Elucidating the Ionic Liquid-Induced Mixed Inhibition of GH1 -Glucosidase H0HC94
The journal of physical chemistry. B(2023)
摘要
Deciphering the ionic liquid (IL) tolerance of glycoside hydrolases (GHs) to improve their hydrolysis efficiency for fermentable sugar synthesis in the "one-pot" process has long been a hurdle for researchers. In this work, we employed experimental and theoretical approaches to investigate the 1-ethyl-3-methylimidazolium acetate ([C(2)C(1)im][MeCO2])-induced inhibition of GH1 beta-glucosidase (H0HC94) from Agrobacterium tumefaciens 5A. At 10-15% [C(2)C(1)im][MeCO2] concentration, H0HC94 experiences competitive inhibition (R-2 = 0.97, alpha = 2.8). As the IL content increased to 20-25%, the inhibition pattern shifted to mixed-type inhibition (R-2 = 0.98, alpha = 3.4). These findings were further confirmed through characteristic inhibition plots using Lineweaver-Burk plots. Atomistic molecular dynamics simulations conducted with 0% [C(2)C(1)im][MeCO2], 10% [C(2)C(1)im][MeCO2], and 25% [C(2)C(1)im][MeCO2] revealed the accumulation of [C(2)C(1)im](+) at the negatively charged active site of H0HC94 in 10% [C(2)C(1)im][MeCO2], supporting the occurrence of competitive inhibition at lower IL concentrations. At higher IL concentrations, the cations and anions bound to the secondary binding sites (SBSs) of H0HC94, leading to a tertiary conformational change, as captured by the principal component analysis based on the free-energy landscape and protein structure networks. The altered conformation of H0HC94 affected the interaction with [C(2)C(1)im][MeCO2], which could possibly shift the inhibition from competitive to more mixed-type (competitive + noncompetitive) inhibition, as observed in the experiments. For the first time, we report a combined experimental and theoretical insight behind the mixed inhibition of a GH1 beta-glucosidase. Our findings indicated the role of SBS in IL-induced inhibition, which could aid in developing more IL-tolerant beta-glucosidases for biorefinery applications.
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关键词
liquid-induced
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