Journal article
Functional diversity of three tandem C-terminal carbohydrate-binding modules of a β-mannanase
Section for Protein Chemistry and Enzyme Technology, Department of Biotechnology and Biomedicine, Technical University of Denmark1
Enzyme and Protein Chemistry, Section for Protein Chemistry and Enzyme Technology, Department of Biotechnology and Biomedicine, Technical University of Denmark2
Department of Biotechnology and Biomedicine, Technical University of Denmark3
Technical University of Denmark4
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.
Language: | English |
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Year: | 2021 |
Pages: | 100638 |
ISSN: | 1083351x , 00219258 and 10678816 |
Types: | Journal article |
DOI: | 10.1016/j.jbc.2021.100638 |
ORCIDs: | Møller, Marie Sofie , 0000-0002-3434-8588 and Svensson, Birte |
Affinity gel electrophoresis Enzyme kinetics Galactomannan Glucomannan Green fluorescent protein-domain fusion Multimodular enzyme truncation Protein carbohydrate-interaction Pull-down assays Substrate specificity
(1-6)-alpha-glucomannan Amino Acid Sequence Catalytic Domain Cellulose GFP–domain fusion Galactose Gammaproteobacteria Mannans Protein Conformation Sequence Homology Substrate Specificity affinity gel electrophoresis beta-Mannosidase enzyme kinetics galactomannan glucomannan mannan multimodular enzyme truncation protein–carbohydrate interaction pull-down assays substrate specificity