Serial femtosecond X-ray crystallography of an anaerobically formed catalytic intermediate of copper amine oxidase
Serial femtosecond X-ray crystallography of an anaerobically formed catalytic intermediate of copper amine oxidase
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DOI：
10.1107/s2059798322010385
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发表时间：
2022-12-01
影响因子：
2.2
通讯作者：
Okajima, Toshihide
中科院分区：
文献类型：
--
作者：
Murakawa, Takeshi;Suzuki, Mamoru;Okajima, Toshihide
酶通过其结构变化有效地促进催化反应的机制仍未完全阐明。利用X射线自由电子激光(XFELs)的连续飞秒X射线结晶学(SFX)的最新进展使解决这些问题成为可能。特别是，混合注入序列结晶学(MISC)在直接观察与正在进行的酶反应相关的结构变化方面很有希望。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。在1.94埃分辨率下测定的结构表明，在厌氧单周转条件下，氨基间苯二酚和半醌自由基中间体之间的肽基辅助因子处于平衡状态。这些结果表明，在整个液体喷射SFX测量过程中，厌氧条件保持得很好，防止了催化中间体与氧气反应。这些结果也为进行时间分辨MISC研究厌氧条件下的酶反应机理提供了必要的框架。
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 angstrom 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.