Role of Conformational Change and Glucose Binding Sites in the Enhanced Glucose Tolerance of Agrobacterium tumefaciens 5A GH1 β-Glucosidase Mutants

Abstract

β-Glucosidases are often inhibited by their reaction product glucose and a barrier to the efficient lignocellulosic biomass hydrolysis to glucose. We had previously reported the mutants, C174V, and H229S, with a nearly 2-fold increased glucose tolerance over the wild type (WT), H0HC94, encoded in Agrobacterium tumefaciens 5A (apparent K i , Glc = 686 mM). We report our steady−state and time-resolved intrinsic fluorescence spectroscopy, circular dichroism, and isothermal titration calorimetry (ITC) studies to further understand increased glucose tolerance. Changes in the mutants' emission intensity and the differential change in quenching rate in the absence and presence of glucose reflect changes in protein conformation by glucose. Time-resolved lifetime and anisotropy measurements further indicated the microenvironment differences across solvent-exposed tryptophan residues and a higher hydrodynamic radius due to glucose binding, respectively. ITC measurements confirmed the increase of glucose binding sites in the mutants. The experiment results were supported by molecular dynamics simulations, which revealed significant variations in the glucose−protein hydrogen-bonding profiles. Protein structure network analysis of the simulated structures further indicates the mutants' conformation change than the WT. Computational studies also indicated additional glucose binding sites in mutants. Our results indicate the role of glucose binding in modulating the enzyme response to glucose.

Rational engineering of a β-glucosidase (H0HC94) from glycosyl family I (GH1) to improve catalytic performance on cellobiose

“…We previously reported that glucose might form multiple hydrogen bonds to H0HC94 through hydroxyl groups. 20,21 Our current simulations show that the average number of inter-hydrogen bonds between H0HC94 and substrates were 10 ± 3, 20 ± 4, 9 ± 3, and 15 ± 3 in S1, S2, S3, and S4, respectively (Supplementary Table S1; Figure 4a). Cellobiose formed comparatively more hydrogen bonds with both WT and mutant than pNPGlc.…”

“…A similar rmsd pattern was previously reported, signifying the overall structural stability of the enzyme under the studied timescales. 20, 21…”

“…A similar rmsd pattern was previously reported, signifying the overall structural stability of the enzyme under the studied timescales. 20,21 To understand the role of amino acid residues, the residue flexibility was measured by the Root-Mean-Square fluctuation (rmsf) profiles during the simulations (Figure 3b). The active site catalytic residues, active site tunnel residues, and gatekeeper residues exhibited low average rmsf (≤ 1.0734 Å) across the H0HC94 variants indicating no significant changes due to the Q319 mutation (Supplementary Table S3).…”

“…15 AMBER20 19 was utilized to carry out the simulations. The residue numbering of the enzyme is as per previous reports 20, 21 . The AMBERff14SB 22 force field was employed for the protein, whereas the GLYCAM06 23 and General Amber Force Field (GAFF) were assigned to cellobiose and p NPGlc molecules, respectively.…”

“…In recent years, dynamic changes in the conformation of enzymes from different glycoside hydrolases (GHs) such as endoglucanase Cel12A, 32 xylanase, 31 and β-Glucosidase 20,21 was investigated by PSN to provide global characteristics of protein structures during MD simulations. We examined the conformational changes of WT and Q319A in the presence of pNPGlc and cellobiose by constructing the PSN on each enzyme structure from the simulation trajectories and evaluating it using novel network parameters, i.e., dynamically stable network communities (connected cliques) and hubs (nodes with ≥ 4 edges).…”