循环酶域的冷电磁结构和双功能II类合成酶中的基质道的评估
Matthew N Gaynes1, Kollin Schultz2,3, Eliott S Wenger1
1Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania, 19104-6323, USA.
bioRxiv : the preprint server for biology
|September 2, 2025
概括
我们确定了真菌二酸合成酶 (PvCPS) 的冷EM结构,揭示了实验室核形成和明显的催化机制的保存残留物. 此外,它还证明了其前基转移酶和循环酶域之间的基质道.
科学领域:
- 生物化学和分子生物学
- 结构生物学
- 自然产品生物合成
背景情况:
- 二酸合成酶 (CPS) 是与labdane相关的天然产品的生物合成中的关键酶,具有重要的药物和环境相关性.
- 了解二级二烯合成酶的结构功能关系,特别是真菌酶,对于探索新生物活性化合物至关重要.
- 具有前基转移酶和循环酶活动的双功能CPS尚未在结构上进行表征,特别是在基质道方面.
研究的目的:
- 通过高分辨率的冷电子显微镜 (cryo-EM) 来确定来自Penicillium verruculosum* (PvCPS) 的二酸二酶类域.
- 通过比较PvCPS与已知的细菌和植物正方形来研究真菌CPS的结构功能关系.
- 阐明双功能PvCPS中prenyltransferase和cyclase域之间的基质道化机制.
主要方法:
- 以2.9 Å分辨率对PvCPS的63kDII类循环酶域进行净化和冷电磁结构的确定.
- 对PvCPS与来自细菌和植物的同类酶进行比较的结构分析.
- 基质竞争实验,以评估 prenyltransferase 和 cyclase 域之间的相互作用和基质转移.
主要成果:
- 与其他CPS相比,冷-EM结构显示了对形成双循环实验室核至关重要的保存残留物,以及参与最终脱质化步骤的分离催化残留物.
- 结构比较突出了二烯循环的保存动机,但也突出了真菌酶的独特特征.
- 基质竞争测试表明,即使在单独的结构中,也可以从前转移酶转移基酸盐 (GGPP),从而支持高效的基质道化.
结论:
- PvCPS环酶域的结构和功能特征为真菌II类二烯合成酶的演变和机制提供了洞察力.
- 这些发现表明,前转移酶和循环酶域之间的短暂关联促进了基质通道,优化了酸的产生.
- 这项研究为蛋白质工程的努力奠定了基础,以产生具有潜在治疗应用的新型实验室基架.
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