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

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 bacteri...

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Published in:	The Journal of biological chemistry Vol. 296; p. 100638
Main Authors:	Møller, Marie Sofie, El Bouaballati, Souad, Henrissat, Bernard, Svensson, Birte
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 01-01-2021 American Society for Biochemistry and Molecular Biology
Subjects:	affinity gel electrophoresis Amino Acid Sequence beta-Mannosidase Catalytic Domain Cellulose enzyme kinetics galactomannan Gammaproteobacteria GFP–domain fusion glucomannan Life Sciences mannan Mannans multimodular enzyme truncation Protein Conformation protein–carbohydrate interaction pull-down assays Sequence Homology Substrate Specificity galactomannan LPMO DNS INM mannan affinity gel electrophoresis GFP–domain fusion CGM-lv CBM pull-down assays multimodular enzyme truncation substrate specificity AGE GG CD GH CBM10 glucomannan PASC GH5_8 HEC enzyme kinetics KGM BMCC protein–carbohydrate interaction green fluorescent protein-domain fusion protein carbohydrate-interaction
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Summary:	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, 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 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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 PMCID: PMC8121702
ISSN:	0021-9258 1083-351X
DOI:	10.1016/j.jbc.2021.100638