Functional diversity of three tandem C-terminal carbohydrate-binding modules of a β-mannanase

Abstract

Carbohydrate active enzymes, such as those involved in plant cell wall and storage polysaccharide biosynthesis and deconstruction, often contain repeating noncatalytic carbohydrate-binding modules (CBMs) to compensate for low-affinity binding typical of protein-carbohydrate interactions. The bacterium Saccharophagus degradans produces an endo-β-mannanase of glycoside hydrolase family 5 subfamily 8 with three phylogenetically distinct family 10 CBMs located C-terminally from the catalytic domain (SdGH5_8-CBM10x3). However, the functional roles and cooperativity of these CBM domains in polysaccharide binding are not clear. To learn more, we studied the full-length enzyme, three stepwise CBM family 10 (CBM10) truncations, and GFP fusions of the individual CBM10s and all three domains together by pull-down assays, affinity gel electrophoresis, and activity assays. Only the C-terminal CBM10-3 was found to bind strongly to microcrystalline cellulose (dissociation constant, K_d = 1.48 μM). CBM10-3 and CBM10-2 bound galactomannan with similar affinity (K_d = 0.2-0.4 mg/ml), but CBM10-1 had 20-fold lower affinity for this substrate. CBM10 truncations barely affected specific activity on carob galactomannan and konjac glucomannan. Full-length SdGH5_8-CBM10x3 was twofold more active on the highly galactose-decorated viscous guar gum galactomannan and crystalline ivory nut mannan at high enzyme concentrations, but the specific activity was fourfold to ninefold reduced at low enzyme and substrate concentrations compared with the enzyme lacking CBM10-2 and CBM10-3. Comparison of activity and binding data for the different enzyme forms indicates unproductive and productive polysaccharide binding to occur. We conclude that the C-terminal-most CBM10-3 secures firm binding, with contribution from CBM10-2, which with CBM10-1 also provides spatial flexibility.

Keywords: GFP–domain fusion; affinity gel electrophoresis; enzyme kinetics; galactomannan; glucomannan; mannan; multimodular enzyme truncation; protein–carbohydrate interaction; pull-down assays; substrate specificity.

Figure 1

Structures of the included polysaccharides. Mannose (Man), galactose (Gal), and glucose (Glc) ratios are given for galactomannans and glucomannan.

Figure 2

Domain architecture of the native Saccharophagus degradans protein (GenBank accession no.: ABD79918) and the different recombinant full-length and truncated enzyme forms and CBM10 domains produced and characterized. The residues at domain borders are numbered.

Figure 3

Phylogenetic tree including CBM10(s) isolated from full-length enzyme sequences (i.e., without CD and other modules). CBM10s originating from characterized enzymes are labeled (see Table 1 for detailed information). See Figure S1 for the multiple sequence alignment used to generate the phylogenetic tree.

Figure 4

Comparison of characterized CBM10s.A, structure-based multiple sequence alignment including characterized CBM10s. Cysteines involved in disulphide bridges in the Cj_GH10CBM10 structure (Protein Data Bank ID: 1E8R) are denoted by a “C,” whereas binding residues of Cj_GH10CBM10 (Y8, W22, and W24) are indicated by asterisks. The insertion differentiating the subgroup containing Sd_GH5CBM10-2 from the other regions of the phylogenetic tree is indicated by a dotted line. B–D, superimposition of the structure of Cj_GH10CBM10 (orange; Protein Data Bank ID: 1E8R) and homology models of the three Sd_GH5CBM10s (B. Sd_GH5CBM10-1, green; C. Sd_GH5CBM10-2, blue; D. Sd_GH5CBM10-3, purple) with binding residues shown as sticks. The numbering is according to Cj_GH10CBM10, and the equivalent residue of the Sd_GH5CBM10s is given. The region of the insertion in Sd_GH5CBM10-2 (C) is encircled.

Figure 5

Kinetics of the full-length enzyme SdGH5_8-CBM10x3. The three stepwise truncated forms on ivory nut mannan (INM) (A) and guar gum galactomannan (GG) (B). SdGH5_8-CBM10x3 (■), SdGH5_8-CBM10x2 (•), SdGH5_8-CBM10x1 (▲), and SdGH5_8 (◆). The Michaelis–Menten fit to the SdGH5_8-CBM10x3 data is shown as dashed line in A.

Figure 6

Qualitative pull-down assay with Sd_GH5CBM10–GFP fusions of each of the three domains Sd_GH5CBM10-1, Sd_GH5CBM10-2, and Sd_GH5CBM10-3 and all three domains combined Sd_GH5CBM10x3_GFP by the insoluble polysaccharides microcrystalline cellulose (Avicel) and ivory nut mannan (INM), respectively. SDS-PAGE of samples from supernatants (SN) and pellets (P) as well as the protein stocks used for the pull-down assay. GFP alone is included as a control (right panel). Molecular mass values (kDa) of marker proteins are indicated.

Figure 7

Quantitative pull-down assay with microcrystalline cellulose (Avicel). Binding isotherms of the C-terminal domain, Sd_GH5CBM10-3_GFP (A) and all three domains together, Sd_GH5CBM10x3_GFP (B). The filled and open symbols represent replicates from different days. No significant binding was observed of the first and the middle domains, Sd_GH5CBM10-1_GFP, Sd_GH5CBM10-2_GFP, or of GFP alone (data not shown).