Characterization of the Fast and Promiscuous Macrocyclase from Plant PCY1 Enables the Use of Simple Substrates.,ACS Chemical Biology

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Characterization of the Fast and Promiscuous Macrocyclase from Plant PCY1 Enables the Use of Simple Substrates.
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2018-02-12 , DOI: 10.1021/acschembio.8b00050
Hannes Ludewig ₁ , Clarissa M Czekster ₁ , Emilia Oueis ₁ , Elizabeth S Munday ₂ , Mohammed Arshad ₃ , Silvia A Synowsky ₁ , Andrew F Bent ₁ , James H Naismith _{1,

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Affiliation

Cyclic ribosomally derived peptides possess diverse bioactivities and are currently of major interest in drug development. However, it can be chemically challenging to synthesize these molecules, hindering the diversification and testing of cyclic peptide leads. Enzymes used in vitro offer a solution to this; however peptide macrocyclization remains the bottleneck. PCY1, involved in the biosynthesis of plant orbitides, belongs to the class of prolyl oligopeptidases and natively displays substrate promiscuity. PCY1 is a promising candidate for in vitro utilization, but its substrates require an 11 to 16 residue C-terminal recognition tail. We have characterized PCY1 both kinetically and structurally with multiple substrate complexes revealing the molecular basis of recognition and catalysis. Using these insights, we have identified a three residue C-terminal extension that replaces the natural recognition tail permitting PCY1 to operate on synthetic substrates. We demonstrate that PCY1 can macrocyclize a variety of substrates with this short tail, including unnatural amino acids and nonamino acids, highlighting PCY1's potential in biocatalysis.

中文翻译：

通过对植物PCY1中快速混杂环氧化物酶的表征，可以使用简单的底物。

环状核糖体衍生的肽具有多种生物活性，目前在药物开发中引起了人们的极大兴趣。然而，合成这些分子可能在化学上具有挑战性，从而阻碍了环肽引线的多样化和测试。体外使用的酶为此提供了解决方案。然而，肽的大环化仍然是瓶颈。参与植物Orbitides生物合成的PCY1属于脯氨酰寡肽酶类，天然显示底物混杂。PCY1是体外应用的有希望的候选者，但其底物需要11至16个残基的C末端识别尾巴。我们已经表征了PCY1在动力学和结构上具有多种底物配合物，揭示了识别和催化的分子基础。利用这些见解，我们确定了三个残基的C末端延伸，该延伸取代了自然识别尾巴，从而使PCY1可以在合成底物上运行。我们证明了PCY1可以通过这种短尾巴使各种底物发生大环化，包括非天然氨基酸和非氨基酸，从而突出了PCY1在生物催化中的潜力。

更新日期：2018-01-29

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