Artificial Metalloenzymes Research Articles

Intermolecular Abiological Hetero-Diels–Alder Reaction Catalyzed by an Artificial Metalloenzyme

Key words hetero-Diels–Alder reaction - artificial enzyme - metalloenzyme mimic - enzymatic Lewis acid catalysis

Prodrug Activation by Gold Artificial Metalloenzyme-Catalyzed Synthesis of Phenanthridinium Derivatives via Hydroamination.

An emerging approach in the field of targeted drug delivery is the establishment of abiotic metal-triggered prodrug mechanisms that can control the release of bioactive drugs. Currently, the design of prodrugs that use abiotic metals as a trigger relies heavily on uncaging strategies. Here, we introduce a strategy based on the gold-catalyzed activation of a phenanthridinium-based prodrug via hydroamination under physiological conditions. To make the prodrug strategy biocompatible, a gold artificial metalloenzyme (ArM) based on human serum albumin, rather than the free gold metal complex, was used as a trigger for prodrug activation. The albumin-based gold ArM protected the catalytic activity of the bound gold metal even in the presence of up to 1 mM glutathione in vitro. The drug synthesized via the gold ArM exerted a therapeutic effect in cell-based assays, highlighting the potential usefulness of the gold ArM in anticancer applications.

Prodrug Activation by Gold Artificial Metalloenzyme‐Catalyzed Synthesis of Phenanthridinium Derivatives via Hydroamination

AbstractAn emerging approach in the field of targeted drug delivery is the establishment of abiotic metal‐triggered prodrug mechanisms that can control the release of bioactive drugs. Currently, the design of prodrugs that use abiotic metals as a trigger relies heavily on uncaging strategies. Here, we introduce a strategy based on the gold‐catalyzed activation of a phenanthridinium‐based prodrug via hydroamination under physiological conditions. To make the prodrug strategy biocompatible, a gold artificial metalloenzyme (ArM) based on human serum albumin, rather than the free gold metal complex, was used as a trigger for prodrug activation. The albumin‐based gold ArM protected the catalytic activity of the bound gold metal even in the presence of up to 1 mM glutathione in vitro. The drug synthesized via the gold ArM exerted a therapeutic effect in cell‐based assays, highlighting the potential usefulness of the gold ArM in anticancer applications.

Design of artificial metalloenzymes for the reduction of nicotinamide cofactors

Artificial metalloenzymes result from the insertion of a catalytically active metal complex into a biological scaffold, generally a protein devoid of other catalytic functionalities. As such, their design requires efforts to engineer substrate binding, in addition to accommodating the artificial catalyst. Here we constructed and characterised artificial metalloenzymes using alcohol dehydrogenase as starting point, an enzyme which has both a cofactor and a substrate binding pocket. A docking approach was used to determine suitable positions for catalyst anchoring to single cysteine mutants, leading to an artificial metalloenzyme capable to reduce both natural cofactors and the hydrophobic 1-benzylnicotinamide mimic. Kinetic studies revealed that the new construct displayed a Michaelis-Menten behaviour with the native nicotinamide cofactors, which were suggested by docking to bind at a surface exposed site, different compared to their native binding position. On the other hand, the kinetic and docking data suggested that a typical enzyme behaviour was not observed with the hydrophobic 1-benzylnicotinamide mimic, with which binding events were plausible both inside and outside the protein. This work demonstrates an extended substrate scope of the artificial metalloenzymes and provides information about the binding sites of the nicotinamide substrates, which can be exploited to further engineer artificial metalloenzymes for cofactor regeneration. Synopsis about graphical abstractThe manuscript provides information on the design of artificial metalloenzymes based on the bioconjugation of rhodium complexes to alcohol dehydrogenase, to improve their ability to reduce hydrophobic substrates. The graphical abstract presents different binding modes and results observed with native cofactors as substrates, compared to the hydrophobic benzylnicotinamide.

Controlled Ligand Exchange Between Ruthenium Organometallic Cofactor Precursors and a Naïve Protein Scaffold Generates Artificial Metalloenzymes Catalysing Transfer Hydrogenation

AbstractMany natural metalloenzymes assemble from proteins and biosynthesised complexes, generating potent catalysts by changing metal coordination. Here we adopt the same strategy to generate artificial metalloenzymes (ArMs) using ligand exchange to unmask catalytic activity. By systematically testing RuII(η6‐arene)(bipyridine) complexes designed to facilitate the displacement of functionalised bipyridines, we develop a fast and robust procedure for generating new enzymes via ligand exchange in a protein that has not evolved to bind such a complex. The resulting metal cofactors form peptidic coordination bonds but also retain a non‐biological ligand. Tandem mass spectrometry and 19F NMR spectroscopy were used to characterise the organometallic cofactors and identify the protein‐derived ligands. By introduction of ruthenium cofactors into a 4‐helical bundle, transfer hydrogenation catalysts were generated that displayed a 35‐fold rate increase when compared to the respective small molecule reaction in solution.

Controlled Ligand Exchange Between Ruthenium Organometallic Cofactor Precursors and a Naïve Protein Scaffold Generates Artificial Metalloenzymes Catalysing Transfer Hydrogenation.

Many natural metalloenzymes assemble from proteins and biosynthesised complexes, generating potent catalysts by changing metal coordination. Here we adopt the same strategy to generate artificial metalloenzymes (ArMs) using ligand exchange to unmask catalytic activity. By systematically testing RuII(η6‐arene)(bipyridine) complexes designed to facilitate the displacement of functionalised bipyridines, we develop a fast and robust procedure for generating new enzymes via ligand exchange in a protein that has not evolved to bind such a complex. The resulting metal cofactors form peptidic coordination bonds but also retain a non‐biological ligand. Tandem mass spectrometry and 19F NMR spectroscopy were used to characterise the organometallic cofactors and identify the protein‐derived ligands. By introduction of ruthenium cofactors into a 4‐helical bundle, transfer hydrogenation catalysts were generated that displayed a 35‐fold rate increase when compared to the respective small molecule reaction in solution.

Engineering Thermostability in Artificial Metalloenzymes to Increase Catalytic Activity

Protein engineering has shown widespread use in improving the industrial application of enzymes and broadening the conditions they are able to operate under by increasing their thermostability and solvent tolerance. Here, we show that protein engineering can be used to increase the thermostability of an artificial metalloenzyme. Thermostable variants of the human steroid carrier protein 2L, modified to bind a metal catalyst, were created by rational design using structural data and a 3DM database. These variants were tested to identify mutations that enhanced the stability of the protein scaffold, and a significant increase in melting temperature was observed with a number of modified metalloenzymes. The ability to withstand higher reaction temperatures resulted in an increased activity in the hydroformylation of 1-octene, with more than fivefold improvement in turnover number, whereas the selectivity for linear aldehyde remained high up to 80%.

Dipeptides of S-Substituted Dehydrocysteine as Artzyme Building Blocks: Synthesis, Complexing Abilities and Antiproliferative Properties

Background: Dehydropeptides are analogs of peptides containing at least one conjugate double bond between α,β-carbon atoms. Its presence provides unique structural properties and reaction centre for chemical modification. In this study, the series of new class of dipeptides containing S-substituted dehydrocysteine with variety of heterocyclic moieties was prepared. The compounds were designed as the building blocks for the construction of artificial metalloenzymes (artzymes). Therefore, the complexing properties of representative compounds were also evaluated. Furthermore, the acknowledged biological activity of natural dehydropeptides was the reason to extend the study for antiproliferative action of against several cancer cell lines. Methods: The synthetic strategy involves glycyl and phenylalanyl-(Z)-β-bromodehydroalanine as a substrate in one pot addition/elimination reaction of thiols. After deprotection of N-terminal amino group the compounds with triazole ring were tested as complexones for copper(II) ions using potentiometric titration and spectroscopic techniques (UV-Vis, CD, EPR). Finally, the antiproliferative activity was evaluated by sulforhodamine B assay. Results and Conclusions: A simple and efficient procedure for preparation of dipeptides containing S-substituded dehydrocysteine was provided. The peptides containing triazole appeared to be strong complexones of copper(II) ions. Some of the peptides exhibited promising antiproliferative activities against number of cancer cell lines, including cell lines resistant to widely used anticancer agent.

Alternative Strategy to Obtain Artificial Imine Reductase by Exploiting Vancomycin/D-Ala-D-Ala Interactions with an Iridium Metal Complex.

Based on the supramolecular interaction between vancomycin (Van), an antibiotic glycopeptide, and D-Ala-D-Ala (DADA) dipeptides, a novel class of artificial metalloenzymes was synthesized and characterized. The presence of an iridium(III) ligand at the N-terminus of DADA allowed the use of the metalloenzyme as a catalyst in the asymmetric transfer hydrogenation of cyclic imines. In particular, the type of link between DADA and the metal-chelating moiety was found to be fundamental for inducing asymmetry in the reaction outcome, as highlighted by both computational studies and catalytic results. Using the [IrCp*(m-I)Cl]Cl ⊂ Van complex in 0.1 M CH3COONa buffer at pH 5, a significant 70% (S) e.e. was obtained in the reduction of quinaldine B.

Biodegradation of aromatic pollutants by metalloenzymes: A structural-functional-environmental perspective

• Biodegradation of environmental pollutants has been favored for decades. • Significant progress has been made in the biodegradation of aromatic pollutants by metalloenzymes. • Both native and artificial metalloenzymes have been applied for biodegradation. • The progress sheds light on the structure-function relationship of metalloenzymes. • The progress provides clues for applications of metalloenzymes in environmental protection. Environmental security is closely related to public health. Aromatic pollutants constitute a class of hazardous environmental chemicals, such as halophenols and polycyclic aromatic hydrocarbons. Biodegradation of environmental pollutants has been favored for decades. As reviewed herein, significant progress has been made in biodegradation of aromatic pollutants by native metalloenzymes, including heme enzymes (peroxidases and cytochrome P450s), non-heme iron-containing enzymes (Rieske dioxygenases), and copper-containing enzymes (catechol oxidase, tyrosinase, and laccase), and artificial metalloenzymes, including engineered myoglobins, designed manganese peroxidase and de novo designed metalloenzymes, such as those in helical bundles and peptide assemblies, as well as synthetic structural and functional models of natural enzymes. These advances shed light on the structure–function relationship of metalloenzymes, providing clues for potential applications in environmental protection. Future directions for making full use of native and artificial metalloenzymes in environmental science are also prospected.

In Vivo Assembly of Artificial Metalloenzymes and Application in Whole-Cell Biocatalysis*.

We report the supramolecular assembly of artificial metalloenzymes (ArMs), based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneous copper(II)–phenanthroline complex, in the cytoplasm of E. coli cells. A combination of catalysis, cell‐fractionation, and inhibitor experiments, supplemented with in‐cell solid‐state NMR spectroscopy, confirmed the in‐cell assembly. The ArM‐containing whole cells were active in the catalysis of the enantioselective Friedel–Crafts alkylation of indoles and the Diels–Alder reaction of azachalcone with cyclopentadiene. Directed evolution resulted in two different improved mutants for both reactions, LmrR_A92E_M8D and LmrR_A92E_V15A, respectively. The whole‐cell ArM system required no engineering of the microbial host, the protein scaffold, or the cofactor to achieve ArM assembly and catalysis. We consider this a key step towards integrating abiological catalysis with biosynthesis to generate a hybrid metabolism.

Efficient Lewis acid catalysis of an abiological reaction in a de novo protein scaffold.

New enzyme catalysts are usually engineered by repurposing the active sites of natural proteins. Here we show that design and directed evolution can be used to transform a non-natural, functionally naive zinc-binding protein into a highly active catalyst for an abiological hetero-Diels-Alder reaction. The artificial metalloenzyme achieves >104 turnovers per active site, exerts absolute control over reaction pathway and product stereochemistry, and displays a catalytic proficiency (1/KTS = 2.9 × 1010 M-1) that exceeds all previously characterized Diels-Alderases. These properties capitalize on effective Lewis acid catalysis, a chemical strategy for accelerating Diels-Alder reactions common in the laboratory but so far unknown in nature. Extension of this approach to other metal ions and other de novo scaffolds may propel the design field in exciting new directions.

In Vivo Assembly of Artificial Metalloenzymes and Application in Whole‐Cell Biocatalysis**

AbstractWe report the supramolecular assembly of artificial metalloenzymes (ArMs), based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneous copper(II)–phenanthroline complex, in the cytoplasm of E. coli cells. A combination of catalysis, cell‐fractionation, and inhibitor experiments, supplemented with in‐cell solid‐state NMR spectroscopy, confirmed the in‐cell assembly. The ArM‐containing whole cells were active in the catalysis of the enantioselective Friedel–Crafts alkylation of indoles and the Diels–Alder reaction of azachalcone with cyclopentadiene. Directed evolution resulted in two different improved mutants for both reactions, LmrR_A92E_M8D and LmrR_A92E_V15A, respectively. The whole‐cell ArM system required no engineering of the microbial host, the protein scaffold, or the cofactor to achieve ArM assembly and catalysis. We consider this a key step towards integrating abiological catalysis with biosynthesis to generate a hybrid metabolism.

Systematic engineering of artificial metalloenzymes for new-to-nature reactions.

Artificial metalloenzymes (ArMs) catalyzing new-to-nature reactions could play an important role in transitioning toward a sustainable economy. While ArMs have been created for various transformations, attempts at their genetic optimization have been case specific and resulted mostly in modest improvements. To realize their full potential, methods to rapidly discover active ArM variants for ideally any reaction of interest are required. Here, we introduce a reaction-independent, automation-compatible platform, which relies on periplasmic compartmentalization in Escherichia coli to rapidly and reliably engineer ArMs based on the biotin-streptavidin technology. We systematically assess 400 ArM mutants for five bioorthogonal transformations involving different metals, reaction mechanisms, and reactants, which include novel ArMs for gold-catalyzed hydroamination and hydroarylation. Activity enhancements up to 15-fold highlight the potential of the systematic approach. Furthermore, we suggest smart screening strategies and build machine learning models that accurately predict ArM activity from sequence, which has crucial implications for future ArM development.

Enzyme/Nanocopper Hybrid Nanozymes: Modulating Enzyme-like Activity by the Protein Structure for Biosensing and Tumor Catalytic Therapy.

Artificial enzymes with modulated enzyme-mimicking activities of natural systems represent a challenge in catalytic applications. Here, we show the creation of artificial Cu metalloenzymes based on the generation of Cu nanoparticles in an enzyme matrix. Different enzymes were used, and the structural differences between the enzymes especially influenced the controlled the size of the nanoparticles and the environment that surrounds them. Herein, we demonstrated that the oxidase-like catalytic activity of these copper nanozymes was rationally modulated by enzyme used as a scaffold, with a special role in the nanoparticle size and their environment. In this sense, these nanocopper hybrids have confirmed the ability to mimic a unique enzymatic activity completely different from the natural activity of the enzyme used as a scaffold, such as tyrosinase-like activity or as Fenton catalyst, which has extremely higher stability than natural mushroom tyrosinase. More interestingly, the oxidoreductase-like activity of nanocopper hybrids was cooperatively modulated with the synergistic effect between the enzyme and the nanoparticles improving the catalase activity (no peroxidase activity). Additionally, a novel dual (metallic and enzymatic activity) of the nanozyme made the highly improved catechol-like activity interesting for the design of 3,4-dihydroxy-l-phenylalanine (l-DOPA) biosensor for detection of tyrosinase. These hybrids also showed cytotoxic activity against different tumor cells, interesting in biocatalytic tumor therapy.

Recent advance of chemoenzymatic catalysis for the synthesis of chemicals: Scope and challenge

Chemoenzymatic catalysis can give full play to the advantages of versatile reactivity of chemocatalysis and excellent chemo-, regio-, and stereoselectivities of biocatalysis. These chemoenzymatic methods can not only save resource, cost, and operating time but also reduce the number of reaction steps, and avoid separating unstable intermediates, leading to the generation of more products under greener circumstances and thereby playing an indispensable role in the fields of medicine, materials and fine chemicals. Although incompatible challenges between chemocatalyst and biocatalyst remain, strategies such as biphasic system, artificial metalloenzymes, immobilization or supramolecular host, and protein engineering have been designed to overcome these issues. In this review, chemoenzymatic catalysis according to different chemocatalysis types was classifiably described, and in particular, the classic dynamic kinetic resolutions (DKR) and cofactor regeneration were summarized. Finally, the bottlenecks and development of chemoenzymatic catalysis were summarized, and future development was prospected.

Intracellular Unnatural Catalysis Enabled by an Artificial Metalloenzyme.

Artificial metalloenzymes, constructed by incorporating a synthetic catalyst into the internal spaces of a protein scaffold, can perform noncanonical chemical transformations that are not possible using natural enzymes. The addition of cell-permeable modules to artificial metalloenzymes allows for noncanonical catalysis to be implemented as a function of mammalian cells. In this chapter, we describe a protocol for controlling cellular function through a cascade consisting of an artificial metalloenzyme and a gene-circuit engineered via synthetic biology.

Myoglobins engineered with artificial cofactors serve as artificial metalloenzymes and models of natural enzymes.

Metalloenzymes naturally achieve various reactivities by assembling limited types of cofactors with endogenous amino acid residues. Enzymes containing metal porphyrinoid cofactors such as heme, cobalamin and F430 exert precise control over the reactivities of the cofactors with protein matrices. This perspective article focuses on our recent efforts to assemble metal complexes of non-natural porphyrinoids within the protein matrix of myoglobin, an oxygen storage hemoprotein. Engineered myoglobins with suitable metal complexes as artificial cofactors demonstrate unique reactivities toward C-H bond hydroxylation, olefin cyclopropanation, methyl group transfer and methane generation. In these cases, the protein matrix enhances the catalytic activities of the cofactors and allows us to monitor the active intermediates. The present findings indicate that placing artificial cofactors in protein matrices provides a useful strategy for creating artificial metalloenzymes that catalyse otherwise unfavourable reactions and providing enzyme models for elucidating the complicated reaction mechanisms of natural enzymes.

A protein scaffold enables hydrogen evolution for a Ni-bisdiphosphine complex.

An artificial metalloenzyme acting as a functional biomimic of hydrogenase enzymes was activated by assembly via covalent attachment of the molecular complex, [Ni(PNglycineP)2]2-, within a structured protein scaffold. Electrocatalytic H2 production was observed from pH 3.0 to 10.0 for the artificial enzyme, while no electrocatalytic activity was observed for similar [Ni(PNP)2]2+ systems.

Tandem catalysis using an enzyme and a polymeric ruthenium-based artificial metalloenzyme

A Ru-containing single-chain nanoparticle (SCNP) performs allylcarbamate cleavage reactions in biologically relevant environments more efficiently than free catalyst and works in synergy with a natural enzyme to perform tandem catalysis.