Substrate binding and cleavage mechanism of rubber oxygenase RoxA

本课题旨在研究橡胶加氧酶RoxA对天然橡胶（聚异戊二烯）的生化裂解机理。这将通过(i)鉴定参与双氧结合的RoxA活性位点的催化重要残基，（ii）鉴定负责聚合物（疏水）底物与RoxA特异性结合的残基，以及（iii）测定底物、底物类似物或抑制剂结合时RoxA分子的变化（UVvis光谱、EPR信号、产物模式、3D结构）来完成。我们将构建、纯化一组RoxA突变蛋白，并对其进行生物化学和生物物理表征，并在与自制备的底物类似物共结晶后确定RoxA和RoxA Phe317Tyr突变蛋白的结构。结合我们已经资助的关于乳胶清除蛋白（Lcp）功能的项目，我们希望比较RoxA和Lcp的生化裂解机制，以了解大自然如何为相同的生化任务开发两种不同的解决方案，即RoxA和Lcp对天然橡胶的氧化裂解。

The aim of this proposal is to determine the biochemical cleavage mechanism of natural rubber (polyisoprene) by rubber oxygenase RoxA. This will be done by (i) identification of catalytically important residues of the active site of RoxA that participate in dioxygen binding, (ii) by identification of residues that are responsible for specific binding of the polymeric (hydrophobic) substrate to RoxA and by (iii) determination of changes of the RoxA molecule upon binding of substrate, substrate analogs or inhibitors (UVvis spectroscopy, EPR signals, product pattern, 3D structure). We will construct, purify and biochemically and biophysically characterize a set of RoxA muteins and will determine the structures of RoxA and RoxA Phe317Tyr mutein after co-crystallization with self-prepared substrate analogs. Together with our already funded project on the function of latex clearing protein (Lcp) we want to compare the biochemical cleavage mechanisms of RoxA and Lcp in order to understand how nature developed two different solutions for the same biochemical task, namely the oxidative cleavage of natural rubber by RoxA and Lcp.