Key Laboratory of Medical Molecule Science and Pharmaceutics Engineering, Ministry of Industry and Information Technology, Institute of Biochemical Engineering, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing, PR China.
Key Laboratory for Industrial Biocatalysis, Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing, PR China.
Biotechnol Bioeng. 2021 May;118(5):1962-1972. doi: 10.1002/bit.27710. Epub 2021 Feb 19.
Glycoside hydrolase family 2 (GH2) enzymes are generally composed of three domains: TIM-barrel domain (TIM), immunoglobulin-like β-sandwich domain (ISD), and sugar-binding domain (SBD). The combination of these three domains yields multiple structural combinations with different properties. Theoretically, the drawbacks of a given GH2 fold may be circumvented by efficiently reassembling the three domains. However, very few successful cases have been reported. In this study, we used six GH2 β-glucuronidases (GUSs) from bacteria, fungi, or humans as model enzymes and constructed a series of mutants by reassembling the domains from different GUSs. The mutants PGUS-At, GUS-PAA, and GUS-PAP, with reassembled domains from fungal GUSs, showed improved expression levels, activity, and thermostability, respectively. Specifically, compared to the parental enzyme, the mutant PGUS-At displayed 3.8 times higher expression, the mutant GUS-PAA displayed 1.0 time higher catalytic efficiency (k /K ), and the mutant GUS-PAP displayed 7.5 times higher thermostability at 65°C. Furthermore, two-hybrid mutants, GUS-AEA and GUS-PEP, were constructed with the ISD from a bacterial GUS and SBD and TIM domain from fungal GUSs. GUS-AEA and GUS-PEP showed 30.4% and 23.0% higher thermostability than GUS-PAP, respectively. Finally, molecular dynamics simulations were conducted to uncover the molecular reasons for the increased thermostability of the mutant.
糖苷水解酶家族 2 (GH2) 酶通常由三个结构域组成：TIM 桶状结构域 (TIM)、免疫球蛋白样β-折叠结构域 (ISD) 和糖结合结构域 (SBD)。这三个结构域的组合产生了具有不同性质的多种结构组合。从理论上讲，通过有效地重新组装这三个结构域，可以克服给定 GH2 折叠的缺点。然而，报道的成功案例很少。在这项研究中，我们使用了来自细菌、真菌或人类的六种 GH2 β-葡萄糖醛酸酶 (GUS) 作为模型酶，并通过重新组装来自不同 GUS 的结构域构建了一系列突变体。具有来自真菌 GUS 重新组装结构域的突变体 PGUS-At、GUS-PAA 和 GUS-PAP 分别表现出更高的表达水平、活性和热稳定性。具体而言，与亲本酶相比，突变体 PGUS-At 的表达水平提高了 3.8 倍，突变体 GUS-PAA 的催化效率 (k /K )提高了 1.0 倍，突变体 GUS-PAP 在 65°C 时的热稳定性提高了 7.5 倍。此外，还构建了具有来自细菌 GUS 的 ISD 和 SBD 以及来自真菌 GUS 的 TIM 结构域的二杂交突变体 GUS-AEA 和 GUS-PEP。GUS-AEA 和 GUS-PEP 的热稳定性分别比 GUS-PAP 高 30.4%和 23.0%。最后，进行了分子动力学模拟，以揭示突变体热稳定性提高的分子原因。
