PDB model fitting to SAXS data for the octamer.

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，对来自频繁斯图策菌（Stutzerimonas frequens）GOM2的邻苯二酚1,2-双加氧酶（catechol 1,2 dioxygenase，SfC12DO）开展结构分析。尺寸排阻色谱-小角X射线散射（Size Exclusion Chromatography-Small Angle X-ray Scattering，SEC-SAXS）实验结果显示，SfC12DO经冻干（lyophilization）与复溶处理后，可形成二聚体、四聚体、八聚体等多种具有酶催化活性的寡聚体形式。该发现与既往研究结论存在差异：既往报道中，具备可用晶体结构的同源酶均以活性二聚体形式存在；另有研究仅在低离子强度条件下的溶液中，观察到SfC12DO及其来自抗辐射不动杆菌（Acinetobacter radioresistens）的同源同工酶isoA C12DO以三聚体形式存在。部分研究也曾报道四聚体形式的C12DO，例如红球菌（Rodococcus erythropolis）的C12DO。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）实验的联合结果，进一步揭示了上述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，证明其可根据实验所处溶液的理化性质，以不同寡聚体形式存在。值得注意的是，无论处于何种寡聚体状态，SfC12DO在经冻干处理后仍可维持其酶催化活性。所有上述特性均使SfC12DO成为污染土壤或水体生物修复（bioremediation）应用的潜在候选酶。