Ammonia monooxygenase of Nitrosomonas europaea: Structure, function and catalytic mechanism

亚硝化单胞菌在好氧或缺氧的化能无机自养条件下将氨氧化成亚硝酸盐。两种酶在这种能量产生反应中具有关键功能：氨单加氧酶（AMO），催化氨氧化为羟胺，以及羟胺氧化还原酶（HAO），将羟胺氧化为亚硝酸盐。Igarashi等人（1997）解析了羟胺氧化还原酶的2.8碱基结构。本项目的目的是从N. europaea，在E.大肠杆菌和纯化的蛋白质，建立一个协议，从两个来源的蛋白质的结晶，并解决四级结构，包括其催化中心和辅因子存在的架构。相关距离将通过X射线吸收光谱法进行验证，氧化还原状态将通过电子顺磁共振（EPR）光谱法进行鉴定。酶测定将进一步优化以表征AMO的催化活性。我们的策略，在缺氧条件下，在NO和铜的存在下，纯化酶已经导致了第一个有希望的结果，并结合异源表达的AMO蛋白，成功实施该项目的基础。最终目标是将联合收割机的结构和生物化学数据结合起来，在分子水平上建立AMO催化功能的详细模型。

Nitrosomonas europaea oxidizes ammonia to nitrite under either oxic or anoxic chemolithoautotrophic conditions. Two enzymes have key functions in this energy yielding reaction: The ammonia monooxygenase (AMO), catalyzing the oxidation of ammonia to hydroxylamine, and the hydroxylamine oxidoreductase (HAO), oxidizing hydroxylamine to nitrite. The 2.8 Å structure of the hydroxylamine oxidoreductase was resolved by Igarashi et al. (1997). The objectives of this project are to purify the AMO from N. europaea, to heterologously express the AMO in E. coli and to purify the protein, to establish a protocol for the crystallization of the protein from both sources, and to solve the quaternary structure including the architecture of its catalytic center and co-factors present. Relevant distances will be verified by X-ray absorption spectroscopy, and redox states will be identified by electron paramagnetic resonance (EPR) spectroscopy. The enzyme assay will be further optimized to characterize the catalytic activity of the AMO. Our strategy to purify the enzyme under anoxic conditions in the presence of NO and copper led already to first promising results and is, in combination with the heterologous expression of the AMO proteins, the fundament for a successful implementation of this project. The ultimate goal is to combine structural and biochemical data to develop a detailed model of the catalytic function of the AMO at the molecular level.