FIG 4.
PsGH105A and PsGH105B act on the unsaturated oligogalacturonates produced by PsPL1 with different efficiencies. (A) FACE gel showing the shift in the banding patterns of oligogalacturonates produced by PsPL1 from polygalacturonate (PGA) following treatment with PsGH105A, PsGH105B, or both. Galacturonate (GalUA), unsaturated digalacturonate (ΔGalUA2), and unsaturated trigalacturonate (ΔGalUA3) were included as standards. + and −, presence and absence, respectively, of substrate or enzyme. (B) Removal of unsaturated uronyl residues from the nonreducing end of PsPL1-generated oligogalacturonates by GH105As as detected by a decrease in OD230. PsPL1 was added to PGA in all reaction mixtures at time zero, and either PsGH105A, PsGH105B, or buffer was added to separate reaction mixtures after 30 min (indicated by the arrow). Data shown are the mean of three replicates, and error bars, where visible, represent the SEM. (C and D) Michaelis-Menten kinetics for PsGH105A and PsGH105B against ΔGalUA2 (C) and a mixture of unsaturated oligogalacturonates with an estimated degree of polymerization of ≥4 (D). Data shown are the mean of three replicates, and error bars represent the SEM. The enzyme concentrations used were 250 nM for both enzymes against ΔGalUA2 and 1 nM PsGH105A and 10 nM PsGH105B against ΔGalUAn≥4.