ABSTRACT
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 ([C2C1im][MeCO2])-induced inhibition of GH1 ß-glucosidase (H0HC94) from Agrobacterium tumefaciens 5A. At 10-15% [C2C1im][MeCO2] concentration, H0HC94 experiences competitive inhibition (R2 = 0.97, alpha = 2.8). As the IL content increased to 20-25%, the inhibition pattern shifted to mixed-type inhibition (R2 = 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% [C2C1im][MeCO2], 10% [C2C1im][MeCO2], and 25% [C2C1im][MeCO2] revealed the accumulation of [C2C1im]+ at the negatively charged active site of H0HC94 in 10% [C2C1im][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 [C2C1im][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 ß-glucosidase. Our findings indicated the role of SBS in IL-induced inhibition, which could aid in developing more IL-tolerant ß-glucosidases for biorefinery applications.
