Structural basis for the different cleavage mechanisms of poly(1,4-cis-isoprene) by rubber oxygenases RoxA and RoxB

橡胶加氧酶 RoxA 和 RoxB 聚（1,4-顺式异戊二烯）不同裂解机制的结构基础

• 批准号: 462261492
• 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
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/462261492?language=en
• 关键词: Structural; basis; different; cleavage; mechanisms

Steroidobacter cummioxidans 35Y (previously Xanthomonas sp. 35Y) forms two related, synergistically acting, extracellular haem-containing rubber (di)oxygenases, RoxA and RoxB, during growth on rubber. RoxA cleaves rubber processively to the C15 oligoisoprenoid 12-oxo-4,8-dimethyl-tri-deca-4,8-diene-1-al (ODTD) in an exo-type fashion, and RoxB cleaves rubber to C20 and higher oligoisoprenoids randomly in an endo-type manner. The 3D structure of RoxA was used to model the structure of RoxB. While the cores of RoxA and RoxB are almost identical they differ in three major loops that form a “dome” above the active haem site. The dome must undergo a conformational change to allow access of the substrate (polyisoprene chain) to the active site haem. We postulate that the differences in the dome-loops between RoxA and RoxB are responsible for the different cleavage modes and cleavage products in RoxA and RoxB. We will address this hypothesis by (i) determination of the (true) RoxB 3D structure, (ii) by biochemical and biophysical analysis of chimeric RoxA-RoxB variants (both constructed chimers and naturally occurring RoxA/RoxB hybrids (RoxCs) and sited-directed mutated rubber oxygenase variants) and (iii) by analysis of the substrate-protein interaction with Bio Layer Interferometrie.

Steroidobacter cummioxidans 35 Y（以前称为Xanthomonas sp. 35 Y）在橡胶上生长期间形成两种相关的协同作用的细胞外含血红素橡胶（二）加氧酶RoxA和RoxB。RoxA以外向型方式将橡胶裂解为C15低聚类异戊二烯12-氧代-4，8-二甲基-十三碳-4，8-二烯-1-醛（ODTD），RoxB以内向型方式将橡胶随机裂解为C20和更高的低聚类异戊二烯。使用RoxA的3D结构来模拟RoxB的结构。虽然RoxA和RoxB的核心几乎是相同的，但它们在三个主要环中不同，这些环在活性血红素位点上方形成“圆顶”。圆顶必须经历构象变化，以允许底物（聚异戊二烯链）进入活性位点血红素。我们推测，RoxA和RoxB之间的圆顶环的差异是负责不同的切割模式和切割产物在RoxA和RoxB。我们将通过（i）确定（真实）RoxB 3D结构，（ii）通过嵌合RoxA-RoxB变体（构建的嵌合体和天然存在的RoxA/RoxB杂交体（RoxC）和定点突变橡胶加氧酶变体）的生物化学和生物物理学分析，以及（iii）通过生物层干涉法分析底物-蛋白质相互作用来解决这一假设。