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bioRxiv · 10.64898/2026.04.23.720277

Zero-shot design of a de novo metalloenzyme

El Nesr, G.·Duerr, S. L.·Mathews, I. I.·Wen, Q.·Zhao, K.·Sarangi, R.·Roethlisberger, U.·Sunden, F.·Huang, P.
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Abstract

The de novo design of enzymes remains a central challenge, requiring consideration of catalytic mechanism and optimization across biochemical and biophysical criteria. Here we present dEVA (design by EVolutionary Algorithm), a multi-objective protein design framework built on principles drawn from evolutionary biology. We apply dEVA to the zero-shot, de novo design of metalloenzymes by optimizing the coordination sphere of catalytic metals. We characterize a bizinc metalloenzyme that exhibits promiscuous hydrolytic activity towards both phosphomonoesters and phosphodiesters. This design achieves a rate enhancement ((kcat/KM)/kw) up to 3 x 1013, comparable to characterized natural phosphatases. dEVA offers a general and modular strategy for the programmable design of protein function without dependence on natural templates or evolutionary information.

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El Nesr, G., Duerr, S. L., Mathews, I. I., Wen, Q., Zhao, K., Sarangi, R., Roethlisberger, U., Sunden, F., Huang, P.. 2026-04-24. Zero-shot design of a de novo metalloenzyme. https://doi.org/10.64898/2026.04.23.720277

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