Substrate binding and cleavage mechanism of rubber oxygenase RoxA

橡胶加氧酶RoxA的底物结合和裂解机制

• 批准号: 281987579
• 负责人: Professor Dr. Dieter Jendrossek
• 金额: --
• 依托单位: Institut für Mikrobiologie (IMB)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/281987579?language=en
• 关键词: Substrate; binding; cleavage; mechanism; rubber

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.

本提案的目的是确定橡胶加氧酶RoxA对天然橡胶（聚异戊二烯）的生物化学裂解机理。这将通过（i）鉴定参与分子氧结合的RoxA活性位点的催化重要残基，（ii）鉴定负责聚合物特异性结合的残基，（iii）鉴定与分子氧结合的活性位点的催化重要残基，（iv）鉴定与分子氧结合的活性位点的催化重要残基，（iv）鉴定与分子氧结合的活性位点的催化重要残基。通过（iii）测定底物、底物类似物或抑制剂结合后RoxA分子的变化，（紫外可见光谱、EPR信号、产物图案、3D结构）。我们将构建，纯化和生化和生物药理学特性的RoxA突变蛋白，并确定RoxA和RoxA Phe317Tyr突变蛋白的结构后，与自制的底物类似物共结晶。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，以了解自然界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。

项目成果

RoxB Is a Novel Type of Rubber Oxygenase That Combines Properties of Rubber Oxygenase RoxA and Latex Clearing Protein (Lcp)

• DOI: 10.1128/aem.00721-17
• 发表时间: 2017-07-01
• 期刊: APPLIED AND ENVIRONMENTAL MICROBIOLOGY
• 影响因子: 4.4
• 作者: Birke, Jakob;Roether, Wolf;Jendrossek, Dieter
• 通讯作者: Jendrossek, Dieter

Rhizobacter gummiphilus NS21 has two rubber oxygenases (RoxA and RoxB) acting synergistically in rubber utilisation

• DOI: 10.1007/s00253-018-9341-6
• 发表时间: 2018-12-01
• 期刊: APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
• 影响因子: 5
• 作者: Birke, Jakob;Roether, Wolf;Jendrossek, Dieter
• 通讯作者: Jendrossek, Dieter