Serial femtosecond X-ray crystallography of an anaerobically formed catalytic intermediate of copper amine oxidase,Acta Crystallographica Section D

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Serial femtosecond X-ray crystallography of an anaerobically formed catalytic intermediate of copper amine oxidase
Acta Crystallographica Section D ( IF 2.6 ) Pub Date : 2022-11-25 , DOI: 10.1107/s2059798322010385
Takeshi Murakawa ₁ , Mamoru Suzuki ₂ , Kenji Fukui ₁ , Tetsuya Masuda ₃ , Michihiro Sugahara ₄ , Kensuke Tono ₄ , Tomoyuki Tanaka ₄ , So Iwata ₄ , Eriko Nango ₄ , Takato Yano ₁ , Katsuyuki Tanizawa ₅ , Toshihide Okajima ₅

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The mechanisms by which enzymes promote catalytic reactions efficiently through their structural changes remain to be fully elucidated. Recent progress in serial femtosecond X-ray crystallography (SFX) using X-ray free-electron lasers (XFELs) has made it possible to address these issues. In particular, mix-and-inject serial crystallography (MISC) is promising for the direct observation of structural changes associated with ongoing enzymic reactions. In this study, SFX measurements using a liquid-jet system were performed on microcrystals of bacterial copper amine oxidase anaerobically premixed with a substrate amine solution. The structure determined at 1.94 Å resolution indicated that the peptidyl quinone cofactor is in equilibrium between the aminoresorcinol and semiquinone radical intermediates, which accumulate only under anaerobic single-turnover conditions. These results show that anaerobic conditions were well maintained throughout the liquid-jet SFX measurements, preventing the catalytic intermediates from reacting with dioxygen. These results also provide a necessary framework for performing time-resolved MISC to study enzymic reaction mechanisms under anaerobic conditions.

中文翻译：

厌氧形成的铜胺氧化酶催化中间体的连续飞秒 X 射线晶体学

酶通过其结构变化有效促进催化反应的机制仍有待充分阐明。使用 X 射线自由电子激光器 (XFEL) 的串行飞秒 X 射线晶体学 (SFX) 的最新进展使解决这些问题成为可能。特别是，混合和注入系列晶体学 (MISC) 有望直接观察与正在进行的酶反应相关的结构变化。在这项研究中，使用液体喷射系统对厌氧预混合底物胺溶液的细菌铜胺氧化酶微晶进行了 SFX 测量。以 1.94 Å 分辨率确定的结构表明，肽基醌辅助因子在氨基间苯二酚和半醌自由基中间体之间处于平衡状态，仅在厌氧单周转条件下积累。这些结果表明，在整个液体喷射 SFX 测量过程中，厌氧条件得到了很好的维持，从而防止催化中间体与分子氧发生反应。这些结果还为执行时间分辨 MISC 以研究厌氧条件下的酶反应机制提供了必要的框架。

更新日期：2022-11-25

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