The ability of unevolved amino acid sequences to become biological catalysts was key to the emergence of life on Earth. However, billions of years of evolution separate complex modern enzymes from their simpler early ancestors. To probe how unevolved sequences can develop new functions, we use ultrahigh-throughput droplet microfluidics to screen for phosphoesterase activity amidst a library of more than one million sequences based on a de novo designed 4-helix bundle. Characterization of hits revealed that acquisition of function involved a large jump in sequence space enriching for truncations that removed >40% of the protein chain. Biophysical characterization of a catalytically active truncated protein revealed that it dimerizes into an α-helical structure, with the gain of function accompanied by increased structural dynamics. The identified phosphodiesterase is a manganese-dependent metalloenzyme that hydrolyses a range of phosphodiesters. It is most active towards cyclic AMP, with a rate acceleration of ~10⁹ and a catalytic proficiency of >10¹⁴ M⁻¹, comparable to larger enzymes shaped by billions of years of evolution.

未进化的氨基酸序列成为生物催化剂的能力是地球上生命出现的关键。然而，数十亿年的进化将复杂的现代酶与其简单的早期祖先分开。为了探究未进化的序列如何开发新功能，我们使用超高通量液滴微流体在基于从头设计的 4 螺旋束的超过一百万个序列的文库中筛选磷酸酯酶活性。命中的表征表明，功能的获取涉及序列空间的大幅跳跃，富集截断，去除了> 40%的蛋白质链。催化活性截短蛋白的生物物理表征表明，它二聚成α螺旋结构，功能的获得伴随着结构动力学的增强。鉴定出的磷酸二酯酶是一种锰依赖性金属酶，可水解一系列磷酸二酯。它对环 AMP 最活跃，速率加速度约为 10 ⁹，催化效率 >10 ¹⁴  M ^-1，可与数十亿年进化形成的较大酶相媲美。