Understanding how the complex molecular architecture of mannan-degrading hydrolases contributes to plant cell wall degradation

Abstract

Microbial degradation of plant cell walls is a central component of the carbon cycle and is of increasing importance in environmentally significant industries. Plant cell wall-degrading enzymes have a complex molecular architecture consisting of catalytic modules and, frequently, multiple non-catalytic carbohydrate binding modules (CBMs). It is currently unclear whether the specificities of the CBMs or the topology of the catalytic modules are the primary drivers for the specificity of these enzymes against plant cell walls. Here, we have evaluated the relationship between CBM specificity and their capacity to enhance the activity of GH5 and GH26 mannanases and CE2 esterases against intact plant cell walls. The data show that cellulose and mannan binding CBMs have the greatest impact on the removal of mannan from tobacco and Physcomitrella cell walls, respectively. Although the action of the GH5 mannanase was independent of the context of mannan in tobacco cell walls, a significant proportion of the polysaccharide was inaccessible to the GH26 enzyme. The recalcitrant mannan, however, was fully accessible to the GH26 mannanase appended to a cellulose binding CBM. Although CE2 esterases display similar specificities against acetylated substrates in vitro, only CjCE2C was active against acetylated mannan in Physcomitrella. Appending a mannan binding CBM27 to CjCE2C potentiated its activity against Physcomitrella walls, whereas a xylan binding CBM reduced the capacity of esterases to deacetylate xylan in tobacco walls. This work provides insight into the biological significance for the complex array of hydrolytic enzymes expressed by plant cell wall-degrading microorganisms.

Keywords: Carbohydrate-binding Protein; Glycoside Hydrolases; Microscopic Imaging; Plant Cell Wall; Polysaccharide.

FIGURE 1.

Schematic of the mannanases and esterases used in this study. The line joining the catalytic modules and CBMs represents a 15-residue Thr-Pro linker. The restriction sites used to clone the DNAs encoding the catalytic modules and CBMs are indicated.

FIGURE 2.

The activity of CjMan5A_CM against tobacco cell walls. Sections were incubated with CjMan5A_CM at the concentrations indicated for 20 min at 25 °C. The mannan remaining after enzymatic treatment was determined by probing with the mannan-specific antibody LM21. Panel A displays representative immunofluorescence micrographs of LM21 binding to enzyme-treated sections in which the scale bar is 50 μm. Panel B shows the fluorescence intensities of the enzyme treated sections. Each datum point presented in this figure and Figs. 3–7 is derived from three or four different sections. The scale bar is 50 mm.

FIGURE 3.

The activity of CjMan26A against tobacco cell walls. Stem sections were incubated with wild type CjMan26A (lacks a CBM) and the enzyme fused to cellulose (CBM3a) and mannan (CBM27) binding CBMs at the concentrations indicated for 20 min at 25 °C. The mannan remaining after enzymatic treatment was determined using the mannan-specific antibody LM21. Panels A and B display representative immunofluorescence micrographs and the quantified fluorescence intensities of all the sections, respectively. The scale bar is 50 μm.

FIGURE 4.

The activity of CjMan26A and CjMan5A against Physcomitrella cell walls. The experiments were performed as described in Fig. 3 with panel A showing representative immunofluorescence micrographs and panel B showing the quantified fluorescence intensities of the cells in the perimeter of all the sections. Panel C shows immunofluorescence micrographs in the red boxes of the representative examples of the inner cells (the pith area), magnified 5 times, of Physcomitrella treated with CjMan5A and derivatives of the enzyme containing the various CBMs. The scale bar is 25 μm in A and 5 mm in the red boxed images in C.

FIGURE 5.

The activity of the CE2 esterases against Physcomitrella cell walls. In panel A the four CE2 esterases (lacking a CBM) at 6 μm were incubated with Physcomitrella sections for 1 h as described under “Materials and Methods.” The presence of acetylated mannan was detected using the antibody CCRC-M170, which specifically binds to acetylated mannans, and immunofluorescence microscopy. In panels B and C Physcomitrella sections were treated with 0.13 μm CjCE2C, CjCE2C-CBM27, and CjCE2C-CBM3a for 70 min. The presence of acetylated mannan was detected by immunofluorescence microscopy using CCRC-M170 antibody as the probe. Panel B shows the fluorescence images of the sections, and Panel C shows the quantified fluorescence intensities. The scale bar is 25 μm.

FIGURE 6.

The activity of the CE2 esterases against tobacco and Miscanthus cell walls. The figure displays the immunofluorescence images of sections of tobacco (panels A and C) and Miscanthus (panel B) stems incubated with 7.6 μm CE2 esterases for 2 h and probed with LM23, which binds to undecorated xylans but not to acetylated xylan (21). In panels A and B the esterase-treated sections were probed directly with LM23, whereas in panel C the esterase-treated sections were incubated with the xylanases Xyn10A (CjCE2B/Xyn10A) or Xyn11A (CjCE2B/Xyn10B) before probing with the antibody. The scale bar is 25 μm in A, B, and C.

FIGURE 7.

The influence of CBMs on the activity of CjMan5A against tobacco cell walls. Sections of tobacco stems were incubated with 0.02 μm CjMan5A and derivatives of the enzyme containing CBMs for 5 min. Representative fluorescence images of enzyme-treated tobacco sections labeled by the mannan-specific antibody LM21 are shown in panel A. Quantification of the fluorescence intensities of the equivalent images are shown in panel B. The scale bar is 50 μm.