The discovery of lytic polysaccharide monooxygenases (LPMOs) has fundamentally changed our understanding of microbial lignocellulose degradation. Cellulomonas bacteria have a rich history of study due to their ability to degrade recalcitrant cellulose, yet little is known about the predicted LPMOs that they encode from Auxiliary Activity Family 10 (AA10). Here, we present the comprehensive biochemical characterization of three AA10 LPMOs from Cellulomonas flavigena (CflaLPMO10A, CflaLPMO10B, and CflaLPMO10C) and one LPMO from Cellulomonas fimi (CfiLPMO10). We demonstrate that these four enzymes oxidize insoluble cellulose with C1 regioselectivity and show a preference for substrates with high surface area. In addition, CflaLPMO10B, CflaLPMO10C, and CfiLPMO10 exhibit limited capacity to perform mixed C1/C4 regioselective oxidative cleavage. Thermostability analysis indicates that these LPMOs can refold spontaneously following denaturation dependent on the presence of copper coordination. Scanning and transmission electron microscopy revealed substrate-specific surface and structural morphological changes following LPMO action on Avicel and phosphoric acid-swollen cellulose (PASC). Further, we demonstrate that the LPMOs encoded by Cellulomonas flavigena exhibit synergy in cellulose degradation, which is due in part to decreased autoinactivation. Together, these results advance understanding of the cellulose utilization machinery of historically important Cellulomonas species beyond hydrolytic enzymes to include lytic cleavage. This work also contributes to the broader mapping of enzyme activity in Auxiliary Activity Family 10 and provides new biocatalysts for potential applications in biomass modification.

中文翻译：

---

来自纤维单胞菌属的四种纤维素活性裂解多糖单加氧酶


裂解多糖单加氧酶（LPMO）的发现从根本上改变了我们对微生物木质纤维素降解的理解。纤维单胞菌具有丰富的研究历史，因为它们具有降解顽固纤维素的能力，但人们对它们编码的辅助活性家族 10 (AA10) 的预测 LPMO 知之甚少。在这里，我们介绍了来自黄色纤维单胞菌的三种 AA10 LPMO（CflaLPMO10A、CflaLPMO10B 和 CflaLPMO10C）和一种来自纤维单胞菌 fimi（CfiLPMO10）的 LPMO 的全面生化特征。我们证明这四种酶以 C1 区域选择性氧化不溶性纤维素，并显示出对高表面积底物的偏好。此外，CflaLPMO10B、CflaLPMO10C 和 CfiLPMO10 执行混合 C1/C4 区域选择性氧化裂解的能力有限。热稳定性分析表明，这些 LPMO 可以在变性后自发地重新折叠，具体取决于铜配位的存在。扫描和透射电子显微镜揭示了 LPMO 对 Avicel 和磷酸溶胀纤维素 (PASC) 作用后底物特异性表面和结构形态的变化。此外，我们证明由黄纤维单胞菌编码的 LPMO 在纤维素降解中表现出协同作用，部分原因是自失活减少。总之，这些结果促进了对历史上重要的纤维单胞菌属物种的纤维素利用机制的理解，除了水解酶之外，还包括裂解。这项工作还有助于更广泛地绘制辅助活性家族 10 中的酶活性，并为生物质改性的潜在应用提供新的生物催化剂。