Fig. 5: Affinity for natural substrates or derived soluble fragments.
From: The structural basis of fungal glucuronoyl esterase activity on natural substrates
a Ligand-binding affinities of soluble ligands to various CuGE variants as determined by LabelFree microscale thermophoresis (MST). Legend details and estimated Kd-values are listed in the panel and error bars represents standard deviation. b ITC enthalpogram of the interaction between WT CuGE and the enzyme’s natural insoluble lignin-rich substrate (LRP) at 5 °C. The titration curve is depicted as a function of the molar ratio (n) between enzyme and the calculated concentration of 4-O-methyl-glucuronoyl in the assay. The latter is an estimation of the concentration of recognition sites in the substrate. c Chemical structures of small ligands 4-O-methyl-d-glucuronic acid (MeGlcA) and the methyl ester of 4-O-methyl-d-glucuronic acid (MeMeGlcA) used in MST measurements of binding affinity in a. The structures are depicted to illustrate that equilibrium between the α- and β-anomers exists in an aqueous solution. d Qualitative SDS-PAGE-based “pull-down” assay illustrating the effect of CBM1 on the binding to the insoluble biopolymers xylan, cellulose and LRP, respectively. 1: Molecular size ladder, 2: WT without substrate, 3: Supernatant after binding between WT CuGE and insoluble substrate, 4: Pellet after binding between WT CuGE and insoluble substrate, 5: Insoluble substrate without enzyme, 6: ΔWT CuGE without substrate, 7: Supernatant after binding between ΔWT CuGE and insoluble substrate, 8: Pellet after binding between ΔWT CuGE and insoluble substrate. The description of lane content applies to all three gel pictures. Source data for panel a, b, and d are provided as a Source Data file.
