Structure and activity of Paenibacillus polymyxa xyloglucanase from glycoside hydrolase family 44

Abstract

The enzymatic degradation of plant polysaccharides is emerging as one of the key environmental goals of the early 21st century, impacting on many processes in the textile and detergent industries as well as biomass conversion to biofuels. One of the well known problems with the use of nonstarch (nonfood)-based substrates such as the plant cell wall is that the cellulose fibers are embedded in a network of diverse polysaccharides, including xyloglucan, that renders access difficult. There is therefore increasing interest in the "accessory enzymes," including xyloglucanases, that may aid biomass degradation through removal of "hemicellulose" polysaccharides. Here, we report the biochemical characterization of the endo-β-1,4-(xylo)glucan hydrolase from Paenibacillus polymyxa with polymeric, oligomeric, and defined chromogenic aryl-oligosaccharide substrates. The enzyme displays an unusual specificity on defined xyloglucan oligosaccharides, cleaving the XXXG-XXXG repeat into XXX and GXXXG. Kinetic analysis on defined oligosaccharides and on aryl-glycosides suggests that both the -4 and +1 subsites show discrimination against xylose-appended glucosides. The three-dimensional structures of PpXG44 have been solved both in apo-form and as a series of ligand complexes that map the -3 to -1 and +1 to +5 subsites of the extended ligand binding cleft. Complex structures are consistent with partial intolerance of xylosides in the -4' subsites. The atypical specificity of PpXG44 may thus find use in industrial processes involving xyloglucan degradation, such as biomass conversion, or in the emerging exciting applications of defined xyloglucans in food, pharmaceuticals, and cellulose fiber modification.

FIGURE 1.

Common side-chain decorations of xyloglucans. Xyloglucan fragments are named according to Ref. .

FIGURE 2.

pH rate profile of PpXG44. The dotted line represents the fit of Equation 1 to the experimental data, and the solid line represents a smooth Bezier curve through the data.

FIGURE 3.

Time-dependent depolymerization of xyloglucan. The incubation time of PpXG44 with xyloglucan is indicated above each chromatogram. The “0-min” time point is for native tamarind xyloglucan without enzyme, and in the “0 + PpXG44” enzyme is added just before heating the sample at 95 °C. The dotted line at 10⁷ Da indicates the cutoff value of the column.

FIGURE 4.

Initial rate kinetics of PpXG44 hydrolysis of β-aryl oligosaccharides. a, GGG-CNP; b, GGGG-CNP (inset, low substrate concentration regime); c, XXXG-CNP (inset, low substrate concentration regime); d, XLLG-CNP.

FIGURE 5.

Structures of inhibitors. a, β-1,4-glucosyl-noeuromycin (Glc-noeuromycin); b, β-1,4-cellobiosyl-oxazine (Glc-Glc-oxazine).

FIGURE 6.

Three-dimensional structure of PpXG44 and its ligand complexes. a, stereo view of a ribbon diagram of PpXG44, color-ramped from the N terminus (red) to the C terminus (magenta), showing the positions of Glc-Glc-oxazine and GXGGG from their respective complexes with PpXG44 as cylinders. A calcium ion is shown in tan, and chloride ion is shown in gray. The +5 subsite position on GXGGG is labeled. Stereo views are shown of the 2F_o − F_c electron density maps, contoured at 1.0 σ, around the ligand in complexes of PpXG44 with Glc-Glc-oxazine (b), Glc-noeuromycin (c), and GXGGG (d) (for the latter complex, atoms in the subsite +1 glucose are modeled with an occupancy of 0.5; all other atoms are fully occupied). Hydrogen bonds are shown as dashed lines, and their approximate length in Ångstroms is indicated. This figure was drawn using CCP4mg (65).

FIGURE 7.

Overlay of PpGH44 enzyme complexes. Stereo view of overlay of cellotetraose from CtGH44A (PDB code 2E0P) (in coral) with Glc-Glc-oxazine from the PpXG44 complex (in green) shows residues from both structures neighboring the −4 subsite region (which is occupied in PDB code 2E0P but unoccupied in the Glc-Glc-oxazine complex). This figure was drawn using CCP4mg (65).