São Carlos Institute of Physics, University of São Paulo, São Carlos, São Paulo, Brazil.
São Carlos Institute of Physics, University of São Paulo, São Carlos, São Paulo, Brazil.
Biochimie. 2019 Oct;165:275-284. doi: 10.1016/j.biochi.2019.08.016. Epub 2019 Aug 28.
Glycoside hydrolase (GH) family 45 is one of the smallest and poorly studied endoglucanase family with a broad biotechnological application ranging from treatment of textiles to conversion of complex cell wall polysaccharides into simple oligo- and monosaccharides. In a present study, GH45 cellulase from Neurospora crassa OR74A (NcCel45A) was characterized both biochemically and structurally. HPLC analysis of the hydrolytic products confirmed the endo-β(1,4) mode of action of the enzyme. Moreover, such pattern revealed that NcCel45A cannot hydrolyze efficiently oligosaccharides with a degree of polymerization smaller than six. The crystal structure of NcCel45A catalytic domain in the apo-form was determined at 1.9 Å resolution and the structure of the enzyme bound to cellobiose was solved and refined to 1.8 Å resolution. Comparative structural analyses and molecular dynamics simulations show that the enzyme dynamics is affected by substrate binding. Taken together, MD simulations and statistical coupling analysis revealed previously unknown correlation of a loop 6 with the breakdown of cellulose substrates by GH45.
糖苷水解酶（GH）家族 45 是最小且研究较差的内切葡聚糖酶家族之一，具有广泛的生物技术应用，包括处理纺织品到将复杂的细胞壁多糖转化为简单的寡糖和单糖。在本研究中，对粗糙脉孢菌 OR74A 的 GH45 纤维素酶（NcCel45A）进行了生化和结构表征。水解产物的 HPLC 分析证实了酶的内切-β(1,4)作用模式。此外，这种模式表明 NcCel45A 不能有效地水解聚合度小于六的低聚糖。以 1.9 Å 的分辨率测定了无配体形式的 NcCel45A 催化结构域的晶体结构，并解决了与纤维二糖结合的酶的结构，并将其精修至 1.8 Å 的分辨率。比较结构分析和分子动力学模拟表明，酶动力学受底物结合的影响。总之，MD 模拟和统计耦合分析揭示了以前未知的 GH45 环 6 与纤维素底物断裂之间的相关性。
