Department of Macromolecular Biochemistry, Leiden Institute of Chemistry, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
Chembiochem. 2021 May 14;22(10):1743-1749. doi: 10.1002/cbic.202000856. Epub 2021 Mar 16.
Glycoside hydrolases (GHs) are attractive tools for multiple biotechnological applications. In conjunction with their hydrolytic function, GHs can perform transglycosylation under specific conditions. In nature, oligosaccharide synthesis is performed by glycosyltransferases (GTs); however, the industrial use of GTs is limited by their instability in solution. A key difference between GTs and GHs is the flexibility of their binding site architecture. We have used the xylanase from Bacillus circulans (BCX) to study the interplay between active-site flexibility and transglycosylation. Residues of the BCX "thumb" were substituted to increase the flexibility of the enzyme binding site. Replacement of the highly conserved residue P116 with glycine shifted the balance of the BCX enzymatic reaction toward transglycosylation. The effects of this point mutation on the structure and dynamics of BCX were investigated by NMR spectroscopy. The P116G mutation induces subtle changes in the configuration of the thumb and enhances the millisecond dynamics of the active site. Based on our findings, we propose the remodelling of the GH enzymes glycon site flexibility as a strategy to improve the transglycosylation efficiency of these biotechnologically important catalysts.
糖苷水解酶（GHs）是多种生物技术应用的有吸引力的工具。在特定条件下，GHs 可以与水解功能相结合进行转糖苷作用。在自然界中，寡糖的合成是由糖基转移酶（GTs）完成的；然而，GTs 的工业用途受到其在溶液中不稳定的限制。GTs 和 GHs 的一个关键区别在于其结合位点结构的灵活性。我们使用来自环状芽孢杆菌（BCX）的木聚糖酶来研究活性位点灵活性和转糖苷作用之间的相互作用。取代 BCX“拇指”的残基可以增加酶结合位点的灵活性。用甘氨酸取代高度保守的残基 P116 会使 BCX 酶反应的平衡向转糖苷作用转移。通过 NMR 光谱研究了该点突变对 BCX 结构和动力学的影响。P116G 突变会引起拇指构象的细微变化，并增强活性位点的毫秒级动力学。基于我们的发现，我们提出了重塑 GH 酶糖基位点灵活性的策略，以提高这些生物技术上重要的催化剂的转糖苷效率。
