Non-proteinaceous hydrolase comprised of a phenylalanine metallo-supramolecular amyloid-like structure

Abstract

Enzymatic activity is crucial for various technological applications, yet the complex structures and limited stability of enzymes often hinder their use. Hence, de novo design of robust biocatalysts that are much simpler than their natural counterparts and possess enhanced catalytic activity has long been a goal in biotechnology. Here, we present evidence for the ability of a single amino acid to self-assemble into a potent and stable catalytic structural entity. Spontaneously, phenylalanine (F) molecules coordinate with zinc ions to form a robust, layered, supramolecular amyloid-like ordered architecture (F–Zn(ii)) and exhibit remarkable carbonic anhydrase-like catalytic activity. Notably, amongst the reported artificial biomolecular hydrolases, F–Zn(ii) displays the lowest molecular mass and highest catalytic efficiency, in addition to reusability, thermal stability, substrate specificity, stereoselectivity and rapid catalytic CO2 hydration ability. Thus, this report provides a rational path towards future de novo design of minimalistic biocatalysts for biotechnological and industrial applications.

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Fig. 1: Design of a minimalistic F–Zn(ii) biocatalyst through bioinspiration from structural insights of CA II.
Fig. 2: Characterization of F–Zn(ii).
Fig. 3: Catalytic esterase activity of F–Zn(ii).
Fig. 4: F–Zn(ii) catalytic esterase reaction mechanism and the chemical structures along the reaction pathway.
Fig. 5: F–Zn(ii) catalytic carbon dioxide hydration and sequestration.

Data availability

The X-ray crystallographic coordinates for the structure reported in this study have been deposited at the Cambridge Crystallographic Data Centre (CCDC), under deposition 1850564. Other data that support the plots within this paper and other finding of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was partially supported by a grant from the European Research Council under the European Union’s Horizon 2020 research and innovation programme (BISON, Advanced ERC grant, no. 694426) (to E.G.). P.M. gratefully acknowledges the Center for Nanoscience and Nanotechnology of Tel Aviv University for a postdoctoral fellowship, C. M. Dobson, University of Cambridge, and B. Rosen, Tel Aviv University, for stimulating discussions. S.S.R.K.C.Y. and B.M.W. acknowledge the support of the US Army Research Office under grant no. W911NF-17-1-0340 and the National Science Foundation for the use of supercomputing resources through the Extreme Science and Engineering Discovery Environment (XSEDE), project no. TG-ENG160024. D.S.E and M.R.S. acknowledge the Northeastern Collaborative Access Team beamline 24-ID-C, which is funded by the National Institute of General Medical Sciences from the National Institutes of Health (grant no. P41 GM103403) and uses resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility, operated under contract no. DE-AC02-06CH11357. The Pilatus 6M detector is funded by an NIH-ORIP HEI grant (no. S10 RR029205). We also thank S. Rencus-Lazar for linguistic editing and all the members of the Gazit laboratories for helpful discussions.

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Authors

Contributions

P.M. and E.G. conceived and designed the experiments. P.M. grew the single crystals of catalytic complex and performed all experiments. P.M. and K.T. conducted the Fourier transform infrared and scanning electron microscopy measurements. D.S.E., M.R.S. and L.J.W.S. collected the single-crystal diffraction data and solved the crystal structure. S.S.R.K.C.Y. and B.M.W. performed the computational studies. P.M. and E.G. wrote and edited the manuscript. All authors discussed and commented on the manuscript.

Corresponding author

Correspondence to Ehud Gazit.

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The authors declare no competing interests.

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Supplementary information

41929_2019_348_MOESM3_ESM.avi

F–Zn(ii) crystallization. In-situ optical microscopy observation of phenylalanine-coordinated zinc ions (F–Zn(ii)) crystallization kinetics. The video was recorded at every 1-s interval.

41929_2019_348_MOESM4_ESM.mp4

Esterase activity. Real-time monitoring of pNPA hydrolysis in the presence and absence of F–Zn(ii) catalyst.

41929_2019_348_MOESM5_ESM.avi

In-situ esterase activity. In-situ optical microscopy experiment describing the effective esterase activity of F–Zn(ii) crystals in water. The video was recorded at every 5-s interval. The change in reaction solution colour with time indicating the formation of chromogenic hydrolysed product pNP.

Supplementary Information

Supplementary methods, Tables 1–3, Figs. 1–17, discussion and references

Supplementary Dataset 1

Atomic coordinates of the optimized computational models.

Supplementary Video 1

F–Zn(ii) crystallization. In-situ optical microscopy observation of phenylalanine-coordinated zinc ions (F–Zn(ii)) crystallization kinetics. The video was recorded at every 1-s interval.

Supplementary Video 2

Esterase activity. Real-time monitoring of pNPA hydrolysis in the presence and absence of F–Zn(ii) catalyst.

Supplementary Video 3

In-situ esterase activity. In-situ optical microscopy experiment describing the effective esterase activity of F–Zn(ii) crystals in water. The video was recorded at every 5-s interval. The change in reaction solution colour with time indicating the formation of chromogenic hydrolysed product pNP.

Compound F-Zn(II)

Crystallographic data of compound F-Zn(ii).

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Makam, P., Yamijala, S.S.R.K.C., Tao, K. et al. Non-proteinaceous hydrolase comprised of a phenylalanine metallo-supramolecular amyloid-like structure. Nat Catal 2, 977–985 (2019). https://doi.org/10.1038/s41929-019-0348-x

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