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  • Review Article
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The importance of catalytic promiscuity for enzyme design and evolution

Abstract

The ability of one enzyme to catalyse multiple, mechanistically distinct transformations likely played a crucial role in organisms’ abilities to adapt to changing external stimuli in the past and can still be observed in extant enzymes. Given the importance of catalytic promiscuity in nature, enzyme designers have recently begun to create catalytically promiscuous enzymes in order to expand the canon of transformations catalysed by proteins. This article aims to both critically review different strategies for the design of enzymes that display catalytic promiscuity for new-to-nature reactions and highlight the successes of subsequent directed-evolution efforts to fine-tune these novel reactivities. For the former, we put a particular emphasis on the creation, stabilization and repurposing of reaction intermediates, which are key for unlocking new activities in an existing or designed active site. For the directed evolution of the resulting catalysts, we contrast approaches for enzyme design that make use of components found in nature and those that achieve new reactivities by incorporating synthetic components. Following the critical analysis of selected examples that are now available, we close this Review by providing a set of considerations and design principles for enzyme engineers, which will guide the future generation of efficient artificial enzymes for synthetically useful, abiotic transformations.

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Fig. 1: Catalytic promiscuity of a computationally designed and evolved enzyme.
Fig. 2: Catalytic promiscuity in P450BM3 from Bacillus megaterium.
Fig. 3: Catalytic promiscuity in cytochrome c from Rhodothermus marinus.
Fig. 4: Unlocking new reactivities in nicotinamide-dependent ketoreductases by photoactivation.
Fig. 5: Catalytic promiscuity in carbonic anhydrase results from exchanging the native, catalytic zinc ion (blue sphere).
Fig. 6: Catalytic promiscuity as a result of replacing haem with synthetic analogues in haem-binding proteins.
Fig. 7: Catalytic promiscuity in streptavidin-based artificial metalloenzymes.
Fig. 8: Catalytic promiscuity in the lactococcal multidrug resistance regulatory protein from Lactococcus lactis.

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Acknowledgements

The authors gratefully acknowledge financial support from the Netherlands Organisation for Scientific Research (NWO, Vici grant 724.013.003 and Veni grant 722.017.007). G.R. acknowledges support from the Ministry of Education, Culture and Science (Gravitation programme no. 024.001.035).

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R.B.L.-G researched data for the article, wrote the manuscript and prepared the figures, with contributions from C.M. All authors contributed to the discussion, reviewing and editing of the manuscript before submission.

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Correspondence to Clemens Mayer or Gerard Roelfes.

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Glossary

Theozyme

A theoretical structure produced during computational enzyme design, where stabilizing amino acid residues are positioned around a computed transition state (complex).

Whole-cell catalysis

Catalytic (enzymatic) conversions which employ intact cells, exploiting either the native metabolism of the organism or by using the organism to (over)produce a particular enzyme.

Site-directed mutagenesis

Altering (a) particular amino acid(s) in a protein through mutation of the gene that encodes it.

Apo-form

In cofactor-dependent enzymes, the form of the enzyme without its cofactor.

Holoenzyme

In cofactor-dependent enzymes, the form of the enzyme complete with its cofactor.

Periplasm

Principally in Gram-negative bacteria, the region of the cell between the outer and inner cytoplasmic membrane.

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Leveson-Gower, R.B., Mayer, C. & Roelfes, G. The importance of catalytic promiscuity for enzyme design and evolution. Nat Rev Chem 3, 687–705 (2019). https://doi.org/10.1038/s41570-019-0143-x

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