An Artificial Metalloenzyme for Olefin Metathesis
Chem. Commun. 2011, 47, 12068, 10.1039/c1cc15005g
A Grubbs–Hoveyda type olefin metathesis catalyst, equipped with an electrophilic bromoacetamide group, was used to modify a cysteine-containing variant of a small heat shock protein from Methanocaldococcus jannaschii. The resulting artificial metalloenzyme was found to be active under acidic conditions in a benchmark ring closing metathesis reaction.
Metal: RuLigand type: CarbeneHost protein: Small heat shock protein from M. jannaschiiAnchoring strategy: CovalentOptimization: ---Reaction: Olefin metathesisMax TON: 25ee: ---PDB: ---Notes: RCM
A Noncanonical Proximal Heme Ligand Affords an Efficient Peroxidase in a Globin Fold
J. Am. Chem. Soc. 2018, 140, 1535-1543, 10.1021/jacs.7b12621
Expanding the range of genetically encoded metal coordination environments accessible within tunable protein scaffolds presents excellent opportunities for the creation of metalloenzymes with augmented properties and novel activities. Here, we demonstrate that installation of a noncanonical Nδ-methyl histidine (NMH) as the proximal heme ligand in the oxygen binding protein myoglobin (Mb) leads to substantial increases in heme redox potential and promiscuous peroxidase activity. Structural characterization of this catalytically modified myoglobin variant (Mb NMH) revealed significant changes in the proximal pocket, including alterations to hydrogen-bonding interactions involving the prosthetic porphyrin cofactor. Further optimization of Mb NMH via a combination of rational modification and several rounds of laboratory evolution afforded efficient peroxidase biocatalysts within a globin fold, with activities comparable to those displayed by nature’s peroxidases.
Ligand type: N-methyl histidine; PorphyrinAnchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: Oxidation of amplex red
Capture and Characterization of a Reactive Haem– Carbenoid Complex in an Artificial Metalloenzyme
Nat. Catal. 2018, 1, 578-584, 10.1038/s41929-018-0105-6
Non-canonical amino acid ligands are useful for fine-tuning the catalytic properties of metalloenzymes. Here, we show that recombinant replacement of the histidine ligand proximal to haem in myoglobin with Nδ-methylhistidine enhances the protein’s promiscuous carbene-transfer chemistry, enabling efficient styrene cyclopropanation in the absence of reductant, even under aerobic conditions. The increased electrophilicity of the modified Fe(iii) centre, combined with subtle structural adjustments at the active site, allows direct attack of ethyl diazoacetate to produce a reactive carbenoid adduct, which has an unusual bridging Fe(iii)–C–N(pyrrole) configuration as shown by X-ray crystallography. Quantum chemical calculations suggest that the bridged complex equilibrates with the more reactive end-on isomer, ensuring efficient cyclopropanation. These findings underscore the potential of non-canonical ligands for extending the capabilities of metalloenzymes by opening up new reaction pathways and facilitating the characterization of reactive species that would not otherwise accumulate.
Ligand type: Nδ-methylhistidine; PorphyrinAnchoring strategy: ---Max TON: 1000ee: 99PDB: 6F17Notes: Structure of the Mb*(NMH) haem-iron complex
Ligand type: Nδ-methylhistidine; PorphyrinAnchoring strategy: ---Max TON: 1000ee: 99PDB: 6G5BNotes: Structure of the Mb*(NMH) haem-iron–carbenoid complex
Efficient Lewis Acid Catalysis of an Abiological Reaction in a De Novo Protein Scaffold
Nat. Chem. 2021, 13, 231-235, 10.1038/s41557-020-00628-4
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.
Metal: ZnLigand type: Amino acidHost protein: De novo-designed proteinAnchoring strategy: DativeReaction: Diels-Alder reactionMax TON: >10000ee: 99PDB: ---Notes: PDB: 3V1C, 7BWW
Engineered Metalloenzymes with Non-Canonical Coordination EnvironmentsReview
Chem. - Eur. J. 2018, 24, 11821-11830, 10.1002/chem.201800975
Nature employs a limited number of genetically encoded, metal‐coordinating residues to create metalloenzymes with diverse structures and functions. Engineered components of the cellular translation machinery can now be exploited to encode non‐canonical ligands with user‐defined electronic and structural properties. This ability to install “chemically programmed” ligands into proteins can provide powerful chemical probes of metalloenzyme mechanism and presents excellent opportunities to create metalloprotein catalysts with augmented properties and novel activities. In this Concept article, we provide an overview of several recent studies describing the creation of engineered metalloenzymes with interesting catalytic properties, and reveal how characterization of these systems has advanced our understanding of nature's bioinorganic mechanisms. We also highlight how powerful laboratory evolution protocols can be readily adapted to allow optimization of metalloenzymes with non‐canonical ligands. This approach combines beneficial features of small molecule and protein catalysis by allowing the installation of a greater variety of local metal coordination environments into evolvable protein scaffolds, and holds great promise for the future creation of powerful metalloprotein catalysts for a host of synthetically valuable transformations.
Noncanonical Heme Ligands Steer Carbene Transfer Reactivity in an Artificial Metalloenzyme
Angew. Chem. Int. Ed. 2021, 60, 15063-15068, 10.1002/anie.202103437
Changing the primary metal coordination sphere is a powerful strategy for tuning metalloprotein properties. Here we used amber stop codon suppression with engineered pyrrolysyl-tRNA synthetases, including two newly evolved enzymes, to replace the proximal histidine in myoglobin with Nδ-methylhistidine, 5-thiazoylalanine, 4-thiazoylalanine and 3-(3-thienyl)alanine. In addition to tuning the heme redox potential over a >200 mV range, these noncanonical ligands modulate the protein's carbene transfer activity with ethyl diazoacetate. Variants with increased reduction potential proved superior for cyclopropanation and N–H insertion, whereas variants with reduced Eo values gave higher S–H insertion activity. Given the functional importance of histidine in many enzymes, these genetically encoded analogues could be valuable tools for probing mechanism and enabling new chemistries.
Max TON: ---ee: >99PDB: ---Notes: yield: styrene cyclopropanation 71% max, cf free heme <5%
Reaction: N-H InsertionMax TON: ---ee: ---PDB: ---Notes: Yield: aniline insertion 74-93%
Reaction: S-H insertionMax TON: ---ee: ---PDB: ---Notes: Yield: thiophenol insertion 18-36% but still outperforms heme