Primordial sequence signatures in modern proteins imply ancestral origins tracing back to simple peptides. Although short peptides seldom adopt unique folds, metal ions might have templated their assembly into higher-order structures in early evolution and imparted useful chemical reactivity. Recapitulating such a biogenetic scenario, we have combined design and laboratory evolution to transform a zinc-binding peptide into a globular enzyme capable of accelerating ester cleavage with exacting enantiospecificity and high catalytic efficiency (kcat/KM~ 106M−1s−1). The simultaneous optimization of structure and function in a naïve peptide scaffold not only illustrates a plausible enzyme evolutionary pathway from the distant past to the present but also proffers exciting future opportunities for enzyme design and engineering.

New enzyme catalysts are usually engineered by repurposing the active sites of natural proteins. Here we show that design and directed evolution can be used to transform a nonnatural, functionally naïve zinc-binding protein into a highly active catalyst for an abiological hetero-Diels-Alder reaction. The artificial metalloenzyme achieves >10 4 turnovers per active site, exerts absolute control over reaction pathway and product stereochemistry, and displays a chemical proficiency (1/K TS = 2.9 x 10 10 M -1 ) that exceeds all previously characterised Diels-Alderases. These properties capitalise on effective Lewis acid catalysis, a chemical strategy for accelerating Diels-Alder reactions common in the lab but so far unknown in nature. Extension of this approach to other metal ions and other de novo scaffolds may propel the design field in exciting new directions.

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