Abstract
Enzymes play an increasingly important role in improving the benignity and efficiency of chemical production, yet the diversity of their applications lags heavily behind chemical catalysts as a result of the relatively narrow range of reaction mechanisms of enzymes. The creation of enzymes containing non-biological functionalities facilitates reaction mechanisms outside nature’s canon and paves the way towards fully programmable biocatalysis1,2,3. Here we present a completely genetically encoded boronic-acid-containing designer enzyme with organocatalytic reactivity not achievable with natural or engineered biocatalysts4,5. This boron enzyme catalyses the kinetic resolution of hydroxyketones by oxime formation, in which crucial interactions with the protein scaffold assist in the catalysis. A directed evolution campaign led to a variant with natural-enzyme-like enantioselectivities for several different substrates. The unique activation mode of the boron enzyme was confirmed using X-ray crystallography, high-resolution mass spectrometry (HRMS) and 11B NMR spectroscopy. Our study demonstrates that genetic-code expansion can be used to create evolvable enantioselective enzymes that rely on xenobiotic catalytic moieties such as boronic acids and access reaction mechanisms not reachable through catalytic promiscuity of natural or engineered enzymes.
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Data availability
All data are available in the main text or the Supplementary Information. The crystallographic data for the structures of BOS and BOS_EHL have been deposited in the Protein Data Bank with accession numbers 8QDF and 8QDH, respectively. Source data are provided with this paper.
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Acknowledgements
We thank J. Kemmink, P. van der Meulen and J. Hekelaar for analytical support. We also thank I. Drienovská for the preparation of some of the plasmids used in this work. This work was supported by The Netherlands Ministry of Education, Culture and Science (Gravitation programme no. 024.001.035) and the European Research Council (ERC advanced grant 885396). L.L. acknowledges the support of the Leopoldina - National Academy of Sciences for a postdoctoral fellowship (LPDS 2021-11). The European Synchrotron Radiation Facility (ESRF) is acknowledged for provision of synchrotron radiation facilities and we are grateful to M. Bowler, D. Flot and D. Nurizzo for their assistance and support in using ESRF beamline MASSIF-1.
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L.L., R.B.L.-G. and G.R. conceived the project. L.L. developed, optimized and performed the scope of the kinetic resolution reaction. R.B.L.-G. expressed designer enzymes and performed the evolution campaign. H.J.R. performed crystal-growing experiments. A.-M.W.H.T. analysed the X-ray data. G.R. directed the project. All authors discussed the results and L.L., R.B.L.-G. and G.R. wrote the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 96-well UV/Vis plate assay results from the first round of directed evolution.
Top left, amino-acid positions chosen for saturation mutagenesis with β-carbons visualized as spheres. Top right, colour scale for rate of (R)-benzoin consumption observed in the lysates. Bottom, rate of benzoin consumption observed in 96-well-format lysates for the library members, screened in quadruplet. Some mutants were not produced owing to unsuccessful QuikChange reactions; these are labelled in grey. Two colonies of F93D were accidentally picked into positions intended for F93E, and so four more colonies were picked into otherwise blank wells.
Extended Data Fig. 2 96-well UV/Vis plate assay results from the second round of directed evolution.
Top left, amino-acid positions chosen for saturation mutagenesis with β-carbons visualized as spheres. Second-round positions are in green and the position of the first-round hit is in orange. Top right, colour scale for rate of (R)-benzoin consumption observed in the lysates. Bottom, rate of benzoin consumption observed in 96-well-format lysates for the library members, screened in quadruplet. Some mutants were not produced owing to unsuccessful QuikChange reactions; these are labelled in grey.
Extended Data Fig. 3 96-well UV/Vis plate assay results from the third round of directed evolution.
Top left, amino-acid positions chosen for saturation mutagenesis with β-carbons visualized as spheres (magenta). Positions of mutations in the template incorporated from the first and second rounds are shown in orange and green, respectively. Top right, colour scale for rate of (R)-benzoin consumption observed in the lysates. Bottom, rate of benzoin consumption observed in 96-well-format lysates for the library members, screened in quadruplet. Some mutants were not produced owing to unsuccessful QuikChange reactions; these are labelled in grey.
Extended Data Fig. 4 Structural features of BOS (PDB: 8QDF, purple) compared with wild-type LmrR (PDB: 3F8B, grey).
Top, overlay of the two structures shown in cartoon representation, with key residues N19, W96 and M89pBoF shown as sticks. BOS features a large reduction in the distance between the centres of the aromatic rings of the two central tryptophans from 7.2 Å in wild-type LmrR to 4.9 Å. Bottom, surface representation of the two structures showing that BOS features a closed pore, compared with the large open pore found in wild-type LmrR.
Extended Data Fig. 5 Effect of the N19A mutation on cyclic-boronate ester formation observed by 11B NMR.
Comparison of the 11B NMR spectra of BOS and BOS_N19A in phosphate buffer (a,b) and Tris buffer (c,d).
Extended Data Fig. 6 11B NMR spectra of BOS and BOS_N19A in the presence of a strong diol binder (4-nitrocatechol).
Comparison of interaction with 4-nitrocatechol of BOS (a) and BOS_N19A (b).
Extended Data Fig. 7 Structure of the improved variant BOS_EHL (PDB: 8QDH) compared with the parent BOS (PDB: 8QDF).
a, Complete overlay shows minimal changes in the overall structure with a partially collapsed pocket compared with wild-type LmrR (for example, PDB: 3F8B in the ligand-free state, not shown). b, Detailed view of an overlay of the catalytic pocket showing the interactions of N19 as well as the mutations at positions 8, 92 and 93. For clarity, only one of the two modelled binding modes of Tris is shown.
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Longwitz, L., Leveson-Gower, R.B., Rozeboom, H.J. et al. Boron catalysis in a designer enzyme. Nature (2024). https://doi.org/10.1038/s41586-024-07391-3
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DOI: https://doi.org/10.1038/s41586-024-07391-3
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