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Structural basis for proenzyme maturation, substrate recognition, and ligation by a hyperactive peptide asparaginyl ligase.
The Plant Cell ( IF 11.6 ) Pub Date : 2022-11-29 , DOI: 10.1093/plcell/koac281
Side Hu 1, 2 , Abbas El Sahili 1, 2 , Srujana Kishore 1, 2 , Yee Hwa Wong 1, 2 , Xinya Hemu 1 , Boon Chong Goh 2, 3 , Sang Zhipei 1 , Zhen Wang 1 , James P Tam 1 , Chuan-Fa Liu 1 , Julien Lescar 1, 2
Affiliation  

Peptide ligases are versatile enzymes that can be utilized for precise protein conjugation for bioengineering applications. Hyperactive peptide asparaginyl ligases (PALs), such as butelase-1, belong to a small class of enzymes from cyclotide-producing plants that can perform site-specific, rapid ligation reactions after a target peptide asparagine/aspartic acid (Asx) residue binds to the active site of the ligase. How PALs specifically recognize their polypeptide substrates has remained elusive, especially at the prime binding side of the enzyme. Here we report crystal structures that capture VyPAL2, a catalytically efficient PAL from Viola yedoensis, in an activated state, with and without a bound substrate. The bound structure shows one ligase with the N-terminal polypeptide tail from another ligase molecule trapped at its active site, revealing how Asx inserts in the enzyme's S1 pocket and why a hydrophobic residue is required at the P2' position. Besides illustrating the anchoring role played by P1 and P2' residues, these results uncover a role for the Gatekeeper residue at the surface of the S2 pocket in shifting the nonprime portion of the substrate and, as a result, the activity toward ligation or hydrolysis. These results suggest a picture for proenzyme maturation in the vacuole and will inform the rational design of peptide ligases with tailored specificities.

中文翻译:

酶原成熟、底物识别和高活性肽天冬酰胺酰连接酶连接的结构基础。

肽连接酶是多功能酶,可用于生物工程应用中的精确蛋白质缀合。高活性肽天冬酰胺酰连接酶 (PAL),例如 Butelase-1,属于来自产生环肽的植物的一小类酶,可在目标肽天冬酰胺/天冬氨酸 (Asx) 残基结合后进行位点特异性的快速连接反应。连接酶的活性位点。PAL 如何特异性识别其多肽底物仍然难以捉摸,特别是在酶的主要结合侧。在这里,我们报告了捕获 VyPAL2 的晶体结构,VyPAL2 是一种来自紫花地丁的催化高效 PAL,处于激活状态,有或没有结合底物。结合结构显示一种连接酶的活性位点处捕获了另一种连接酶分子的 N 端多肽尾部,揭示了 Asx 如何插入酶的 S1 袋中以及为什么 P2' 位置需要疏水残基。除了说明 P1 和 P2' 残基发挥的锚定作用外,这些结果还揭示了 S2 口袋表面的 Gatekeeper 残基在移动底物非引物部分中的作用,从而导致连接或水解活性。这些结果揭示了液泡中酶原成熟的情况,并将为具有定制特异性的肽连接酶的合理设计提供信息。
更新日期:2022-09-13
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