Evolving artificial metalloenzymes via random mutagenesis


Random mutagenesis has the potential to optimize the efficiency and selectivity of protein catalysts without requiring detailed knowledge of protein structure; however, introducing synthetic metal cofactors complicates the expression and screening of enzyme libraries, and activity arising from free cofactor must be eliminated. Here we report an efficient platform to create and screen libraries of artificial metalloenzymes (ArMs) via random mutagenesis, which we use to evolve highly selective dirhodium cyclopropanases. Error-prone PCR and combinatorial codon mutagenesis enabled multiplexed analysis of random mutations, including at sites distal to the putative ArM active site that are difficult to identify using targeted mutagenesis approaches. Variants that exhibited significantly improved selectivity for each of the cyclopropane product enantiomers were identified, and higher activity than previously reported ArM cyclopropanases obtained via targeted mutagenesis was also observed. This improved selectivity carried over to other dirhodium-catalysed transformations, including N–H, S–H and Si–H insertion, demonstrating that ArMs evolved for one reaction can serve as starting points to evolve catalysts for others.

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Figure 1: Model reaction and ArM structure.
Figure 2: Overview of ArM evolution protocol.
Figure 3: Overview of the directed evolution lineages generated and time-course comparison of several catalysts.
Figure 4: Location of mutations in evolved ArMs.
Figure 5: Combinatorial codon mutagenesis sites and protocol.
Figure 6: Time-course experiments of ArM-catalysed cyclopropanations of styrene with (4-methoxyphenyl)methyldiazoacetate.

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  1. 1

    Mahatthananchai, J., Dumas, A. M. & Bode, J. W. Catalytic selective synthesis. Angew. Chem. Int. Ed. 51, 10954–10990 (2012).

    Google Scholar 

  2. 2

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

    CAS  Article  Google Scholar 

  3. 3

    Romero, P. A. & Arnold, F. H. Exploring protein fitness landscapes by directed evolution. Nat. Rev. Mol. Cell Biol. 10, 866–876 (2009).

    CAS  Article  Google Scholar 

  4. 4

    Lu, Y., Berry, S. & Pfister, T. Engineering novel metalloproteins: design of metal-binding sites into native protein scaffolds. Chem. Rev. 101, 3047–3080 (2001).

    CAS  Article  Google Scholar 

  5. 5

    Gerlt, J. A. & Babbitt, P. C. Enzyme (re)design: lessons from natural evolution and computation. Curr. Opin. Chem. Biol. 13, 10–18 (2009).

    CAS  Article  Google Scholar 

  6. 6

    Chen, K. & Arnold, F. H. Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide. Proc. Natl Acad. Sci. USA 90, 5618–5622 (1993).

    CAS  Article  Google Scholar 

  7. 7

    Stemmer, W. P. C. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391 (1994).

    CAS  Article  Google Scholar 

  8. 8

    Lutz, S. Beyond directed evolution–semi-rational protein engineering and design. Curr. Opin. Biotech. 21, 734–743 (2010).

    CAS  Article  Google Scholar 

  9. 9

    Lewis, J. C. Artificial metalloenzymes and metallopeptide catalysts for organic synthesis. ACS Catal. 3, 2954–2975 (2013).

    CAS  Article  Google Scholar 

  10. 10

    Kazlauskas, R. J. & Bornscheuer, U. T. Finding better protein engineering strategies. Nat. Chem. Biol. 5, 526–529 (2009).

    CAS  Article  Google Scholar 

  11. 11

    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. 2016, 4318–4320 (2006).

    Article  Google Scholar 

  12. 12

    Jeschek, M. et al. Directed evolution of artificial metalloenzymes for in vivo metathesis. Nature 537, 661–665 (2016).

    CAS  Article  Google Scholar 

  13. 13

    Song, W. J. & Tezcan, F. A. A designed supramolecular protein assembly with in vivo enzymatic activity. Science 346, 1525–1528 (2014).

    CAS  Article  Google Scholar 

  14. 14

    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).

    CAS  Article  Google Scholar 

  15. 15

    Hyster, T. K. Genetic optimization of metalloenzymes: enhancing enzymes for non-natural reactions. Angew. Chem. Int. Ed. 55, 7344–7357 (2016).

    CAS  Article  Google Scholar 

  16. 16

    Sreenilayam, G., Moore, E. J., Steck, V. & Fasan, R. Metal substitution modulates the reactivity and extends the reaction scope of myoglobin carbene transfer catalysts. Adv. Synth. Catal. 359, 2076–2089 (2017).

    CAS  Article  Google Scholar 

  17. 17

    Sreenilayam, G., Moore, E. J., Steck, V. & Fasan, R. Stereoselective olefin cyclopropanation under aerobic conditions with an artificial enzyme incorporating an iron-chlorin e6 cofactor. ACS Catal. 7, 7629–7633 (2017).

    CAS  Article  Google Scholar 

  18. 18

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

    CAS  Article  Google Scholar 

  19. 19

    Juhász, T., Szeltner, Z. & Polgár, L. Properties of the prolyl oligopeptidase homologue from Pyrococcus furiosus. FEBS Lett. 580, 3493–3497 (2006).

    Article  Google Scholar 

  20. 20

    Polgár, L. The prolyl oligopeptidase family. Cell. Mol. Life Sci. 59, 349–362 (2002).

    Article  Google Scholar 

  21. 21

    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).

    CAS  Article  Google Scholar 

  22. 22

    Harris, M. N., Madura, J. D., Ming, L.-J. & Harwood, V. J. Kinetic and mechanistic studies of prolyl oligopeptidase from the hyperthermophile Pyrococcus furiosus. J. Biol. Chem. 276, 19310–19317 (2001).

    CAS  Article  Google Scholar 

  23. 23

    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).

    CAS  Article  Google Scholar 

  24. 24

    Reetz, M. T. et al. A robust protein host for anchoring chelating ligands and organocatalysts. ChemBioChem 9, 552–564 (2008).

    CAS  Article  Google Scholar 

  25. 25

    Drummond, D., Iverson, B., Georgiou, G. & Arnold, F. H. Why high-error-rate random mutagenesis libraries are enriched in functional and improved proteins. J. Mol. Biol. 350, 806–816 (2005).

    CAS  Article  Google Scholar 

  26. 26

    Dąbrowski, S. & Kur, J. Cloning and expression in Escherichia coli of the recombinant his-tagged DNA polymerases from Pyrococcus furiosus and Pyrococcus woesei. Protein Expr. Purif. 14, 131–138 (1998).

    Article  Google Scholar 

  27. 27

    Punna, S., Kaltgrad, E. & Finn, M. G. ‘Clickable’ agarose for affinity chromatography. Bioconj. Chem. 16, 1536–1541 (2005).

    CAS  Article  Google Scholar 

  28. 28

    Davies, R. R. et al. Artificial metalloenzymes based on protein cavities: exploring the effect of altering the metal ligand attachment position by site directed mutagenesis. Bioorg. Med. Chem. Lett. 9, 79–84 (1999).

    CAS  Article  Google Scholar 

  29. 29

    Filice, M. et al. Synthesis of a heterogeneous artificial metallolipase with chimeric catalytic activity. Chem. Commun. 51, 9324–9327 (2015).

    CAS  Article  Google Scholar 

  30. 30

    Ball, Z. T. Designing enzyme-like catalysts: a rhodium(II) metallopeptide case study. Acc. Chem. Res. 46, 560–570 (2013).

    CAS  Article  Google Scholar 

  31. 31

    Renata, H. et al. Identification of mechanism-based inactivation in P450-catalyzed cyclopropanation facilitates engineering of improved enzymes. J. Am. Chem. Soc. 138, 12527–12533 (2016).

    CAS  Article  Google Scholar 

  32. 32

    Popp, B. V. & Ball, Z. T. Proximity-driven metallopeptide catalysis: remarkable side-chain scope enables modification of the Fos bZip domain. Chem. Sci. 2, 690–695 (2011).

    CAS  Article  Google Scholar 

  33. 33

    Lewis, J. C. & Arnold, F. H. Catalysts on demand: selective oxidations by laboratory-evolved cytochrome P450 BM3. CHIMIA 63, 309–312 (2009).

    CAS  Article  Google Scholar 

  34. 34

    Lu, Y., Yeung, N., Sieracki, N. & Marshall, N. M. Design of functional metalloproteins. Nature 460, 855–862 (2009).

    CAS  Article  Google Scholar 

  35. 35

    Tokuriki, N. & Tawfik, D. S. Protein dynamism and evolvability. Science 324, 203–207 (2009).

    CAS  Article  Google Scholar 

  36. 36

    Kaushik, S., Etchebest, C. & Sowdhamini, R. Decoding the structural events in substrate-gating mechanism of eukaryotic prolyl oligopeptidase using normal mode analysis and molecular dynamics simulations. Proteins 82, 1428–1443 (2014).

    CAS  Article  Google Scholar 

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This work was supported by, or in part by, the US Army Research Laboratory and the US Army Research Office under contract/grant nos. W911NF-14-1-0334 and 66796-LS-RIP (to J.C.L.), the National Science Foundation (NSF) under CAREER Award CHE-1351991 (to J.C.L.), The David and Lucile Packard Foundation (to J.C.L.), the National Institutes of Health (NIH) National Cancer Institute (R00CA175399 to R.E.M.), the Damon Runyon Cancer Research Foundation (DFS-08-14 to R.E.M.) and the NSF under the Center for Chemical Innovation Center for Selective C–H Functionalization (CHE-1700982, to J.C.L.). K.E.G. and D.M.U. were funded by an NIH Chemistry and Biology Interface Training Grant (T32 GM008720) and G.L. was supported by the Kwanjeong Educational Foundation. MS data were acquired on instruments purchased using an NSF instrumentation grant (CHE-1048528).

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H.Y. and P.S. developed the soluble ArM evolution procedure. C.Z. optimized the soluble ArM evolution procedure. A.M.S. and D.M.U. optimized the soluble ArM evolution procedure and collected conversion and selectivity data for all evolved ArMs. H.J.P. optimized and executed the immobilized ArM evolution procedure. K.B. and Y.G. cloned POP amber mutants and the combinatorial codon mutagenesis library. K.E.-G., G.L. and R.E.M. conducted and analysed the LC-MS/MS experiments. J.C.L. devised the experiments and procedures, designed the ArM variants and libraries, analysed data and wrote the manuscript.

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Correspondence to Jared C. Lewis.

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Yang, H., Swartz, A., Park, H. et al. Evolving artificial metalloenzymes via random mutagenesis. Nature Chem 10, 318–324 (2018). https://doi.org/10.1038/nchem.2927

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