Abstract
L-Proline is a powerful organocatalyst widely applied in asymmetric synthesis due to its secondary amine functionality. However, in proteins, this functional group is locked in peptide bonds, rendering proline catalytically inactive. Natural enzymes that leverage L-proline-based catalysis are exceedingly rare. Here, we engineer the nonenzymatic protein scaffold LmrR into a new-to-nature biocatalyst by exposing its native L-proline residue at the N-terminus to catalyze enantioselective aldol reactions. Through rational design, protein engineering, and reaction optimization, we develop an engineered LmrR variant that achieves up to 99% conversion and >99% enantiomeric excess across a range of aromatic and heteroaromatic aldehyde substrates. Our findings reveal a unique strategy for unlocking dormant catalytic potential in natural amino acids and protein scaffolds, providing an applicable approach to create tailored, L-proline-based enzymes for asymmetric synthesis.
Data availability
All data supporting the findings of this study are available within the article and its Supplementary Information, or available from the corresponding author upon request. Source data are provided with this paper.
References
Kissman, E. N. et al. Expanding chemistry through in vitro and in vivo biocatalysis. Nature 631, 37–48 (2024).
O’Connell, A. et al. Biocatalysis: landmark discoveries and applications in chemical synthesis. Chem. Soc. Rev. 53, 2828–2850 (2024).
Benítez-Mateos, A. I., Roura Padrosa, D. & Paradisi, F. Multistep enzyme cascades as a route towards green and sustainable pharmaceutical syntheses. Nat. Chem. 14, 489–499 (2022).
Huffman, M. A. et al. Design of an in vitro biocatalytic cascade for the manufacture of islatravir. Science 366, 1255–1259 (2019).
Barra, L. et al. β-NAD as a building block in natural product biosynthesis. Nature 600, 754–758 (2021).
Thorpe, T. W. et al. Multifunctional biocatalyst for conjugate reduction and reductive amination. Nature 604, 86–91 (2022).
Lister, T. M. et al. Engineered enzymes for enantioselective nucleophilic aromatic substitutions. Nature 639, 375–381 (2025).
Crawshaw, R. et al. Engineering an efficient and enantioselective enzyme for the Morita–Baylis–Hillman reaction. Nat. Chem. 14, 313–320 (2022).
Jeschek, M. et al. Directed evolution of artificial metalloenzymes for in vivo metathesis. Nature 537, 661–665 (2016).
Mao, R. et al. Biocatalytic, enantioenriched primary amination of tertiary C–H bonds. Nat. Catal. 7, 585–592 (2024).
Brogan, A. P. S., Bui-Le, L. & Hallett, J. P. Non-aqueous homogenous biocatalytic conversion of polysaccharides in ionic liquids using chemically modified glucosidase. Nat. Chem. 10, 859–865 (2018).
Lu, H., Ouyang, J., Liu, W. Q., Wu, C. & Li, J. Enzyme-polymer-conjugate-based Pickering emulsions for cell-free expression and cascade biotransformation. Angew. Chem. Int. Ed. 62, e202312906 (2023).
Lovelock, S. L. et al. The road to fully programmable protein catalysis. Nature 606, 49–58 (2022).
Brouwer, B. et al. Noncanonical amino acids: bringing new-to-nature functionalities to biocatalysis. Chem. Rev. 124, 10877–10923 (2024).
Liang, A. D., Serrano-Plana, J., Peterson, R. L. & Ward, T. R. Artificial metalloenzymes based on the biotin-streptavidin technology: enzymatic cascades and directed evolution. Acc. Chem. Res. 52, 585–595 (2019).
Drienovská, I. & Roelfes, G. Expanding the enzyme universe with genetically encoded unnatural amino acids. Nat. Catal. 3, 193–202 (2020).
Hanreich, S., Bonandi, E. & Drienovská, I. Design of artificial enzymes: insights into protein scaffolds. ChemBioChem 24, e202200566 (2023).
Madoori, P. K., Agustiandari, H., Driessen, A. J. & Thunnissen, A. M. Structure of the transcriptional regulator LmrR and its mechanism of multidrug recognition. EMBO J. 28, 156–166 (2009).
Roelfes, G. LmrR: a privileged scaffold for artificial metalloenzymes. Acc. Chem. Res. 52, 545–556 (2019).
Drienovská, I., Mayer, C., Dulson, C. & Roelfes, G. A designer enzyme for hydrazone and oxime formation featuring an unnatural catalytic aniline residue. Nat. Chem. 10, 946–952 (2018).
Zhou, Z. & Roelfes, G. Synergistic catalysis in an artificial enzyme by simultaneous action of two abiological catalytic sites. Nat. Catal. 3, 289–294 (2020).
Leveson-Gower, R. B., Zhou, Z., Drienovská, I. & Roelfes, G. Unlocking iminium catalysis in artificial enzymes to create a Friedel-Crafts alkylase. ACS Catal. 11, 6763–6770 (2021).
Liu, Y., Ba, F., Liu, W. Q., Wu, C. & Li, J. Plug-and-play functionalization of protein−polymer conjugates for tunable catalysis enabled by genetically encoded “click” chemistry. ACS Catal. 12, 4165–4174 (2022).
Sun, N. et al. Enantioselective [2+2]-cycloadditions with triplet photoenzymes. Nature 611, 715–720 (2022).
Longwitz, L., Leveson-Gower, R. B., Rozeboom, H. J., Thunnissen, A. W. H. & Roelfes, G. Boron catalysis in a designer enzyme. Nature 629, 824–829 (2024).
Mou, S. B., Chen, K. Y., Kunthic, T. & Xiang, Z. Design and evolution of an artificial Friedel-Crafts alkylation enzyme featuring an organoboronic acid residue. J. Am. Chem. Soc. 146, 26676–26686 (2024).
Veen, M. J. et al. Artificial gold enzymes using a genetically encoded thiophenol-based noble-metal-binding ligand. Angew. Chem. Int. Ed. 64, e202421912 (2025).
List, B., Lerner, R. A. & Barbas, C. F. Proline-catalyzed direct asymmetric aldol reactions. J. Am. Chem. Soc. 122, 2395–2396 (2000).
Torii, H., Nakadai, M., Ishihara, K., Saito, S. & Yamamoto, H. Asymmetric direct aldol reaction assisted by water and a proline-derived tetrazole catalyst. Angew. Chem. Int. Ed. 43, 1983–1986 (2004).
List, B., Pojarliev, P. & Martin, H. J. Efficient proline-catalyzed Michael additions of unmodified ketones to nitro olefins. Org. Lett. 3, 2423–2425 (2001).
Bae, D., Lee, J. W. & Ryu, D. H. Enantio- and diastereoselective Michael addition of cyclic ketones/aldehydes to nitroolefins in water as catalyzed by proline-derived bifunctional organocatalysts. J. Org. Chem. 87, 16532–16541 (2022).
List, B. The direct catalytic asymmetric three-component Mannich reaction. J. Am. Chem. Soc. 122, 9336–9337 (2000).
Yang, J. W., Chandler, C., Stadler, M., Kampen, D. & List, B. Proline-catalysed Mannich reactions of acetaldehyde. Nature 452, 453–455 (2008).
List, B. Proline-catalyzed asymmetric reactions. Tetrahedron 58, 5573–5590 (2002).
Notz, W., Tanaka, F. & Barbas, C. F. III Enamine-based organocatalysis with proline and diamines: the development of direct catalytic asymmetric aldol, Mannich, Michael, and Diels−Alder reactions. Acc. Chem. Res. 37, 580–591 (2004).
Mukherjee, S., Yang, J. W., Hoffmann, S. & List, B. Asymmetric enamine catalysis. Chem. Rev. 107, 5471–5569 (2007).
Gruttadauria, M., Giacalone, F. & Noto, R. Supported proline and proline-derivatives as recyclable organocatalysts. Chem. Soc. Rev. 37, 1666–1688 (2008).
Zandvoort, E., Baas, B. J., Quax, W. J. & Poelarends, G. J. Systematic screening for catalytic promiscuity in 4-oxalocrotonate tautomerase: enamine formation and aldolase activity. ChemBioChem 12, 602–609 (2011).
Zandvoort, E., Geertsema, E. M., Baas, B. J., Quax, W. J. & Poelarends, G. J. Bridging between organocatalysis and biocatalysis: asymmetric addition of acetaldehyde to β-nitrostyrenes catalyzed by a promiscuous proline-based tautomerase. Angew. Chem. Int. Ed. 51, 1240–1243 (2012).
Guo, C., Saifuddin, M., Saravanan, T., Sharifi, M. & Poelarends, G. J. Biocatalytic asymmetric Michael additions of nitromethane to α,β-unsaturated aldehydes via enzyme-bound iminium ion intermediates. ACS Catal. 9, 4369–4373 (2019).
Saifuddin, M. et al. Enantioselective aldol addition of acetaldehyde to aromatic aldehydes catalyzed by proline-based carboligases. ACS Catal. 10, 2522–2527 (2020).
Xu, G., Crotti, M., Saravanan, T., Kataja, K. M. & Poelarends, G. J. Enantiocomplementary epoxidation reactions catalyzed by an engineered cofactor-independent non-natural peroxygenase. Angew. Chem. Int. Ed. 59, 10374–10378 (2020).
Kunzendorf, A., Xu, G., Saifuddin, M., Saravanan, T. & Poelarends, G. J. Biocatalytic asymmetric cyclopropanations via enzyme-bound iminium ion intermediates. Angew. Chem. Int. Ed. 60, 24059–24063 (2021).
Xu, G. et al. Gene fusion and directed evolution to break structural symmetry and boost catalysis by an oligomeric C-C bond-forming enzyme. Angew. Chem. Int. Ed. 61, e202113970 (2022).
Lorentzen, E., Siebers, B., Hensel, R. & Pohl, E. Mechanism of the Schiff base forming fructose-1,6-bisphosphate aldolase: structural analysis of reaction intermediates. Biochemistry 44, 4222–4229 (2005).
Clapés, P. & Garrabou, X. Current trends in asymmetric synthesis with aldolases. Adv. Synth. Catal. 353, 2263–2283 (2011).
Hernández, K., Szekrenyi, A. & Clapés, P. Nucleophile promiscuity of natural and engineered aldolases. ChemBioChem 19, 1353–1358 (2018).
Kunzendorf, A. et al. Unlocking asymmetric Michael additions in an archetypical class I aldolase by directed evolution. ACS Catal. 11, 13236–13243 (2021).
Zhou, H. et al. Engineering 2-deoxy-D-ribose-5-phosphate aldolase for anti- and syn-selective epoxidations of α,β-unsaturated aldehydes. Angew. Chem. Int. Ed. 64, e202503054 (2025).
Hélaine, V. et al. Recent advances in the substrate selectivity of aldolases. ACS Catal. 12, 733–761 (2022).
Poddar, H., Rahimi, M., Geertsema, E. M., Thunnissen, A. M. & Poelarends, G. J. Evidence for the formation of an enamine species during aldol and Michael-type addition reactions promiscuously catalyzed by 4-oxalocrotonate tautomerase. ChemBioChem 16, 738–741 (2015).
List, B. Enamine catalysis is a powerful strategy for the catalytic generation and use of carbanion equivalents. Acc. Chem. Res. 37, 548–557 (2004).
Bock, D. A., Lehmann, C. W. & List, B. Crystal structures of proline-derived enamines. Proc. Natl. Acad. Sci. USA 107, 20636–20641 (2010).
Schmid, M. B., Zeitler, K. & Gschwind, R. M. The elusive enamine intermediate in proline-catalyzed aldol reactions: NMR detection, formation pathway, and stabilization trends. Angew. Chem. Int. Ed. 49, 4997–5003 (2010).
Lowther, W. T. & Matthews, B. W. Structure and function of the methionine aminopeptidases. Biochim. Biophys. Acta 1477, 157–167 (2000).
Xiao, Q., Zhang, F., Nacev, B. A., Liu, J. O. & Pei, D. Protein N-terminal processing: substrate specificity of Escherichia coli and human methionine aminopeptidases. Biochemistry 49, 5588–5599 (2010).
Moatsou, D. et al. Self-assembly of temperature-responsive protein-polymer bioconjugates. Bioconjug. Chem. 26, 1890–1899 (2015).
Phillips, R. S. Temperature modulation of the stereochemistry of enzymatic catalysis: prospects for exploitation. Trends Biotechnol. 14, 13–16 (1996).
Magnusson, A. O., Takwa, M., Hamberg, A. & Hult, K. An S-selective lipase was created by rational redesign and the enantioselectivity increased with temperature. Angew. Chem. Int. Ed. 44, 4582–4585 (2005).
Röllig, R., Paul, C. E., Duquesne, K., Kara, S. & Alphand, V. Exploring the temperature effect on enantioselectivity of a Baeyer-Villiger biooxidation by the 2,5-DKCMO module: the SLM approach. ChemBioChem 23, e202200293 (2022).
Secundo, F. & Phillips, R. S. Effects of pH on enantiospecificity of alcohol dehydrogenases from Thermoanaerobacter ethanolicus and horse liver. Enzym. Microb. Technol. 19, 487–492 (1996).
Gordon, J. C. et al. H++: a server for estimating pKas and adding missing hydrogens to macromolecules. Nucleic Acids Res. 33, W368–W371 (2005).
Anandakrishnan, R., Aguilar, B. & Onufriev, A. V. H++ 3.0: automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulations. Nucleic Acids Res. 40, W537–W541 (2012).
Williams, T. L. et al. Secondary amine catalysis in enzyme design: broadening protein template diversity through genetic code expansion. Angew. Chem. Int. Ed. 63, e202403098 (2024).
Zhu, Z., Hu, Q., Fu, Y., Tong, Y. & Zhou, Z. Design and evolution of an enzyme for the asymmetric Michael addition of cyclic ketones to nitroolefins by enamine catalysis. Angew. Chem. Int. Ed. 63, e202404312 (2024).
Acknowledgements
This work was supported by the National Natural Science Foundation of China (grant no. 32571664 to J.L.), the National Key Research and Development Program of China (grant no. 2023YFA0914000 to J.L.), the Shanghai Science and Technology Committee (grant no. 24ZR1451000 to J.L.), and the School of Physical Science and Technology of ShanghaiTech University (grant no. SPST-YSFZ-2024-01 to J.L.). H.L. acknowledges support from the Shanghai Post-doctoral Excellence Program (grant no. 2025520). The authors also acknowledge the High-Performance Computing (HPC) Platform of ShanghaiTech University.
Author information
Authors and Affiliations
Contributions
J.L., H.L., and W.-Q.L. designed the experiments. H.L. performed the experiments. X.J. and W.-Q.L. carried out MALDI-TOF-MS analysis. X.Z. performed NMR analysis. Y.Z., Y.L., and Y.G. performed HPLC analysis. H.L. and J.L. analyzed the data and prepared the illustrations. H.L. and J.L. wrote the manuscript with input from all authors. J.L. contributed to project conception and supervised the project.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Communications thanks anonymous reviewers for their contribution to the peer review of this work. [A peer review file is available.]
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Lu, H., Liu, WQ., Ji, X. et al. Engineering LmrR protein for L-proline-based asymmetric aldol biocatalysis. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69968-y
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41467-026-69968-y
