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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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.
Vendruscolo, M. & Dobson, C. M. Structural biology: dynamic visions of enzymatic reactions. Science 313, 1586–1587 (2006).
DeGrado, W., Wasserman, Z. & Lear, J. Protein design, a minimalist approach. Science 243, 622–628 (1989).
Benkovic, S. J. & Hammes-Schiffer, S. A perspective on enzyme catalysis. Science 301, 1196–1202 (2003).
Aldridge, S. Industry backs biocatalysis for greener manufacturing. Nat. Biotechnol. 31, 95–96 (2013).
Schmid, A. et al. Industrial biocatalysis today and tomorrow. Nature 409, 258–268 (2001).
Arnold, F. H. Combinatorial and computational challenges for biocatalyst design. Nature 409, 253–257 (2001).
Christianson, D. W. & Fierke, C. A. Carbonic anhydrase: evolution of the zinc binding site by nature and by design. Acc. Chem. Res. 29, 331–339 (1996).
Boone, C., Habibzadegan, A., Gill, S. & McKenna, R. Carbonic anhydrases and their biotechnological applications. Biomolecules 3, 553–562 (2013).
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).
Rufo, C. M. et al. Short peptides self-assemble to produce catalytic amyloids. Nat. Chem. 6, 303–309 (2014).
Guler, M. O. & Stupp, S. I. A self-assembled nanofiber catalyst for ester hydrolysis. J. Am. Chem. Soc. 129, 12082–12083 (2007).
Friedmann, M. P. et al. Towards prebiotic catalytic amyloids using high throughput screening. PLoS ONE 10, e0143948 (2015).
Zhang, C. et al. Switchable hydrolase based on reversible formation of supramolecular catalytic site using a self-assembling peptide. Angew. Chem. Int. Ed. Engl. 56, 14511–14515 (2017).
Singh, N., Conte, M. P., Ulijn, R. V., Miravet, J. F. & Escuder, B. Insight into the esterase like activity demonstrated by an imidazole appended self-assembling hydrogelator. Chem. Commun. 51, 13213–13216 (2015).
Huang, Z. et al. Self-assembly of amphiphilic peptides into bio-functionalized nanotubes: a novel hydrolase model. J. Mater. Chem. B 1, 2297–2304 (2013).
Zhang, C. et al. Self-assembled peptide nanofibers designed as biological enzymes for catalyzing ester hydrolysis. ACS Nano 8, 11715–11723 (2014).
Al-Garawi, Z. S. et al. The amyloid architecture provides a scaffold for enzyme-like catalysts. Nanoscale 9, 10773–10783 (2017).
Lee, M. et al. Zinc-binding structure of a catalytic amyloid from solid-state NMR. Proc. Natl Acad. Sci. USA 114, 6191–6196 (2017).
Lengyel, Z., Rufo, C. M., Moroz, Y. S., Makhlynets, O. V. & Korendovych, I. V. Copper-containing catalytic amyloids promote phosphoester hydrolysis and tandem reactions. ACS Catal. 8, 59–62 (2018).
Gazit, E. Self-assembled peptide nanostructures: the design of molecular building blocks and their technological utilization. Chem. Soc. Rev. 36, 1263–1269 (2007).
Tao, K., Makam, P., Aizen, R. & Gazit, E. Self-assembling peptide semiconductors. Science 358, eaam9756 (2017).
Makam, P. & Gazit, E. Minimalistic peptide supramolecular co-assembly: expanding the conformational space for nanotechnology. Chem. Soc. Rev. 47, 3406–3420 (2018).
Adler-Abramovich, L. et al. Phenylalanine assembly into toxic fibrils suggests amyloid etiology in phenylketonuria. Nat. Chem. Biol. 8, 701–706 (2012).
Mossou, E. et al. The self-assembling zwitterionic form of l-phenylalanine at neutral pH. Acta Crystallogr. C 70, 326–331 (2014).
Shaham-Niv, S., Adler-Abramovich, L., Schnaider, L. & Gazit, E. Extension of the generic amyloid hypothesis to nonproteinaceous metabolite assemblies. Sci. Adv. 1, e1500137 (2015).
Aizen, R., Tao, K., Rencus-Lazar, S. & Gazit, E. Functional metabolite assemblies—a review. J. Nanopart. Res. 20, 125 (2018).
Eisenberg, D. & Jucker, M. The amyloid state of proteins in human diseases. Cell 148, 1188–1203 (2012).
Carny, O. & Gazit, E. A model for the role of short self-assembled peptides in the very early stages of the origin of life. FASEB J. 19, 1051–1055 (2005).
Rout, S. K., Friedmann, M. P., Riek, R. & Greenwald, J. A prebiotic template-directed peptide synthesis based on amyloids. Nat. Commun. 9, 234 (2018).
Greenwald, J. & Riek, R. On the possible amyloid origin of protein folds. J. Mol. Biol. 421, 417–426 (2012).
Omosun, T. O. et al. Catalytic diversity in self-propagating peptide assemblies. Nat. Chem. 9, 805–809 (2017).
Zozulia, O., Dolan, M. A. & Korendovych, I. V. Catalytic peptide assemblies. Chem. Soc. Rev. 47, 3621–3639 (2018).
Singh, N., Kumar, M., Miravet, J. F., Ulijn, R. V. & Escuder, B. Peptide-based molecular hydrogels as supramolecular protein mimics. Chem. Eur. J. 23, 981–993 (2017).
Greenwald, J., Kwiatkowski, W. & Riek, R. Peptide amyloids in the origin of life. J. Mol. Biol. 430, 3735–3750 (2018).
Elius Hossain, M., Mahmudul Hasan, M., Halim, M. E., Ehsan, M. Q. & Halim, M. A. Interaction between transition metals and phenylalanine: a combined experimental and computational study. Spectrochim. Acta A 138, 499–508 (2015).
Makin, O. S., Atkins, E., Sikorski, P., Johansson, J. & Serpell, L. C. Molecular basis for amyloid fibril formation and stability. Proc. Natl Acad. Sci. USA 102, 315–320 (2005).
Riek, R. & Eisenberg, D. S. The activities of amyloids from a structural perspective. Nature 539, 227–235 (2016).
Konar, S. et al. Structural determination and characterization of copper and zinc bis-glycinates with X-ray crystallography and mass spectrometry. J. Coord. Chem. 63, 3335–3347 (2010).
Song, R. et al. Principles governing catalytic activity of self-assembled short peptides. J. Am. Chem. Soc. 141, 223–231 (2019).
Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).
Sheldrick, G. M. SHELXT – integrated space-group and crystal-structure determination. Acta Crystallogr. A 71, 3–8 (2015).
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).
Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).
Verpoorte, J. A., Mehta, S. & Edsall, J. T. Esterase activities of human carbonic anhydrases B and C. J. Biol. Chem. 242, 4221–4229 (1967).
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.
The authors declare no competing interests.
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Supplementary methods, Tables 1–3, Figs. 1–17, discussion and references
Atomic coordinates of the optimized computational models.
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.
Esterase activity. Real-time monitoring of pNPA hydrolysis in the presence and absence of F–Zn(ii) catalyst.
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.
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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