Structural and Biochemical Characterization of a New Phage-Encoded Muramidase, KTN6 Gp46.

PHAGE (New Rochelle, N.Y.) Pub Date : 2024-06-21 eCollection Date: 2024-06-01 DOI:10.1089/phage.2023.0040

Marta Sanz-Gaitero, Vincent De Maesschalck, Ankur Patel, Hannelore Longin, Vera Van Noort, Lorena Rodriguez-Rubio, Michael van Ryne, Katarzyna Danis-Wlodarczyk, Zuzanna Drulis-Kawa, Stephane Mesnage, Mark van Raaij, Rob Lavigne

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Abstract

Background: Endolysins are phage-encoded lytic enzymes that degrade bacterial peptidoglycan at the end of phage lytic cycles to release new phage particles. These enzymes are being explored as an alternative to small-molecule antibiotics.

Methods: The crystal structure of KTN6 Gp46 was determined and compared with a ColabFold model. Cleavage specificity was examined using a peptidoglycan digest and reversed-phase high-performance liquid chromatography coupled to mass spectrometry (HPLC/MS).

Results: The structure of KTN6 Gp46 could be determined at 1.4 Å resolution, and key differences in loops of the putative peptidoglycan binding domain were identified in comparison with its closest known homologue, the endolysin of phage SPN1S. Reversed-phase HPLC/MS analysis of the reaction products following peptidoglycan digestion confirmed the muramidase activity of Gp46, consistent with structural predictions.

Conclusion: These insights into the structure and function of endolysins further expand the toolbox for endolysin engineering and explore their potential in enzyme-based antibacterial design strategies.

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一种新的噬菌体编码喃喃苷酶 KTN6 Gp46 的结构和生化特征。

背景：内溶酶是噬菌体编码的溶解酶，可在噬菌体溶解周期结束时降解细菌的肽聚糖，释放出新的噬菌体颗粒。这些酶正被探索用作小分子抗生素的替代品：方法：测定了 KTN6 Gp46 的晶体结构，并与 ColabFold 模型进行了比较。方法：测定了 KTN6 Gp46 的晶体结构，并将其与 ColabFold 模型进行了比较；使用肽聚糖消化法和反相高效液相色谱-质谱联用技术（HPLC/MS）检测了裂解特异性：结果：以1.4埃的分辨率确定了KTN6 Gp46的结构，并确定了假定肽聚糖结合域环路与其最接近的已知同源物--噬菌体SPN1S的内溶菌素--的关键差异。肽聚糖消化后反应产物的反相 HPLC/MS 分析证实了 Gp46 的喃喃苷酶活性，这与结构预测一致：这些对内溶菌素结构和功能的见解进一步扩大了内溶菌素工程的工具箱，并探索了它们在基于酶的抗菌设计策略中的潜力。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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PHAGE (New Rochelle, N.Y.)

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