Dias Artur H S, Skaf Munir S, Silveira Rodrigo L
Institute of Chemistry, University of Campinas, Rua Monteiro Lobato 270, 13083-862, Campinas, Sao Paulo, Brazil.
Center for Computing in Engineering and Sciences, University of Campinas, Rua Monteiro Lobato 270, 13083-862, Campinas, Sao Paulo, Brazil.
J Chem Inf Model. 2025 Jul 14;65(13):7102-7112. doi: 10.1021/acs.jcim.5c00922. Epub 2025 Jun 30.
β-Glucosidases catalyze the hydrolysis of cellobiose to glucose during lignocellulosic biomass depolymerization. A significant limitation of many β-glucosidases is product inhibition by glucose, leading to reduced conversion efficiency. However, certain β-glucosidases exhibit tolerance or even stimulation by glucose. The mechanisms underlying this remarkable feature remain poorly elucidated. Here, we employ molecular dynamics simulations to investigate the molecular basis of glucose tolerance and stimulation within the family 1 β-glucosidase from (Bgl). Potential of mean force calculations reveal a substantial difference in binding free energies between cellobiose (-12.5 kcal/mol) and glucose (-4.3 kcal/mol) at the Bgl active site, indicating that the glucose product is a considerably weaker ligand than the cellobiose substrate. These findings are consistent with our observations that Bgl undergoes conformational changes in its substrate binding site, specifically involving the Trp349 side chain, in the presence of glucose, potentially facilitating glucose expulsion and mitigating product inhibition. Simulations of Bgl solvated in a 200 mM aqueous glucose environment show that glucose molecules from the bulk solution are capable of penetrating and widening the substrate binding pocket, forming direct interactions with cellobiose in the active site, which may contribute to catalytic stimulation. Additionally, we identify seven distinct secondary glucose binding sites located on the Bgl surface, spatially distant from the active site, implying a potential role in allosteric regulation. Finally, we demonstrate that glucose at subsite +1 can adopt multiple orientations relative to glucose at subsite -1, a prerequisite for transglycosylation reactions in Bgl. Our findings elucidate the molecular mechanisms governing Bgl's glucose tolerance and stimulation, thereby enabling the design of site-directed mutagenesis experiments to improve enzyme efficiency for industrial applications, particularly in biofuel production and oligosaccharide synthesis.
β-葡萄糖苷酶在木质纤维素生物质解聚过程中催化纤维二糖水解为葡萄糖。许多β-葡萄糖苷酶的一个显著局限性是受葡萄糖的产物抑制，导致转化效率降低。然而，某些β-葡萄糖苷酶对葡萄糖表现出耐受性甚至受到葡萄糖的刺激。这一显著特性背后的机制仍未得到充分阐明。在此，我们采用分子动力学模拟来研究来自[具体来源未给出]的1型β-葡萄糖苷酶（Bgl）中葡萄糖耐受性和刺激作用的分子基础。平均力势计算表明，在Bgl活性位点，纤维二糖（-12.5千卡/摩尔）和葡萄糖（-4.3千卡/摩尔）之间的结合自由能存在显著差异，这表明葡萄糖产物作为配体比纤维二糖底物弱得多。这些发现与我们的观察结果一致，即在葡萄糖存在下，Bgl的底物结合位点会发生构象变化，特别是涉及色氨酸349侧链，这可能有助于葡萄糖排出并减轻产物抑制。在200 mM葡萄糖水溶液环境中对Bgl进行的模拟表明，来自本体溶液的葡萄糖分子能够穿透并拓宽底物结合口袋，与活性位点中的纤维二糖形成直接相互作用，这可能有助于催化刺激。此外，我们在Bgl表面识别出七个不同的二级葡萄糖结合位点，它们在空间上远离活性位点，这意味着其在变构调节中可能发挥作用。最后，我们证明亚位点+1处的葡萄糖相对于亚位点-1处的葡萄糖可以采取多种取向，这是Bgl中糖基转移反应的一个先决条件。我们的发现阐明了控制Bgl葡萄糖耐受性和刺激作用的分子机制，从而能够设计定点诱变实验以提高酶在工业应用中的效率，特别是在生物燃料生产和寡糖合成中。
