National Research Center «Kurchatov Institute» - GOSNIIGENETIKA, Kurchatov Genomic Center, 1-st Dorozhniy pr. 1, 117545, Moscow, Russian Federation.
National Research Center «Kurchatov Institute», Kurchatov Sq. 1, 123182, Moscow, Russian Federation.
Appl Microbiol Biotechnol. 2022 Feb;106(4):1493-1509. doi: 10.1007/s00253-022-11811-7. Epub 2022 Feb 7.
In this study, we compared the properties and structures of three fungal GH12 enzymes: the strict endoglucanase Bgh12A and the xyloglucanase Xgh12B from Aspergillus cervinus, and the endoglucanase Egh12 from Thielavia terrestris combining activity on linear β-glucan and branched xyloglucan. Egh12 from T. terrestris was produced in Pichia pastoris, purified, and characterized as a thermostable enzyme with maximal activity at 70 ºC and a half-life time of 138 min at 65 °C. We for the first time demonstrated that the GH12 endoglucanases Egh12 and Bgh12A, but not the strict xyloglucanase Xgh12B, hydrolyzed (1,3)-β-linkages in (1,3;1,4)-β-D-glucooligosaccharides and had transglycosylase activity on (1,3)-β-D-glucooligosaccharides. Phylogenetic analysis indicated that Egh12 from T. terrestris and Bgh12A from A. cervinus are more related than Bgh12A and Xgh12B isolated from one strain. The X-ray structure of Bgh12A was determined with 2.17 Å resolution and compared with 3D-homology models of Egh12 and Xgh12B. The enzymes have a β-jelly roll structure with a catalytic cleft running across the protein. Comparative analysis and a docking study demonstrated the importance of endoglucanase-specific loop 1 partly covering the catalytic cleft for correct placement of the linear substrates. Variability in substrate specificity between the GH12 endoglucanases is determined by non-conservative residues in structural loops framing the catalytic cleft. A residue responsible for the thermostability of Egh12 was predicted. The key structural elements and residues described in this study may serve as potential targets for modification aimed at the improvement of enzymatic properties. KEY POINTS: • Thermostable endoglucanase Egh12 from T. terrestris was produced in P. pastoris, purified, and characterized • The X-ray structure of GH12 endoglucanase Bgh12A from A. cervinus was resolved • GH12 endoglucanases, but not GH12 xyloglucanases, hydrolyze (1,3)-β-linkages in (1,3;1,4)-β-D-glucooligosaccharides.
在这项研究中，我们比较了三种真菌 GH12 酶的性质和结构：严格的内切葡聚糖酶 Bgh12A 和木葡聚糖酶 Xgh12B 来自曲霉菌，内切葡聚糖酶 Egh12 来自土曲霉。Egh12 来自土曲霉在巴斯德毕赤酵母中生产，经过纯化和特性分析，是一种具有热稳定性的酶，最适活性温度为 70℃，在 65℃下半衰期为 138 分钟。我们首次证明，GH12 内切葡聚糖酶 Egh12 和 Bgh12A，但不是严格的木葡聚糖酶 Xgh12B，可水解（1,3）-β 键在（1,3;1,4）-β-D-葡寡糖中，并具有（1,3）-β-D-葡寡糖的转糖基酶活性。系统发育分析表明，土曲霉中的 Egh12 和曲霉菌中的 Bgh12A 比从同一菌株中分离的 Bgh12A 和 Xgh12B 更为相关。Bgh12A 的 X 射线结构已确定，分辨率为 2.17Å，并与 Egh12 和 Xgh12B 的 3D 同源模型进行了比较。这些酶具有 β-果冻卷结构，催化裂缝贯穿整个蛋白质。比较分析和对接研究表明，内切葡聚糖酶特异性环 1 的重要性，部分覆盖催化裂缝，以正确放置线性底物。GH12 内切葡聚糖酶之间的底物特异性差异是由构成催化裂缝的结构环中的非保守残基决定的。预测了一个负责 Egh12 耐热性的残基。本研究中描述的关键结构元素和残基可能成为修饰的潜在目标，旨在改善酶的性质。要点： • 土曲霉中耐热性内切葡聚糖酶 Egh12 在巴斯德毕赤酵母中生产，经过纯化和特性分析 • 已解析了曲霉菌 GH12 内切葡聚糖酶 Bgh12A 的 X 射线结构 • GH12 内切葡聚糖酶，但不是 GH12 木葡聚糖酶，可水解（1,3）-β 键在（1,3;1,4）-β-D-葡寡糖中。
