Structure and Mechanism of Myo-Inositol Oxygenase

描述（由申请人提供）：肌醇加氧酶（MIOX）在人类已知的唯一途径中催化肌醇（Ml）分解的第一步，肌醇是细胞信号磷酸肌醇的糖主干。有证据表明，MIOX的表达或活性增加可能与糖尿病相关的病理有关，这标志着MIOX是一个潜在的药物靶点。我们的初步数据表明，MIOX反应通过一种前所未有的化学机制进行。非血红素二铁簇在其混合价（ll /ll）氧化态下，可能与底物的一个或多个氧原子配位，与分子氧反应生成形式二铁(ll /ll)-超氧化物复合物，从底物中提取氢。我们试图通过生物物理和生化方法确定：(1)这种独特的酶及其双铁辅因子的结构；(2)蛋白质如何促进活性混合价形式的初始形成和稳定性（这在许多其他非血红素二铁蛋白中是不稳定的）；(3)底物如何与蛋白质和辅因子相互作用；(4)这种相互作用是否以及如何激活辅因子或底物（或两者）以与O2进行后续反应；(5)每个化学步骤的性质和速率，从酶*底物复合物的氧加入到产物的释放，以及我们已经发现的两个活性中间体的结构，以及在这个序列中还有待发现的其他中间体。通过这些研究，我们希望为合理设计抗糖尿病并发症的MIOX抑制剂提供必要的工具。

DESCRIPTION (provided by applicant): Myo-inositol oxygenase (MIOX) catalyzes the first step in the only known pathway in humans for breakdown of myo-inositol (Ml), the sugar backbone of cell-signaling phosphoinositides. Evidence suggests that increased expression or activity of MIOX may contribute to pathologies commonly associated with diabetes mellitus, marking MIOX as a potential drug target. Our preliminary data show that the MIOX reaction proceeds via an unprecedented chemical mechanism. A non-heme diiron cluster in its mixed-valent, ll/lll, oxidation state, which probably coordinates one or more oxygen atoms of the substrate, reacts with molecular oxygen to generate a formally diiron(lll/lll)-superoxide complex, which abstracts hydrogen from the substrate. We seek to determine by biophysical and biochemical methods: (1) the structure of this unique enzyme and its diiron cofactor; (2) how the protein promotes initial formation and stability of the active, mixed-valent form (which is unstable in many other non-heme diiron proteins); (3) how the substrate interacts with the protein and cofactor; (4) whether and how this interaction activates the cofactor or substrate (or both) for subsequent reaction with O2; and (5) the nature and rate of each chemical step leading from addition of oxygen to the enzyme*substrate complex through release of the product along with the structures of two reactive intermediates that we have already discovered and additional intermediates that remain to be discovered in this sequence. By so doing, we hope to provide the necessary tools for rational design of MIOX inhibitors that could be useful in combating complications from diabetes mellitus.