Institut de Science et d'Ingénierie Supramoléculaires (ISIS), International Center for Frontier Research in Chemistry (icFRC), University of Strasbourg, CNRS (UMR 7006), Strasbourg 67000, France.
Integrated Structural Biology Platform, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS (UMR 7104), INSERM (U1258), University of Strasbourg, Illkirch 67404, France.
J Am Chem Soc. 2021 Mar 10;143(9):3330-3339. doi: 10.1021/jacs.0c10053. Epub 2021 Feb 26.
The design of catalytic proteins with functional sites capable of specific chemistry is gaining momentum and a number of artificial enzymes have recently been reported, including hydrolases, oxidoreductases, retro-aldolases, and others. Our goal is to develop a peptide ligase for robust catalysis of amide bond formation that possesses no stringent restrictions to the amino acid composition at the ligation junction. We report here the successful completion of the first step in this long-term project by building a completely de novo protein with predefined acyl transfer catalytic activity. We applied a minimalist approach to rationally design an oxyanion hole within a small cavity that contains an adjacent thiol nucleophile. The N-terminus of the α-helix with unpaired hydrogen-bond donors was exploited as a structural motif to stabilize negatively charged tetrahedral intermediates in nucleophilic addition-elimination reactions at the acyl group. Cysteine acting as a principal catalytic residue was introduced at the second residue position of the α-helix N-terminus in a designed three-α-helix protein based on structural informatics prediction. We showed that this minimal set of functional elements is sufficient for the emergence of catalytic activity in a de novo protein. Using peptide-thioesters as acyl-donors, we demonstrated their catalyzed amidation concomitant with hydrolysis and proved that the environment at the catalytic site critically influences the reaction outcome. These results represent a promising starting point for the development of efficient catalysts for protein labeling, conjugation, and peptide ligation.
设计具有能够进行特定化学催化功能位点的催化蛋白正变得越来越流行，最近已经有许多人工酶被报道，包括水解酶、氧化还原酶、逆醛缩酶等。我们的目标是开发一种肽连接酶，用于稳健地催化酰胺键形成，并且对连接点处的氨基酸组成没有严格的限制。我们在这里报告了该长期项目的第一步的成功完成，即构建了一种具有预定义酰基转移催化活性的完全从头设计的蛋白质。我们采用了一种极简主义的方法，在一个包含相邻硫醇亲核试剂的小空腔内合理设计了一个氧阴离子穴。利用未配对氢键供体的α-螺旋的 N 端作为结构模体，稳定亲核加成-消除反应中酰基的带负电荷的四面体型中间体。半胱氨酸作为主要催化残基，被引入基于结构信息学预测的设计的三个α-螺旋蛋白的α-螺旋 N 端的第二位。我们表明，这种最小的功能元件集足以使从头设计的蛋白质中出现催化活性。我们使用肽硫酯作为酰基供体，证明了它们可以催化酰胺化，同时伴随水解，并证明催化位点的环境对反应结果有重要影响。这些结果为开发用于蛋白质标记、缀合和肽连接的高效催化剂提供了一个有希望的起点。
