Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Engineering new catalytic activities in enzymes

Nature Catalysis volume 3, pages 203–213 (2020)Cite this article

Subjects

Abstract

The efficiency, selectivity and sustainability benefits offered by enzymes are enticing chemists to consider biocatalytic transformations to complement or even supplant more traditional synthetic routes. Increasing demands for efficient and versatile synthetic methods, combined with powerful new discovery and engineering tools, has prompted innovations in biocatalysis, especially the development of new enzymes for precise transformations or ‘molecular editing’. As a result, the past decade has witnessed an impressive expansion of the catalytic repertoire of enzymes to include new and useful transformations not known (or relevant) in the biological world. In this Review we illustrate various ways in which researchers have approached using the catalytic machineries of enzymes for new-to-nature transformations. These efforts have identified genetically encoded catalysts that can be tuned and diversified by engineering the protein sequence, particularly by directed evolution. Discovery and improvement of these new enzyme activities is opening a floodgate that connects the chemistry of the biological world to that invented by humans over the past 100 years.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Strategies for the discovery of new enzyme functions.
Fig. 2: Promiscuous functions enabled by versatile active sites.
Fig. 3: New chemistries with cofactor-dependent enzymes.
Fig. 4: Chemomimetic carbene- and nitrene-transfer chemistries with engineered haem proteins.
Fig. 5: Different strategies for artificial enzyme construction.

Similar content being viewed by others

References

  1. Hönig, M., Sondermann, P., Carreira, E. M. & Turner, N. Enantioselective chemo- and biocatalysis: partners in retrosynthesis. Angew. Chem. Int. Ed. 56, 8942–8973 (2017). This Review covers many examples of designing retrosynthetic routes with enzymes.

    Article  CAS  Google Scholar 

  2. de Souza, R. O. M. A., Miranda, L. S. M. & Bornscheuer, U. T. A retrosynthetic approach for biocatalysis in organic synthesis. Chem. Eur. J. 23, 12040–12063 (2017).

    Article  CAS  PubMed  Google Scholar 

  3. Turner, N. J. & Humphrey, L. Biocatalysis in Organic Synthesis: The Retrosynthesis Approach (Royal Society of Chemistry, 2018).

  4. Huang, P.-S., Boyken, S. E. & Baker, D. The coming of age of de novo protein design. Nature 537, 320–327 (2016).

    Article  CAS  PubMed  Google Scholar 

  5. Zanghellini, A. de novo computational enzyme design. Curr. Opin. Chem. Biol. 29, 132–138 (2014).

    CAS  Google Scholar 

  6. Khersonsky, O. & Tawfik, D. S. Enzyme promiscuity: a mechanistic and evolutionary perspective. Annu. Rev. Biochem. 79, 471–505 (2010). An in-depth Review of how to recognize and understand enzyme promiscuity.

    Article  CAS  PubMed  Google Scholar 

  7. Tawfik, D. S. Messy biology and the origins of evolutionary innovations. Nat. Chem. Bio. 6, 692–696 (2010).

    Article  CAS  Google Scholar 

  8. Busto, E., Gotor-Fernández, V. & Gotor, V. Hydrolases: catalytically promiscuous enzymes for non-conventional reactions in organic synthesis. Chem. Soc. Rev. 39, 4504–4523 (2010).

    Article  CAS  PubMed  Google Scholar 

  9. Campos, K. R. et al. The importance of synthetic chemistry in the pharmaceutical industry. Science 363, eaat0805 (2019).

    Article  CAS  PubMed  Google Scholar 

  10. Bornscheuer, U. T. & Kazlauskas, R. J. Catalytic promiscuity in biocatalysis: using old enzymes to form new bonds and follow new pathways. Angew. Chem. Int. Ed. 43, 6032–6040 (2004).

    Article  CAS  Google Scholar 

  11. Branneby, C. et al. Carbon–carbon bonds by hydrolytic enzymes. J. Am. Chem. Soc. 125, 874–875 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Mayer, C. et al. The E358S mutant of Agrobacterium sp. β-glucosidase is greatly improved glycosynthase. FEBS Lett. 466, 40–44 (2000).

    Article  CAS  PubMed  Google Scholar 

  13. Jahn, M. et al. Thioglycoligases: mutant glycosidases for thioglycoside synthesis. Angew. Chem. Int. Ed. 42, 352–354 (2003).

    Article  CAS  Google Scholar 

  14. Schallmey, A. & Schallmey, M. Recent advances on halohydrin dehalogenases—from enzyme identification to novel biocatalytic applications. Appl. Microbiol. Biotechnol. 100, 7827–7839 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Fox, R. J. et al. Improving catalytic function by ProSAR-driven enzyme evolution. Nat. Biotechnol. 25, 338–344 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Syrén, P.-O. et al. Squalene-hopene cyclases—evolution, dynamics and catalytic scope. Curr. Opin. Chem. Biol. 41, 73–82 (2016).

    Article  CAS  Google Scholar 

  17. Seitz, M. et al. Synthesis of heterocyclic terpenoids by promiscuous squalene–hopene cyclases. ChemBioChem 14, 436–439 (2013).

    Article  CAS  PubMed  Google Scholar 

  18. Hammer, S. C. et al. Squalene hopene cyclases are protonases for stereoselective Brønsted acid catalysis. Nat. Chem. Bio. 11, 121–126 (2015).

    Article  CAS  Google Scholar 

  19. Mangas-Sanchez, J. et al. Imine reductases (IREDs). Curr. Opin. Chem. Biol. 37, 19–25 (2017).

    Article  CAS  PubMed  Google Scholar 

  20. Sharma, M. et al. A mechanism for reductive amination catalyzed by fungal reductive aminases. ACS Catal. 8, 11534–11541 (2018).

    Article  CAS  Google Scholar 

  21. Aleku, G. A. et al. A reductive aminase from Aspergillus oryzae. Nat. Chem. 9, 961–969 (2017).

    Article  CAS  PubMed  Google Scholar 

  22. Emmanuel, M. A., Greenberg, N. R., Oblinsky, D. G. & Hyster, T. K. Accessing non-natural reactivity by irradiating nicotinamide-dependent enzymes with light. Nature 540, 414–417 (2016). This ground-breaking example of photo-induced enzyme promiscuity using the excited states of biological cofactors opens a whole new route to accessing non-natural enzyme activities.

    Article  CAS  PubMed  Google Scholar 

  23. Biegasiewicz, K. F. et al. Catalytic promiscuity enabled by photoredox catalysis in nicotinamide-dependent oxidoreductases. Nat. Chem. 10, 770–775 (2018).

    Article  CAS  PubMed  Google Scholar 

  24. Dockrey, S. A. B. & Narayan, A. R. H. Flavin-dependent biocatalysts in synthesis. Tetrahedron 75, 1115–1121 (2019).

    Article  CAS  Google Scholar 

  25. Sandoval, B. A., Meichan, A. J. & Hyster, T. K. Enantioselective hydrogen atom transfer: discovery of catalytic promiscuity in flavin-dependent ‘ene’-reductases. J. Am. Chem. Soc. 139, 11313–11316 (2017).

    Article  CAS  PubMed  Google Scholar 

  26. Sandoval, B. A. et al. Photoenzymatic catalysis enables radical-mediated ketone reduction in ene-reductases. Angew. Chem. Int. Ed. 58, 8714–8718 (2019).

    Article  CAS  Google Scholar 

  27. Biegasiewicz, K. F. et al. Photoexcitation of flavoenzymes enables a stereoselective radical cyclization. Science 364, 1166–1169 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Sorigué, D. et al. An algal photoenzyme converts fatty acids to hydrocarbons. Science 357, 903–907 (2017).

    Article  CAS  PubMed  Google Scholar 

  29. Zhang, W. et al. Hydrocarbon synthesis via photoenzymatic decarboxylation of carboxylic acids. J. Am. Chem. Soc. 141, 3116–3120 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Meyer, D., Neumann, P., Ficner, R. & Tittmann, K. Observation of a stable carbene at the active site of a thiamin enzyme. Nat. Chem. Bio. 9, 488–490 (2013).

    Article  CAS  Google Scholar 

  31. Breslow, R. On the mechanism of thiamine action. IV.1 Evidence from studies on model systems. J. Am. Chem. Soc. 80, 3719–3726 (1958).

    Article  CAS  Google Scholar 

  32. Loschonsky, S. et al. Catalytic scope of the thiamine-dependent multifunctional enzyme cyclohexane-1,2-dione hydrolase. ChemBioChem 15, 389–392 (2014).

    Article  CAS  PubMed  Google Scholar 

  33. Loschonsky, S. et al. Extended reaction scope of thiamine diphosphate dependent cyclohexane-1,2-dione hydrolase: From C–C bond cleavage to C–C bond ligation. Angew. Chem. Int. Ed. 53, 14402–14406 (2014).

    Article  CAS  Google Scholar 

  34. Bernacchia, G. et al. Enzymatic chemoselective aldehyde–ketone cross-couplings through the polarity reversal of methylacetoin. Angew. Chem. Int. Ed. 54, 7171–7175 (2015).

    Article  CAS  Google Scholar 

  35. Demir, A. S. et al. Enantioselective synthesis of α-hydroxy ketones via benzaldehyde lyase-catalyzed C–C bond formation reaction. Adv. Synth. Catal. 344, 96–103 (2002).

    Article  CAS  Google Scholar 

  36. Dünkelmann, P. et al. Development of a donor–acceptor concept for enzymatic cross-coupling reactions of aldehydes: the first asymmetric cross-benzoin condensation. J. Am. Chem. Soc. 124, 12084–12085 (2002). Excellent example of how enzyme catalysis can solve long-standing chemoselectivity problems in synthetic methodologies.

    Article  CAS  PubMed  Google Scholar 

  37. Dresen, C. et al. The enzymatic asymmetric conjugate umpolung reaction. Angew. Chem. Int. Ed. 49, 6600–6603 (2010).

    Article  CAS  Google Scholar 

  38. Tai, C.-H. & Cook, P. F. Pyridoxal 5′-phosphate-dependent α, β-elimination reactions: mechanism of O-acetylserine sulfhydrylase. Acc. Chem. Res. 34, 49–59 (2001).

    Article  CAS  PubMed  Google Scholar 

  39. Maier, T. H. P. Semisynthetic production of unnatural l-α-amino acids by metabolic engineering of the cysteine-biosynthetic pathway. Nat. Biotechnol. 21, 422–427 (2003).

    Article  CAS  PubMed  Google Scholar 

  40. Phillips, R. S. Synthetic applications of tryptophan synthase. Tetrahedron.: Asymmetry 15, 2787–2792 (2004).

    Article  CAS  Google Scholar 

  41. Buller, A. R. et al. Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation. Proc. Natl. Acad. Sci. USA 112, 14599–14604 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Romney, D. K. et al. Unlocking reactivity of TrpB: a general biocatalytic platform for synthesis of tryptophan analogues. J. Am. Chem. Soc. 139, 10769–10776 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Boville, C. E. et al. Engineered biosynthesis of β-alkyl tryptophan analogs. Angew. Chem. Int. Ed. 57, 14764–14768 (2018).

    Article  CAS  Google Scholar 

  44. Herger, M. et al. Synthesis of β-branched tryptophan analogues using an engineered subunit of tryptophan synthase. J. Am. Chem. Soc. 138, 8388–8391 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Romney, D. K., Sarai, N. S. & Arnold, F. H. Nitroalkanes as versatile nucleophiles for enzymatic synthesis of noncanonical amino acids. ACS Catal. 9, 8726–8730 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Werner, E. R., Blau, N. & Thöny, B. Tetrahydrobiopterin: biochemistry and pathophysiology. Biochem J. 438, 397–414 (2011).

    Article  CAS  PubMed  Google Scholar 

  47. Heberling, M. M., Wu, B., Bartsch, S. & Janssen, D. B. Priming ammonia lyases and aminomutases for industrial and therapeutic applications. Curr. Opin. Chem. Biol. 17, 250–260 (2013).

    Article  CAS  PubMed  Google Scholar 

  48. Marshall, S. A., Payne, K. A. P. & Leys, D. The UbiX–UbiD system: the biosynthesis and use of prenylated flavin (prFMN). Arch. Biochem. Biophys. 632, 209–221 (2017).

    Article  CAS  PubMed  Google Scholar 

  49. Vaillancourt, F. H., Yin, J. & Walsh, C. T. SyrB2 in syringomycin E biosynthesis is a nonheme FeII α-ketoglutarate- and O2-dependent halogenase. Proc. Natl. Acad. Sci. USA 102, 10111–10116 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Wong, S. D. et al. Elucidation of the iron(iv)–oxo intermediate in the non-haem iron halogenase SyrB2. Nature 499, 320–323 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Matthews, M. L. et al. Direct nitration and azidation of aliphatic carbons by an iron-dependent halogenase. Nat. Chem. Bio. 10, 209–215 (2014).

    Article  CAS  Google Scholar 

  52. Neugebauer, M. E. et al. A family of radical halogenases for the engineering of amino-acid-based products. Nat. Chem. Bio. 10, 209–215 (2014).

    Article  CAS  Google Scholar 

  53. de Visser, S. P., Kumar, D. & Shaik, S. How do aldehyde side products occur during alkene epoxidation by cytochrome P450? Theory reveals a state-specific multi-state scenario where the high-spin component leads to all side products. J. Inorg. Biochem. 98, 1183–1193 (2004).

    Article  CAS  PubMed  Google Scholar 

  54. Hammer, S. C. et al. Anti-Markovnikov alkene oxidation by metal-oxo-mediated enzyme catalysis. Science 358, 215–218 (2017). This study demonstrates how a new enzyme activity can be obtained by promoting an alternative reaction pathway.

    Article  CAS  PubMed  Google Scholar 

  55. Prier, C. K. & Arnold, F. H. Chemomimetic biocatalysis: exploiting the synthetic potential of cofactor-dependent enzymes to create new catalysts. J. Am. Chem. Soc. 137, 13992–14006 (2015).

    Article  CAS  PubMed  Google Scholar 

  56. Che, C.-M. & Huang, J.-S. Metalloporphyrin-based oxidation systems: from biomimetic reactions to application in organic synthesis. Chem. Commun. 3996–4015 (2009).

  57. Lu, H. & Zhang, X. P. Catalytic C–H functionalization by metalloporphyrins: recent developments and future directions. Chem. Soc. Rev. 40, 1899–1909 (2011).

    Article  CAS  PubMed  Google Scholar 

  58. Che, C.-M., Lo, V. K.-Y., Zhou, C.-Y. & Huang, J.-S. Selective functionalisation of saturated C–H bonds with metalloporphyrin catalysts. Chem. Soc. Rev. 40, 1950–1975 (2011).

    Article  CAS  PubMed  Google Scholar 

  59. Maxwell, J. L., Brown, K. C., Bartley, D. W. & Kodadek, T. Mechanism of the rhodium porphyrin-catalyzed cyclopropanation of alkenes. Science 256, 1544–1547 (1992).

    Article  CAS  PubMed  Google Scholar 

  60. Wolf, J. R. et al. Shape and stereoselective cyclopropanation of alkenes catalyzed by iron porphyrins. J. Am. Chem. Soc. 117, 9194–9199 (1995).

    Article  CAS  Google Scholar 

  61. Coelho, P. S., Brustad, E. M., Kannan, A. & Arnold, F. H. Olefin cyclopropanation via carbene transfer catalyzed by engineered cytochrome P450 enzymes. Science 339, 307–310 (2013). This report of a non-natural carbene-transfer reaction with an engineered haem enzyme laid the foundation for development of novel carbene and nitrene transferases.

    Article  CAS  PubMed  Google Scholar 

  62. Coelho, P. S. et al. A serine-substituted P450 catalyzes highly efficient carbene transfer to olefins in vivo. Nat. Chem. Biol. 9, 485–487 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Brandenberg, O. F. et al. Stereoselective enzymatic synthesis of heteroatom-substituted cyclopropanes. ACS Catal. 8, 2629–2634 (2018).

    Article  CAS  Google Scholar 

  64. Wang, Z. J. et al. Improved cyclopropanation activity of histidine-ligated cytochrome P450 enables the enantioselective formal synthesis of levomilnacipran. Angew. Chem. Int. Ed. 53, 6810–6813 (2014).

    Article  CAS  Google Scholar 

  65. Knight, A. M. et al. Diverse engineered heme proteins enable stereodivergent cyclopropanation of unactivated alkenes. ACS Cent. Sci. 4, 372–377 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Bordeaux, M., Tyagi, V. & Fasan, R. Highly diastereoselective and enantioselective olefin cyclopropanation using engineered myoglobin-based catalysts. Angew. Chem. Int. Ed. 54, 1744–1748 (2015).

    Article  CAS  Google Scholar 

  67. Chen, K. et al. Enzymatic construction of highly strained carbocycles. Science 360, 71–75 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Farwell, C. C. et al. Enantioselective enzyme-catalyzed aziridination enabled by active-site evolution of a cytochrome P450. ACS Cent. Sci. 1, 89–93 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Tsutsumi, H. et al. Unprecedented cyclization catalyzed by a cytochrome P450 in benzastatin biosynthesis. J. Am. Chem. Soc. 140, 6631–6639 (2018).

    Article  CAS  PubMed  Google Scholar 

  70. Cho, I. et al. Enantioselective aminohydroxylation of styrenyl olefins catalyzed by an engineered hemoprotein. Angew. Chem. Int. Ed. 58, 3138–3142 (2019).

    Article  CAS  Google Scholar 

  71. Svastits, E., Dawson, J. H., Breslow, R. & Gellman, S. H. Functionalized nitrogen atom transfer catalyzed by cytochrome P-450. J. Am. Chem. Soc. 107, 6427–6428 (1985).

    Article  CAS  Google Scholar 

  72. McIntosh, J. A. et al. Enantioselective intramolecular C–H amination catalyzed by engineered cytochrome P450 enzymes in vitro and in vivo. Angew. Chem. Int. Ed. 52, 9309–9312 (2013).

    Article  CAS  Google Scholar 

  73. Yang, Y. et al. An enzymatic platform for the asymmetric amination of primary, secondary and tertiary C(sp 3)–H bonds. Nat. Chem. 11, 987–993 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Singh, R., Bordeaux, M. & Fasan, R. P450-catalyzed intramolecular sp 3 C–H amination with arylsulfonyl azide substrates. ACS Catal. 4, 546–552 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Bordeaux, M., Singh, R. & Fasan, R. Intramolecular C(sp 3)–H amination of arylsulfonyl azides with engineered and artificial myoglobin-based catalysts. Bioorg. Med. Chem. 22, 5697–5704 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Singh, R., Kolev, J. N., Sutera, P. A. & Fasan, R. Enzymatic C(sp 3)–H amination: P450-catalyzed conversion of carbonazidates into oxazolidinones. ACS Catal. 5, 1685–1691 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Hyster, T. K. et al. Enzyme-controlled nitrogen-atom transfer enables regiodivergent C–H amination. J. Am. Chem. Soc. 136, 15505–15508 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Prier, C. K. et al. Enantioselective, intermolecular benzylic C–H amination catalysed by an engineered iron–haem enzyme. Nat. Chem. 9, 629–634 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Zhang, R. K. et al. Enzymatic assembly of carbon–carbon bonds via iron-catalysed sp 3 C–H functionalization. Nature 565, 67–72 (2019). This work highlights the immense potential of natural metalloproteins in addressing important problems in synthetic chemistry, especially those considered to be challenging for earth-abundant transition metal catalysis.

    Article  CAS  PubMed  Google Scholar 

  80. Zhang, J., Huang, X., Zhang, R. K. & Arnold, F. H. Enantiodivergent α-amino C–H fluoroalkylation catalyzed by engineered cytochrome P450s. J. Am. Chem. Soc. 141, 9798–9802 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Farwell, C. C. et al. Enantioselective imidation of sulfides via enzyme-catalyzed intermolecular nitrogen-atom transfer. J. Am. Chem. Soc. 136, 8766–8771 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Tyagi, V. et al. Biocatalytic synthesis of allylic and allenyl sulfides through a myoglobin-catalyzed Doyle–Kirmse reaction. Angew. Chem. Int. Ed. 55, 13562–13566 (2016).

    Article  CAS  Google Scholar 

  83. Prier, C. K. et al. Asymmetric enzymatic synthesis of allylic amines: a sigmatropic rearrangement strategy. Angew. Chem. Int. Ed. 55, 4711–4715 (2016).

    Article  CAS  Google Scholar 

  84. Wang, Z. J., Peck, N. E., Renata, H. & Arnold, F. H. Cytochrome P450-catalyzed insertion of carbenoids into N–H bonds. Chem. Sci. 5, 598–601 (2014).

    Article  CAS  PubMed  Google Scholar 

  85. Sreenilayam, G. & Fasan, R. Myoglobin-catalyzed intermolecular carbene N–H insertion with arylamine substrates. Chem. Commun. 51, 1532–1534 (2015).

    Article  CAS  Google Scholar 

  86. Tyagi, V., Bonn, R. B. & Fasan, R. Intermolecular carbene S–H insertion catalysed by engineered myoglobin-based catalysts. Chem. Sci. 6, 2488–2494 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Chen, K. et al. Alternate heme ligation steers activity and selectivity in engineered cytochrome P450-catalyzed carbene-transfer reactions. J. Am. Chem. Soc. 140, 16402–16407 (2018).

    Article  CAS  PubMed  Google Scholar 

  88. Kan, S. B. J., Lewis, R. D., Chen, K. & Arnold, F. H. Directed evolution of cytochrome c for carbon–silicon bond formation: bringing silicon to life. Science 354, 1048–1051 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Kan, S. B. J. et al. Genetically programmed chiral organoborane synthesis. Nature 552, 132–136 (2017).

    Article  CAS  PubMed  Google Scholar 

  90. Huang, X. et al. A biocatalytic platform for synthesis of chiral α-trifluoromethylated organoborons. ACS Cent. Sci. 5, 270–276 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Chen, K. et al. Engineered cytochrome c-catalyzed lactone-carbene B–H insertion. Synlett 30, 378–382 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Lewis, R. D. et al. Catalytic iron–carbene intermediate revealed in a cytochrome c carbene transferase. Proc. Natl. Acad. Sci. USA 115, 7308–7313 (2018). This report describes the first iron-carbene intermediate captured in a protein structure and is among the very few examples of a carbene intermediate bound to any type of iron complex.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Hayashi, T. et al. Capture and characterization of a reactive haem–carbenoid complex in an artificial metalloenzyme. Nat. Catal. 1, 578–584 (2018).

    Article  CAS  Google Scholar 

  94. Wilson, M. E. & Whitesides, G. M. Conversion of a protein to a homogeneous asymmetric hydrogenation catalyst by site-specific modification with a diphosphinerhodium(i) moiety. J. Am. Chem. Soc. 100, 306–307 (1978).

    Article  CAS  Google Scholar 

  95. Schwizer, F. et al. Artificial metalloenzymes: reaction scope and optimization strategies. Chem. Rev. 118, 142–231 (2018).

    Article  CAS  PubMed  Google Scholar 

  96. Chatterjee, A. et al. An enantioselective artificial Suzukiase based on the biotin–streptavidin technology. Chem. Sci. 7, 673–677 (2016).

    Article  CAS  PubMed  Google Scholar 

  97. Pierron, J. et al. Artificial metalloenzymes for asymmetric allylic alkylation on the basis of the biotin–avidin technology. Angew. Chem. Int. Ed. 47, 701–705 (2008).

    Article  CAS  Google Scholar 

  98. Hyster, T. K., Knörr, L., Ward, T. R. & Rovis, T. Biotinylated Rh(III) complexes in engineered streptavidin for accelerated asymmetric C–H activation. Science 338, 500–503 (2012).

    Article  CAS  PubMed  Google Scholar 

  99. Lo, C. et al. Artificial metalloenzymes for olefin metathesis based on the biotin-(strept)avidin technology. Chem. Commun. 47, 12065–12067 (2011).

    Article  CAS  Google Scholar 

  100. Jeschek, M. et al. Directed evolution of artificial metalloenzymes for in vivo metathesis. Nature 537, 661–665 (2016). This study describes a platform for in vivo artificial metalloenzyme assembly and is one of the rare examples of directed evolution of ArMs.

    Article  CAS  PubMed  Google Scholar 

  101. Hassan, I. S. et al. Asymmetric δ-lactam synthesis with a monomeric streptavidin artificial metalloenzyme. J. Am. Chem. Soc. 141, 4815–4819 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Dürrenberger, M. et al. Artificial transfer hydrogenases for the enantioselective reduction of cyclic imines. Angew. Chem. Int. Ed. 50, 3026–3029 (2011).

    Article  CAS  Google Scholar 

  103. Wu, S. et al. Breaking symmetry: engineering single-chain dimeric streptavidin as host for artificial metalloenzymes. J. Am. Chem. Soc. 15869–15878 (2019).

  104. Raines, D. J. et al. Redox-switchable siderophore anchor enables reversible artificial metalloenzyme assembly. Nat. Catal. 1, 680–688 (2018).

    Article  CAS  Google Scholar 

  105. Eda, S. et al. Biocompatibility and therapeutic potential of glycosylated albumin artificial metalloenzymes. Nat. Catal. 2, 780–792 (2019).

    Article  CAS  Google Scholar 

  106. Mayer, C., Gillingham, D. G., Ward, T. R. & Hilvert, D. An artificial metalloenzyme for olefin metathesis. Chem. Commun. 47, 12068–12070 (2011).

    Article  CAS  Google Scholar 

  107. Jarvis, A. G. et al. Enzyme activity by design: an artificial rhodium hydroformylase for linear aldehydes. Angew. Chem. Int. Ed. 56, 13596–13600 (2017).

    Article  CAS  Google Scholar 

  108. Yang, H., Srivastava, P., Zhang, C. & Lewis, J. C. A general method for artificial metalloenzyme formation through strain-promoted azide–alkyne cycloaddition. ChemBioChem 15, 223–227 (2014).

    Article  CAS  PubMed  Google Scholar 

  109. Srivastava, P., Yang, H., Ellis-Guardiola, K. & Lewis, J. C. Engineering a dirhodium artificial metalloenzyme for selective olefin cyclopropanation. Nat. Commun. 6, 7789 (2015).

    Article  CAS  PubMed  Google Scholar 

  110. Yang, H. et al. Evolving artificial metalloenzymes via random mutagenesis. Nat. Chem. 10, 318–324 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Abe, S. et al. Control of the coordination structure of organometallic palladium complexes in an apo-ferritin cage. J. Am. Chem. Soc. 130, 10512–10514 (2008).

    Article  CAS  PubMed  Google Scholar 

  112. Jing, Q. & Kazlauskas, R. J. Regioselective hydroformylation of styrene using rhodium-substituted carbonic anhydrase. ChemCatChem 2, 953–957 (2010).

    Article  CAS  Google Scholar 

  113. Drienovská, I., Rioz-Martínez, A., Draksharapu, A. & Roelfes, G. Novel artificial metalloenzymes by in vivo incorporation of metal-binding unnatural amino acids. Chem. Sci. 6, 770–776 (2015).

    Article  CAS  PubMed  Google Scholar 

  114. Drienovská, I. et al. Design of an enantioselective artificial metallohydratase enzyme containing an unnatural metal-binding amino acid. Chem. Sci. 8, 7228–7235 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  115. Key, H. M., Dydio, P., Clark, D. S. & Hartwig, J. F. Abiological catalysis by artificial haem proteins containing noble metals in place of iron. Nature 534, 534–537 (2016).

    Article  CAS  PubMed  Google Scholar 

  116. Dydio, P. et al. An artificial metalloenzyme with the kinetics of native enzymes. Science 354, 102–106 (2016).

    Article  CAS  PubMed  Google Scholar 

  117. Key, H. M. et al. Beyond iron: iridium-containing P450 enzymes for selective cyclopropanations of structurally diverse alkenes. ACS Cent. Sci. 3, 302–308 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Dydio, P. et al. Chemoselective, enzymatic C‒H bond amination catalyzed by a cytochrome P450 containing an Ir(Me)–PIX cofactor. J. Am. Chem. Soc. 139, 1750–1753 (2017).

    Article  CAS  PubMed  Google Scholar 

  119. Agostini, F. et al. Biocatalysis with unnatural amino acids: enzymology meets xenobiology. Angew. Chem. Int. Ed. 56, 9680–9703 (2017).

    Article  CAS  Google Scholar 

  120. 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).

    Article  CAS  PubMed  Google Scholar 

  121. Mayer, C. et al. Directed evolution of a designer enzyme featuring an unnatural catalytic amino acid. Angew. Chem. Int. Ed. 58, 2083–2087 (2019).

    Article  CAS  Google Scholar 

  122. Burke, A. J. et al. Design and evolution of an enzyme with a non-canonical organocatalytic mechanism. Nature 570, 219–223 (2019). This artificial enzyme with a novel mechanism from organocatalysis will inspire further development of new enzymes with catalytically functional non-canonical amino acids.

    Article  CAS  PubMed  Google Scholar 

  123. Bornscheuer, U. T. et al. Engineering the third wave of biocatalysis. Nature 485, 185–194 (2012).

    Article  CAS  PubMed  Google Scholar 

  124. Hughes, G. & Lewis, J. C. Introduction: biocatalysis in industry. Chem. Rev. 118, 1–3 (2018).

    Article  CAS  PubMed  Google Scholar 

  125. Bornscheuer, U. T. The fourth wave of biocatalysis is approaching. Philos. Trans. R. Soc. A 376, 20170063 (2017).

    Article  CAS  Google Scholar 

  126. Bornscheuer, U. T. Biocatalysis: successfully crossing boundaries. Angew. Chem. Int. Ed. 55, 4372–4373 (2016).

    Article  CAS  Google Scholar 

  127. Wu., Z. et al. Machine-learning-assisted directed protein evolution with combinatorial libraries. Proc. Natl. Acad. Sci. USA 116, 8852–8858 (2019). Machine learning-guided directed evolution of a haem protein for a non-native function facilitated exploration of sequence space.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  128. O’Hagan, D. & Deng, H. Enzymatic fluorination and biotechnological developments of the fluorinase. Chem. Rev. 115, 634–649 (2015).

    Article  CAS  PubMed  Google Scholar 

  129. Jeon, B., Wang, S.-A., Ruszczycky, M. W. & Liu, H. Natural [4+2]-cyclases. Chem. Rev. 117, 5367–5388 (2017).

    Article  CAS  PubMed  Google Scholar 

  130. Kelly, S. A. et al. Application of ω-transaminases in the pharmaceutical industry. Chem. Rev. 118, 349–367 (2018).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank D. J. Wackelin and Y. Yang (Caltech) for helpful discussions and comments on the manuscript. This work was supported by NSF Division of Molecular and Cellular Biosciences grant no. MCB-1513007, the US Army Research Office Institute for Collaborative Biotechnologies (cooperative agreement no. W911NF-19-2-0026) and the US Army Research Office Institute for Collaborative Biotechnologies (contract no. W911NF-19-D-0001). K.C. thanks the Resnick Sustainability Institute at Caltech for fellowship support. The content of this paper does not necessarily reflect the position or the policy of the funding agencies, and no official endorsement should be inferred.

Author information

Authors and Affiliations

  1. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA

    Kai Chen & Frances H. Arnold

Authors
  1. Kai Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Frances H. Arnold
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors participated in designing and writing the manuscript.

Corresponding author

Correspondence to Frances H. Arnold.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chen, K., Arnold, F.H. Engineering new catalytic activities in enzymes. Nat Catal 3, 203–213 (2020). https://doi.org/10.1038/s41929-019-0385-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41929-019-0385-5

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing