The field of biocatalysis has advanced from harnessing natural enzymes to using directed evolution to obtain new biocatalysts with tailor-made functions1. Several tools have recently been developed to expand the natural enzymatic repertoire with abiotic reactions2,3. For example, artificial metalloenzymes, which combine the versatile reaction scope of transition metals with the beneficial catalytic features of enzymes, offer an attractive means to engineer new reactions. Three complementary strategies exist3: repurposing natural metalloenzymes for abiotic transformations2,4; in silico metalloenzyme (re-)design5,6,7; and incorporation of abiotic cofactors into proteins8,9,10,11. The third strategy offers the opportunity to design a wide variety of artificial metalloenzymes for non-natural reactions. However, many metal cofactors are inhibited by cellular components and therefore require purification of the scaffold protein12,13,14,15. This limits the throughput of genetic optimization schemes applied to artificial metalloenzymes and their applicability in vivo to expand natural metabolism. Here we report the compartmentalization and in vivo evolution of an artificial metalloenzyme for olefin metathesis, which represents an archetypal organometallic reaction16,17,18,19,20,21,22 without equivalent in nature. Building on previous work6 on an artificial metallohydrolase, we exploit the periplasm of Escherichia coli as a reaction compartment for the ‘metathase’ because it offers an auspicious environment for artificial metalloenzymes, mainly owing to low concentrations of inhibitors such as glutathione, which has recently been identified as a major inhibitor15. This strategy facilitated the assembly of a functional metathase in vivo and its directed evolution with substantially increased throughput compared to conventional approaches that rely on purified protein variants. The evolved metathase compares favourably with commercial catalysts, shows activity for different metathesis substrates and can be further evolved in different directions by adjusting the workflow. Our results represent the systematic implementation and evolution of an artificial metalloenzyme that catalyses an abiotic reaction in vivo, with potential applications in, for example, non-natural metabolism.
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Bornscheuer, U. T. et al. Engineering the third wave of biocatalysis. Nature 485, 185–194 (2012)
Renata, H., Wang, Z. J. & Arnold, F. H. Expanding the enzyme universe: accessing non-natural reactions by mechanism-guided directed evolution. Angew. Chem. Int. Ed. 54, 3351–3367 (2015)
Hyster, T. K. & Ward, T. R. Genetic optimization of metalloenzymes: enhancing enzymes for non-natural reactions. Angew. Chem. Int. Ed. 55, 7344–7357 (2016)
Coelho, P. S., Brustad, E. M., Kannan, A. & Arnold, F. H. Olefinic cyclopropanation via carbene transfer catalzyed by engineered cytochrome P450 enzymes. Science 339, 307–310 (2013)
Khare, S. D. et al. Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis. Nat. Chem. Biol. 8, 294–300 (2012)
Song, W. J. & Tezcan, F. A. A designed supramolecular protein assembly with in vivo enzymatic activity. Science 346, 1525–1528 (2014)
Zastrow, M. L., Peacock, A. F. A., Stuckey, J. A. & Pecoraro, V. L. Hydrolytic catalysis and structural stabilization in a designed metalloprotein. Nat. Chem. 4, 118–123 (2012)
Creus, M. et al. X-ray structure and designed evolution of an artificial transfer hydrogenase. Angew. Chem. Int. Ed. 47, 1400–1404 (2008)
Lewis, J. C. Artificial metalloenzymes and metallopeptide catalysts for organic synthesis. ACS Catal. 3, 2954–2975 (2013)
Yu, F. T. et al. Protein design: toward functional metalloenzymes. Chem. Rev. 114, 3495–3578 (2014)
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)
Reetz, M. T., Peyralans, J. J. P., Maichele, A., Fu, Y. & Maywald, M. Directed evolution of hybrid enzymes: evolving enantioselectivity of an achiral Rh-complex anchored to a protein. Chem. Commun. 4318–4320 (2006)
Srivastava, P., Yang, H., Ellis-Guardiola, K. & Lewis, J. C. Engineering a dirhodium artificial metalloenzyme for selective olefin cyclopropanation. Nat. Commun. 6, 7789 (2015)
Sauer, D. F. et al. A highly active biohybrid catalyst for olefin metathesis in water: impact of a hydrophobic cavity in a β-barrel protein. ACS Catal. 5, 7519–7522 (2015)
Wilson, Y. M., Dürrenberger, M., Nogueira, E. S. & Ward, T. R. Neutralizing the detrimental effect of glutathione on precious metal catalysts. J. Am. Chem. Soc. 136, 8928–8932 (2014)
Grubbs, R. H. Olefin-metathesis catalysts for the preparation of molecules and materials (Nobel lecture). Angew. Chem. Int. Ed. 45, 3760–3765 (2006)
Schrock, R. R. Multiple metal–carbon bonds for catalytic metathesis reactions (Nobel lecture). Angew. Chem. Int. Ed. 45, 3748–3759 (2006)
Chauvin, Y. Olefin metathesis: the early days (Nobel lecture). Angew. Chem. Int. Ed. 45, 3740–3747 (2006)
Hoveyda, A. H. & Zhugralin, A. R. The remarkable metal-catalysed olefin metathesis reaction. Nature 450, 243–251 (2007)
Fürstner, A. Teaching metathesis “simple” stereochemistry. Science 341, 1229713 (2013)
Burtscher, D. & Grela, K. Aqueous olefin metathesis. Angew. Chem. Int. Ed. 48, 442–454 (2009)
Lin, Y. A., Chalker, J. M., Floyd, N., Bernardes, G. J. L. & Davis, B. G. Allyl sulfides are privileged substrates in aqueous cross-metathesis: application to site-selective protein modification. J. Am. Chem. Soc. 130, 9642–9643 (2008)
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)
Ward, T. R. Artificial metalloenzymes based on the biotin-avidin technology: enantioselective catalysis and beyond. Acc. Chem. Res. 44, 47–57 (2011)
Ilie, A. & Reetz, M. T. Directed evolution of artificial metalloenzymes. Isr. J. Chem. 55, 51–60 (2015)
Lo, C., Ringenberg, M. R., Gnandt, D., Wilson, Y. & Ward, T. R. Artificial metalloenzymes for olefin metathesis based on the biotin-(strept)avidin technology. Chem. Commun. 47, 12065–12067 (2011)
Kajetanowicz, A., Chatterjee, A., Reuter, R. & Ward, T. R. Biotinylated metathesis catalysts: synthesis and performance in ring closing metathesis. Catal. Lett. 144, 373–379 (2014)
Völker, T., Dempwolff, F., Graumann, P. L. & Meggers, E. Progress towards bioorthogonal catalysis with organometallic compounds. Angew. Chem. Int. Ed. 53, 10536–10540 (2014)
Reetz, M. T., Kahakeaw, D. & Lohmer, R. Addressing the numbers problem in directed evolution. ChemBioChem 9, 1797–1804 (2008)
Reetz, M. T. & Carballeira, J. D. Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat. Protocols 2, 891–903 (2007)
Gallizia, A. et al. Production of a soluble and functional recombinant streptavidin in Escherichia coli. Protein Expr. Purif. 14, 192–196 (1998)
Sambrook, J. F. & Russell, D. W. (eds) Molecular Cloning: A Laboratory Manual 3rd edn (Cold Spring Harbor Laboratory, 2001)
Studier, F. W. Protein production by auto-induction in high-density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005)
Volkmer, B. & Heinemann, M. Condition-dependent cell volume and concentration of Escherichia coli to facilitate data conversion for systems biology modeling. PLoS One 6, e23126 (2011)
Humbert, N., Schürmann, P., Zocchi, A., Neuhaus, J.-M. & Ward, T. R. in Avidin-Biotin Interactions: Methods and Applications (ed. McMahon, R. J. ) 101–110 (Vol. 418 of Methods in Molecular Biology, Humana Press, 2008)
Kada, G., Falk, H. & Gruber, H. J. Accurate measurement of avidin and streptavidin in crude biofluids with a new, optimized biotin-fluorescein conjugate. Biochim. Biophys. Acta 1427, 33–43 (1999)
Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010)
Evans, P. R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr. D 67, 282–292 (2011)
Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)
We thank P. Marlière for discussions. This work was supported by funding from the European Commission Seventh Framework Programme [289572-METACODE] and the Swiss National Science Foundation as part of the NCCR Molecular Systems Engineering. We thank M. Dessing and the single-cell facility (D-BSSE, ETH Zurich) for assistance with flow cytometry.
The authors declare no competing financial interests.
Extended data figures and tables
The activity of the evolved artificial metathase biot-Ru–SAVmut was evaluated for three independently produced and purified protein batches and compared to two independent batches of non-mutated SAV (biot-Ru–SAV). Reactions were performed at 4 mM of substrate 1, 50 μM of biot-Ru and 100 μM of of SAV-variant binding sites, and the product 2 was quantified by fluorescence. The displayed activities represent the initial slopes of the fluorescence signal in the linear reaction phase relative to the activity of free biot-Ru, which was defined as 100%. Bar heights represent the average relative activity of four replicate in vitro experiments (n = 4), with 1 s.d. indicated as error bars.
The activity of the evolved artificial metathase biot-Ru–SAVmut was evaluated for different ratios between the cofactor biot-Ru and SAVmut binding sites (biot-Ru:SAVmutb.s.). Reactions were performed at 4 mM of substrate 1, 5 μM of biot-Ru and varying concentrations of SAVmut binding sites (5–50 μM corresponding to ratios ranging from 1:1 to 1:10). The product 2 was quantified by fluorescence. The displayed activities represent the initial slopes of the fluorescence signal in the linear reaction phase. Bar heights represent the average relative activity of four replicate samples (n = 4), with 1 s.d. indicated as error bars.
a–f, Close-up views of the crystal structure of the complexes biot-Ru–SAVmut (a, c, e) and biot-Ru–SAV (b, d, f) displaying pairs of cis-symmetry-related biot-Ru cofactors (view perpendicular to a two-fold crystal symmetry axis, displayed in red): conformers I–I (severe steric clash; a, b), conformers II–II (c, d) and conformers I–II (e, f). The protein was removed for displaying purposes. The cofactors are contoured with 2Fo − Fc electron density in blue at 1σ and anomalous dispersion density in red at 3.5σ.
a–d, Binding of biot-Ru in complex with SAV (a, b) and SAVmut (c, d). For clarity, only one biot-Ru cofactor (ruthenium represented as green spheres) per opposing SAV or SAVmut monomer pair (grey and pink ribbon traces indicate two monomers facing each other) is depicted in conformations I (a, c) and II (b, d). Key amino acid residues including mutations V47A, N49K, T114Q, A119G and K121R for SAVmut (c, d) are represented in stick models. Hydrogen-bonds are depicted as dashed lines. Water is shown as red spheres.
a, Comparison of mobility in loop-7,8 before (biot-Ru–SAV) and after (biot-Ru–SAVmut) directed evolution as highlighted by Cα atom B factors (colour scale). Mutations are indicated in two opposing monomers by asterisks: *1/*1′, V47A; *2/*2′, N49K; *3/*3′, T114Q; *4/*4′, A119G; and *5/*5′, K121R. b, Normalized (BCα/Baverage) Cα atom B factors of residues within loop-7,8 from different SAV structures that crystalize in the space group I4122 with similar unit cell dimensions. biot-Ru–SAVmut (dark blue) and biot-Ru–SAV (light blue) are compared to PDB reference structures (shades of grey): r1, 2QCB; r2, 3PK2; r3, 2WPU; r4, 4GJV; r5, 4OKA; r6, 2IZJ; and r7, 2IZB.
a, b, The second-generation Hoveyda–Grubbs (HGII) catalyst (a) was obtained from Sigma Aldrich (Buchs) and the Aquamet (AQM) catalyst (b) was purchased from Apeiron Synthesis S.A.
Extended Data Figure 7 Summary of the results obtained by UPLC-MS for the 96-well plate in vivo screen of the dallyl-sulfonamide 5.
Saturation mutagenesis at position 121 (K121X) was performed on SAVmut(47A/49K/114Q/119G/121K). Heights of the navy and blue bars represent the average of three replicates (n = 3), with 1 s.d. indicated as error bars; purple bars represent the value of a single measurement (n = 1).
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Jeschek, M., Reuter, R., Heinisch, T. et al. Directed evolution of artificial metalloenzymes for in vivo metathesis. Nature 537, 661–665 (2016). https://doi.org/10.1038/nature19114
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