SAXS data collection and analysis parameters.

The structural analysis of catechol 1,2 dioxygenase from Stutzerimonas frequens GOM2, SfC12DO, was conducted using various structural techniques. SEC-SAXS experiments revealed that SfC12DO, after lyophilization and reconstitution processes, can form multiple enzymatically active oligomers, including dimers, tetramers, and octamers. These findings differ from previous studies, which reported active dimers in homologous counterparts with available crystallographic structures, or trimers observed exclusively in solution for SfC12DO and its homologous isoA C12DO from Acinetobacter radioresistens under low ionic strength conditions. In some cases, tetramers were also reported, such as for the Rodococcus erythropolis C12DO. The combined results of Small-Angle X-ray Scattering, Dynamic Light Scattering, and Transmission Electron Microscopy experiments provided additional insights into these active oligomers’ shape and molecular organization in an aqueous solution. These results highlight the oligomeric structural plasticity of SfC12DO, proving that it can exist in different oligomeric forms depending on the physicochemical characteristics of the solutions in which the experiments were performed. Remarkably, regardless of its oligomeric state, SfC12DO maintains its enzymatic activity even after prior lyophilization. All these characteristics make SfC12DO a putative candidate for bioremediation applications in polluted soils or waters.

本研究采用多种结构生物学技术，对频临施特策单胞菌（Stutzerimonas frequens）GOM2来源的儿茶酚1,2-双加氧酶（Catechol 1,2-dioxygenase，简称SfC12DO）开展结构分析。尺寸排阻色谱-小角X射线散射（SEC-SAXS）实验结果显示，SfC12DO经冷冻干燥与复溶处理后，可形成多种具有酶活性的寡聚体，包括二聚体、四聚体与八聚体。该研究结果与既往报道存在差异：既往研究中，具备可用晶体结构的同源酶均以活性二聚体形式存在；而此前仅在低离子强度溶液条件下，于SfC12DO及其来自抗辐射不动杆菌（Acinetobacter radioresistens）的同工酶A C12DO中观察到仅在溶液中存在的三聚体，另有部分研究报道红平红球菌（Rodococcus erythropolis）的C12DO可形成四聚体。本研究结合小角X射线散射（Small-Angle X-ray Scattering, SAXS）、动态光散射（Dynamic Light Scattering, DLS）与透射电子显微镜（Transmission Electron Microscopy, TEM）实验结果，进一步揭示了这些活性寡聚体在水溶液中的形态与分子组装模式。上述结果凸显了SfC12DO的寡聚体结构可塑性，证实其可根据实验溶液的理化特性呈现不同的寡聚体形式。值得注意的是，无论处于何种寡聚状态，SfC12DO在预先经过冷冻干燥后仍可维持其酶活性。上述所有特性均使SfC12DO成为污染土壤与水体生物修复应用的潜在候选酶。