Carbohydrate active enzymes, such as those involved in plant cell wall and storage polysaccharide biosynthesis and deconstruction, often contain repeating non-catalytic 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 is not clear. To learn more we studied the full-length enzyme, three stepwise CBM10 truncations, and green fluorescent protein 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, Kd = 1.48 μM). CBM10-3 and CBM10-2 bound galactomannan with similar affinity (Kd = 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 two-fold 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 4- to 9-fold reduced at low enzyme and substrate concentrations compared to the enzyme lacking CBM10-2 and -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.
- Protein carbohydrate-interaction
- Multimodular enzyme truncation
- Substrate specificity
- Enzyme kinetics
- Affinity gel electrophoresis
- Pull-down assays
- Green fluorescent protein-domain fusion