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Emergence of a catalytic tetrad during evolution of a highly active artificial aldolase

Abstract

Designing catalysts that achieve the rates and selectivities of natural enzymes is a long-standing goal in protein chemistry. Here, we show that an ultrahigh-throughput droplet-based microfluidic screening platform can be used to improve a previously optimized artificial aldolase by an additional factor of 30 to give a >109 rate enhancement that rivals the efficiency of class I aldolases. The resulting enzyme catalyses a reversible aldol reaction with high stereoselectivity and tolerates a broad range of substrates. Biochemical and structural studies show that catalysis depends on a Lys-Tyr-Asn-Tyr tetrad that emerged adjacent to a computationally designed hydrophobic pocket during directed evolution. This constellation of residues is poised to activate the substrate by Schiff base formation, promote mechanistically important proton transfers and stabilize multiple transition states along a complex reaction coordinate. The emergence of such a sophisticated catalytic centre shows that there is nothing magical about the catalytic activities or mechanisms of naturally occurring enzymes, or the evolutionary process that gave rise to them.

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Figure 1: Substrates and inhibitor for the computationally designed aldolase.
Figure 2: Microfluidic platform for the directed evolution of aldolases.
Figure 3: Evolution and structural analysis of the optimized RA95.5-8F aldolase.
Figure 4: Hypothetical mechanism for aldol cleavage by the evolved enzyme.

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Acknowledgements

We thank C. Stutz-Ducommun and B. Blattmann from the Protein Crystallization Core Facility at the University of Zurich and the staff at the Swiss Light Source (Paul Scherrer Institute) for outstanding technical support, as well as E. Mayot for surfactant synthesis. We are grateful to the mass spectrometry service of the Laboratory of Organic Chemistry at ETHZ and the Protein Analysis Group at the Functional Genomics Center of the University of Zurich. We thank A. Green, R. Zschoche, M. Reichen, R. Calbrix and all the members of the Hilvert and Griffiths labs for fruitful discussions. This work was generously supported by the Swiss National Science Foundation (D.H.).

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Contributions

D.H., A.D.G., D.B., P.R.E.M., X.G., A.G. and R.O. designed the experiments. R.O. and A.G. developed the assay and evolved retro-aldolases. R.O. and X.G. biochemically characterized the variants. X.G. performed biocatalytic aldol reactions and the synthesis of standards. P.R.E.M. and R.O. crystallized the proteins and solved their structures. The manuscript and figures were prepared by R.O., X.G., A.D.G. and D.H.

Corresponding authors

Correspondence to Andrew D. Griffiths or Donald Hilvert.

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Obexer, R., Godina, A., Garrabou, X. et al. Emergence of a catalytic tetrad during evolution of a highly active artificial aldolase. Nature Chem 9, 50–56 (2017). https://doi.org/10.1038/nchem.2596

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