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Artificial metalloenzymes

Nature Reviews Methods Primers volume 4, Article number: 78 (2024) Cite this article

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Abstract

The development of artificial metalloenzymes (ArMs) aims to expand the capabilities of enzymatic catalysis, most notably towards new reaction mechanisms. Frequently, ArMs harness metal cofactors that are not naturally found in enzymes and embed these in specifically selected or designed protein scaffolds. ArMs have been developed for a wide range of natural and non-natural reactions, underscoring their potential to revolutionize fields such as biocatalysis or metabolic engineering. At the same time, replicating the catalytic prowess of natural enzymes is a highly challenging task, and several limitations need to be overcome to make ArM catalysis widely applicable. In this Primer, we introduce the state of the art in designing and engineering ArMs, describing best practices and important examples and achievements. Moreover, we consider potential applications of ArMs, as well as outstanding challenges, and discuss how these may be addressed in the coming years.

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Fig. 1: Strategies for creating metalloenzymes with new activity.
Fig. 2: Common transition metal ions and their preferred coordination geometry.
Fig. 3: Representative ArMs resulting from cofactor anchoring within natural or de novo-designed protein scaffolds.
Fig. 4: Computational approaches to design and engineer ArMs.
Fig. 5: Examples of ArM-catalysed reactions with potential applications in industrial biocatalysis.
Fig. 6: ArM expression and assembly in Escherichia coli.

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Authors and Affiliations

  1. Department of Chemistry, University of Basel, Basel, Switzerland

    Tobias Vornholt & Thomas R. Ward

  2. Center for Functional Protein Assemblies & Department of Bioscience, TUM School of Natural Sciences, Technical University of Munich (TUM), Garching, Germany

    Florian Leiss-Maier & Cathleen Zeymer

  3. Department of Chemistry, Seoul National University, Seoul, Republic of Korea

    Woo Jae Jeong & Woon Ju Song

  4. Stratingh Institute for Chemistry, University of Groningen, Groningen, The Netherlands

    Gerard Roelfes

Authors
  1. Tobias Vornholt
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  2. Florian Leiss-Maier
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  3. Woo Jae Jeong
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  4. Cathleen Zeymer
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  7. Thomas R. Ward
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The authors contributed equally to all aspects of the article.

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Correspondence to Cathleen Zeymer, Woon Ju Song, Gerard Roelfes or Thomas R. Ward.

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Glossary

Artificial metalloenzyme

A man-made assembly of a protein and a non-native metal cofactor (a metal ion or metal complex).

Cofactor

In the context of artificial metalloenzymes, the cofactor is a metal ion or metal complex that can bind to the protein scaffold and is required for catalysis.

Coiled coil

A structural motif in which several α-helices are coiled together like the strands of a rope.

Density functional theory

(DFT). A quantum-chemical method to calculate the electronic structure and optimized geometry of molecules and transition states.

Directed evolution

Directed evolution mimics natural evolution in the laboratory using cycles of mutagenesis and screening or selection.

Epistasis

The phenomenon that the effect of one mutation is dependent on the presence or absence of a second mutation.

Irving–Williams series

Describes trends in the stability of metal complexes containing divalent first-row transition metal ions, with Cu(II) typically forming the most stable complexes.

Metalloenzyme

An enzyme that requires one or more metal ions for its catalytic activity.

Metalloenzyme repurposing

When natural metalloenzymes are used to catalyse unnatural reactions.

Protein scaffold

In the context of artificial metalloenzymes (ArMs), the protein scaffold refers to a protein that in its apo-form does not have catalytic activity for the desired reaction but can bind a metal cofactor. Following assembly of the ArM, the protein scaffold can influence the metal-catalysed reaction in various ways.

Rosetta

The Rosetta software suite facilitates ab initio protein structure prediction and protein design in a physics-based approach.

Suicide inhibition

An irreversible form of enzyme inhibition wherein a substrate analogue forms a covalent bond with the enzyme.

Theozyme

A theoretical model of a minimal active site comprising the computed transition state and catalytic metal ions or amino acid residues.

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Vornholt, T., Leiss-Maier, F., Jeong, W.J. et al. Artificial metalloenzymes. Nat Rev Methods Primers 4, 78 (2024). https://doi.org/10.1038/s43586-024-00356-w

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