of the enzyme. Here, we designed a novel biomimetic membrane-structured nanovesicle (BNV) to efficiently carry supramolecular enzymes to meet the above requirements. We complexed l-asparaginase (Aase) with hydroxypropyl-β-cyclodextrin (HPCD) to form a supramolecular amphiphile (AS) by self-assembly via noncovalent reversible interactions. We then used the first synthesized polyethylene glycol (PEG 2 kDa)-decorated hyaluronan (12 kDa) and HPCD to self-assemble a semipermeable biomimetic membrane-structured nanovesicle (BNV) together with AS loading. As compared to native Aase, AS@BNV exhibited superior catalytic activity preservation, improved catalytic activity, better pharmacokinetics in rats, enhanced cytotoxic effects, increased antitumor efficacy, and decreased side effects. The underlying mechanisms, such as the autophagy inhibition action against tumor cells, protein-protein docking of the interaction between Aase-serum albumin, and decreased hepatic enzymatic activity, were investigated. This approach paves the way for new types of powerful biomimetic-, supramolecular-, and nanocarrier-based enzymatic therapies.

Antibiotics Targeting the 30S Ribosomal Subunit: A Lesson from Nature to Find and Develop New Drugs.

The use of antibiotics has revolutionized medicine, greatly improving our capacity to save millions of lives from otherwise deadly bacterial infections. Unfortunately, the health-associated benefits provided by antibiotics have been counteracted by bacteria developing or acquiring resistance mechanisms. The negative impact to public health is now considered of high risk due to the rapid spreading of multi-resistant strains. More than 60 % of clinically relevant antibiotics of natural origin target the ribosome, the supramolecular enzyme which translates the genetic information into proteins. Although many of these antibiotics bind the small ribosomal subunit, only a few are reported to inhibit the initiation of protein synthesis, with none reaching commercial availability. Counterintuitively, translation initiation is the most divergent phase of protein synthesis between prokaryotes and eukaryotes, a fact which is a solid premise for the successful identification of drugs with reduced probability of undesired effects to the host. Such a paradox is one of its kind and deserves special attention. In this review, we explore the inhibitors that bind the 30S ribosomal subunit focusing on both the compounds with proved effects on the translation initiation step and the underreported translation initiation inhibitors. In addition, we explore recent screening tests and approaches to discover new drugs targeting translation.

Construction of Artificial Enzymes on aVirus Surface.

Combination of artificial enzyme design and self-assembly strategies leads to a novel way to construct supramolecular enzymes. To address this challenge, auxotrophic expression systems show great potential because they can introduce nonnatural catalytic groups into the subunits of protein assemblies. Among nonnatural amino acids, selenocysteine is the catalytic group of glutathione peroxidase (GPx). With the aid of computer simulation, we have incorporated selenocysteine into natural protein assemblies such as tobacco mosaic virus (TMV) and ferritin by cysteine auxotrophic technology, resulting in the conversion of TMV and ferritin into supramolecular enzymes.

Self-Assembled DNA/Peptide-Based Nanoparticle Exhibiting Synergistic Enzymatic Activity.

Designing enzyme-mimicking active sites in artificial systems is key to achieving catalytic efficiencies rivaling those of natural enzymes and can provide valuable insight in the understanding of the natural evolution of enzymes. Here, we report the design of a catalytic hemin-containing nanoparticle with self-assembled guanine-rich nucleic acid/histidine-rich peptide components that mimics the active site and peroxidative activity of hemoproteins. The chemical complementarities between the folded nucleic acid and peptide enable the spatial arrangement of essential elements in the active site and effective activation of hemin. As a result, remarkable synergistic effects of nucleic acid and peptide on the catalytic performances were observed. The turnover number of peroxide reached the order of that of natural peroxidase, and the catalytic efficiency is comparable to that of myoglobin. These results have implications in the precise design of supramolecular enzyme mimetics, particularly those with hierarchical active sites. The assemblies we describe here may also resemble an intermediate in the evolution of contemporary enzymes from the catalytic RNA of primitive cells.

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channeling is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3-ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an "NADH Sink" was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol.

Supramolecular enzyme engineering in complex nanometer-thin biomimetic organosilica layers

Enzyme shielding at the surface of silica nanoparticles was performed using different mixtures of biomimetic building blocks. The performances of the nanobiocatalysts are strongly impacted by the chemical composition of the shielding layer.

Enabling New Modes of Reactivity via Constrictive Binding in a Supramolecular-Assembly-Catalyzed Aza-Prins Cyclization.

Supramolecular assembly 1 catalyzes a bimolecular aza-Prins cyclization featuring an unexpected transannular 1,5-hydride transfer. This reaction pathway, which is promoted by constrictive binding within the supramolecular cavity of 1, is kinetically disfavored in the absence of 1, as evidenced by the orthogonal reactivity observed in bulk solution. Mechanistic investigation through kinetic analysis and isotopic labeling studies indicates that the rate-limiting step of the transformation is the encapsulation of a transient iminium ion and supports the proposed 1,5-hydride transfer mechanism. This represents a rare example of such an extreme divergence of product selectivity observed within a catalytic metal-ligand supramolecular enzyme mimic.

Construction of a novel guest biomimetic glutathione peroxidase with solvent-dependent catalytic behavior by incorporating the active center into adamantyl molecule

A novel guest biomimetic glutathione peroxidase (3,3'-tellurobis(propane-3,1-diyl) diadamantanecarboxylate, denoted as ADA-Te-ADA) was synthesized. ADA-Te-ADA functioned to overcome the disadvantages in the construction of building block for giant supramolecular biomimetic enzyme. To reveal the catalytic property of hydrophobic ADA-Te-ADA, the catalytic mechanism was investigated using PBS (phosphate buffer (pH 7.0. 50 mM))/methanol solvent mixture as assay solution. Itindicated that ADA-Te-ADA exhibited typical enzyme catalytic behavior by saturation kinetics measurement. Importantly, ADA-Te-ADA exhibited the typical solvent-dependent catalytic behavior. And the highest catalytic rate 4.29 µM x min-1 was obtained when the volume ratio of PBs: methanol was 5 : 5. Especially, the catalytic rates obtained based on various substrates proved that ADA-Te-ADA slightly displayed special substrate selectivity, which was the ideal catalytic characterization of building block for giant supramolecular biomimetic enzyme. The successfully synthesis of ADA-Te-ADA might highlight for the understanding of the catalytic mechanism of hydrophobic guest biomimetic glutathione peroxidase. And it also might provide the basement for the construction of giant supramolecular biomimetic enzyme.

Insights into cellulosome assembly and dynamics: from dissection to reconstruction of the supramolecular enzyme complex

Cellulosomes are multi-enzyme complexes produced by anaerobic bacteria for the efficient deconstruction of plant cell wall polysaccharides. The assembly of enzymatic subunits onto a central non-catalytic scaffoldin subunit is mediated by a highly specific interaction between the enzyme-bearing dockerin modules and the resident cohesin modules of the scaffoldin, which affords their catalytic activities to work synergistically. The scaffoldin also imparts substrate-binding and bacterial-anchoring properties, the latter of which involves a second cohesin-dockerin interaction. Recent structure-function studies reveal an ever-growing array of unique and increasingly complex cohesin-dockerin complexes and cellulosomal enzymes with novel activities. A 'build' approach involving multimodular cellulosomal segments has provided a structural model of an organized yet conformationally dynamic supramolecular assembly with the potential to form higher order structures.

Understanding enzyme catalysis by means of supramolecular artificial enzymes

Enzymes are biomacromolecules responsible for the abundant chemical biotransformations that sustain life. Recently, biochemists have discovered that multiple conformations and numerous parallel paths are involved during the processes catalyzed by enzymes. It is plausible that the entire macromolecular scaffold is involved in catalysis via cooperative motions that result in incredible catalytic efficiency. Moreover, some enzymes can very strongly bind the transition state with an association constant of up to 1024 M−1, suggesting that covalent bond formation is a possible process during the conversion of the transition state in enzyme catalysis, in addition to the concatenation of non-covalent interactions. Supramolecular chemistry provides fundamental knowledge about the relationships between the dynamic structures and functions of organized molecules. By taking advantage of supramolecular concepts, numerous supramolecular enzyme mimics with complex and hierarchical structures have been designed and investigated. Through the study of supramolecular enzyme models, a great deal of information to aid our understanding of the mechanism of catalysis by natural enzymes has been acquired. With the development of supramolecular artificial enzymes, it is possible to replicate the features of natural enzymes with regards to their constitutional complexity and cooperative motions, and eventually decipher the conformation-based catalytic mystery of natural enzymes.