英国剑桥大学Florian Hollfelder和美国普林斯顿大学Michael H. Hecht课题组的一项最新研究，从从头设计的蛋白质库中选择出混杂的极简cAMP磷酸二酯酶。该研究于2024年5月3日发表于国际一流学术期刊《自然—化学》杂志上。

在这项研究中，为了探索未进化的序列如何开发新功能，课题组人员使用超高通量微流体，在超过一百万个基于从头设计的4-螺旋束的序列库中筛选磷酸酯酶活性。命中的蛋白质表征表明，功能的获得涉及到序列空间的一个大跳跃，截断去除超过40%的蛋白质链的序列得到富集。

对具有催化活性的截断蛋白的生物物理表征表明，其二聚体为α-螺旋结构，其功能的增强伴随着结构动力学的增强。所鉴定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，加速速率为~10⁹，催化效率为>10¹⁴ M^-1，与经过数十亿年进化形成的更大的酶相当。

据介绍，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离开来。

附：英文原文

Title: Selection of a promiscuous minimalist cAMP phosphodiesterase from a library of de novo designed proteins

Author: Schnettler, J. David, Wang, Michael S., Gantz, Maximilian, Bunzel, H. Adrian, Karas, Christina, Hollfelder, Florian, Hecht, Michael H.

Issue&Volume: 2024-05-03

Abstract: 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 >1014M1, comparable to larger enzymes shaped by billions of years of evolution.

DOI: 10.1038/s41557-024-01490-4

Source: https://www.nature.com/articles/s41557-024-01490-4