Selection of a Promiscuous Minimalist cAMP Phosphodiesterase from a Library of De Novo Designed Proteins
Selection of a Promiscuous Minimalist cAMP Phosphodiesterase from a Library of De Novo Designed Proteins
复制标题
DOI：
10.1101/2023.02.13.528392
复制
发表时间：
2023-02
期刊：
影响因子：
--
通讯作者：
J. Schnettler;Michael S. Wang;Maximilian Gantz;Christina Karas;F. Hollfelder;M. Hecht
中科院分区：
文献类型：
--
作者：
J. Schnettler;Michael S. Wang;Maximilian Gantz;Christina Karas;F. Hollfelder;M. Hecht
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。为了研究未进化的序列如何开发新功能，我们基于一个从头开始的4-螺旋半随机序列束库，筛选了bb100万个新序列的酶活性。为了反映生物功能的进化选择，我们使用超高通量液滴微流体筛选收集物，以确定产生磷酸酯酶活性的特征。活性命中的表征表明，获得新功能需要在序列空间上有很大的跳跃：筛选富集了去除bb0 - 40%蛋白链并引入重要催化半胱氨酸的截断。截断的蛋白二聚成一个动态的α-螺旋结构，这与功能的获得伴随着相对于亲本4-螺旋束的结构动力学的增加的观点一致。纯化后的蛋白能催化一系列磷酸二酯的水解，对生物第二信使环AMP （cAMP）的活性最大。这种新型的cAMP酶是一种依赖锰的金属酶，其催化cAMP水解的速率为109，催化效率为1014 M−1，可与经过数十亿年进化形成的大型酶相媲美。这些发现表明，分裂成模块化的原始肽可能是将结构和功能多样性引入蛋白质的肥沃途径。图形抽象
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 study how unevolved sequences can develop new functions, we screened for enzymatic activity in a collection of > 1 million novel sequences based on a de novo 4-helix bundle library of semi-random sequences. To mirror evolutionary selection for biological function, we screened the collection using ultrahigh-throughput droplet microfluidics to identify features that yield phosphoesterase activity. Characterization of active hits demonstrated that acquiring new function required a large jump in sequence space: screening enriched for truncations that removed > 40% of the protein chain and introduced a catalytically important cysteine. The truncated protein dimerized into a dynamic α-helical structure, consistent with the idea that gain of function was accompanied by an increase in structural dynamics relative to the parental 4-helix bundle. The purified protein catalyzes the hydrolysis of a range of phosphodiesters, with the greatest activity toward the biological second messenger cyclic AMP (cAMP). The novel cAMPase is a manganese-dependent metalloenzyme and catalyzes cAMP hydrolysis with a rate acceleration on the order of 109 and catalytic proficiency on the order of 1014 M−1, comparable to large enzymes shaped by billions of years of evolution. These findings suggest that fragmentation to modular primordial peptides can be a fertile avenue for introducing structural and functional diversity into proteins. GRAPHICAL ABSTRACT