Department of Chemical Engineering, University of California, Santa Barbara, Santa Barbara, CA, 93106, USA.
The US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA.
Protein Expr Purif. 2023 Oct;210:106323. doi: 10.1016/j.pep.2023.106323. Epub 2023 Jun 16.
Anaerobic fungi (Neocallimastigomycetes) found in the guts of herbivores are biomass deconstruction specialists with a remarkable ability to extract sugars from recalcitrant plant material. Anaerobic fungi, as well as many species of anaerobic bacteria, deploy multi-enzyme complexes called cellulosomes, which modularly tether together hydrolytic enzymes, to accelerate biomass hydrolysis. While the majority of genomically encoded cellulosomal genes in Neocallimastigomycetes are biomass degrading enzymes, the second largest family of cellulosomal genes encode spore coat CotH domains, whose contribution to fungal cellulosome and/or cellular function is unknown. Structural bioinformatics of CotH proteins from the anaerobic fungus Piromyces finnis shows anaerobic fungal CotH domains conserve key ATP and Mg binding motifs from bacterial Bacillus CotH proteins known to act as protein kinases. Experimental characterization further demonstrates ATP hydrolysis activity in the presence and absence of substrate from two cellulosomal P. finnis CotH proteins when recombinantly produced in E. coli. These results present foundational evidence for CotH activity in anaerobic fungi and provide a path towards elucidating the functional contribution of this protein family to fungal cellulosome assembly and activity.
反刍动物肠道中的厌氧真菌（Neocallimastigomycetes）是生物质解构专家，具有从顽固性植物材料中提取糖的非凡能力。厌氧真菌与许多种厌氧细菌一样，利用称为纤维小体的多酶复合物来加速生物质水解，该复合物将水解酶模块化地连接在一起。虽然 Neocallimastigomycetes 中大多数基因组编码的纤维小体基因是生物质降解酶，但第二大亚家族的纤维小体基因编码孢子壳 CotH 结构域，其对真菌纤维小体和/或细胞功能的贡献尚不清楚。来自厌氧真菌 Piromyces finnis 的 CotH 蛋白的结构生物信息学研究表明，厌氧真菌 CotH 结构域保守了来自已知作为蛋白激酶的细菌 Bacillus CotH 蛋白的关键 ATP 和 Mg 结合基序。实验特征进一步证明了在存在和不存在底物的情况下，两种纤维素酶 P. finnis CotH 蛋白在大肠杆菌中重组表达时具有 ATP 水解活性。这些结果为厌氧真菌的 CotH 活性提供了基础证据，并为阐明该蛋白家族对真菌纤维小体组装和活性的功能贡献提供了途径。
