Center of Excellence in Computational Chemistry (CECC), Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
School of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
Biomolecules. 2020 Jun 15;10(6):907. doi: 10.3390/biom10060907.
β-Glucosidases and β-mannosidases hydrolyze substrates that differ only in the epimer of the nonreducing terminal sugar moiety, but most such enzymes show a strong preference for one activity or the other. Rice Os3BGlu7 and Os7BGlu26 β-glycosidases show a less strong preference, but Os3BGlu7 and Os7BGlu26 prefer glucosides and mannosides, respectively. Previous studies of crystal structures with glucoimidazole (GIm) and mannoimidazole (MIm) complexes and metadynamic simulations suggested that Os7BGlu26 hydrolyzes mannosides via the transition state (TS) conformation preferred for mannosides and glucosides via their preferred / TS conformation. However, MIm is weakly bound by both enzymes. In the present study, we found that MIm was not bound in the active site of crystallized Os3BGlu7, but GIm was tightly bound in the -1 subsite in a / conformation via hydrogen bonds with the surrounding residues. One-microsecond molecular dynamics simulations showed that GIm was stably bound in the Os3BGlu7 active site and the glycone-binding site with little distortion. In contrast, MIm initialized in the conformation rapidly relaxed to a / conformation and moved out into a position in the entrance of the active site, where it bound more stably despite making fewer interactions. The lack of MIm binding in the glycone site in protein crystals and simulations implies that the energy required to distort MIm to the conformation for optimal active site residue interactions is sufficient to offset the energy of those interactions in Os3BGlu7. This balance between distortion and binding energy may also provide a rationale for glucosidase versus mannosidase specificity in plant β-glycosidases.
β-葡萄糖苷酶和β-甘露糖苷酶水解的底物仅在非还原末端糖部分的差向异构体上有所不同，但大多数此类酶对一种活性具有强烈的偏好。水稻 Os3BGlu7 和 Os7BGlu26β-糖苷酶的偏好程度较低，但 Os3BGlu7 和 Os7BGlu26 分别优先水解葡萄糖苷和甘露糖苷。先前使用 glucoimidazole (GIm) 和 mannoimidazole (MIm) 复合物的晶体结构研究和元动力学模拟表明，Os7BGlu26 通过有利于甘露糖苷和葡萄糖苷的过渡态 (TS) 构象水解甘露糖苷，而通过其优先的 / TS 构象水解甘露糖苷。然而，MIm 被两种酶的结合较弱。在本研究中，我们发现 MIm 未结合在结晶 Os3BGlu7 的活性部位，但 GIm 通过与周围残基形成氢键以 / 构象紧密结合在-1 亚位点。1 微秒分子动力学模拟表明，GIm 在 Os3BGlu7 活性部位和糖基结合部位稳定结合，几乎没有变形。相比之下，在 / 构象初始化的 MIm 迅速松弛到 / 构象并移出到活性部位的入口处，尽管相互作用较少，但它在该位置结合更稳定。蛋白晶体和模拟中缺乏 MIm 在糖基结合部位的结合表明，将 MIm 扭曲到有利于最佳活性部位残基相互作用的 / 构象所需的能量足以抵消 Os3BGlu7 中这些相互作用的能量。这种扭曲和结合能之间的平衡也可能为植物β-糖苷酶中葡萄糖苷酶与甘露糖苷酶的特异性提供了一个理由。
