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Updated: May 16, 2026
08:59
Defining Substrate Specificities for Lipase and Phospholipase Candidates
Published on: November 23, 2016
How nature can exploit nonspecific catalytic and carbohydrate binding modules to create enzymatic specificity
Fiona Cuskin1, James E Flint, Tracey M Gloster
1Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom.
Summary
Carbohydrate-binding modules (CBMs) enhance enzyme activity. A newly discovered CBM66 specifically targets fructans, conferring exolevanase activity and demonstrating a novel mechanism for enzyme specificity.
Area of Science:
- Enzymology
- Structural Biology
- Carbohydrate Chemistry
Background:
- Noncatalytic carbohydrate-binding modules (CBMs) are known to enhance the activity of glycoside hydrolases, particularly endo-acting enzymes, by increasing substrate proximity.
- The role of CBMs in exo-acting glycoside hydrolases has been less understood due to their typical binding modes not aligning with a targeting function.
Purpose of the Study:
- To investigate the function of CBMs in exo-acting enzymes, specifically the β-fructosidase SacC from Bacillus subtilis.
- To characterize the novel CBM family 66 (CBM66) found in SacC and elucidate its mechanism of action and substrate specificity.
Main Methods:
- Structural analysis of the SacC-derived CBM66 (BsCBM66) in complex with ligands using X-ray crystallography.
- Enzymatic assays to assess the activity of SacC with and without BsCBM66, and to evaluate the effect of appending BsCBM66 to a non-specific β-fructosidase.
Main Results:
- BsCBM66, the founding member of CBM66, specifically targets terminal fructosides of major natural fructans.
- Structural data revealed extensive interactions with terminal fructose moieties, explaining the CBM's broad specificity.
- Removal of BsCBM66 reduced SacC activity against levan by ~100-fold, while its addition to a non-specific enzyme conferred exolevanase activity.
Conclusions:
- BsCBM66 confers specificity for levan, a branched fructan, via an "avidity" mechanism, where the CBM and catalytic module bind different branches of the same molecule.
- This study reveals a unique CBM-mediated enzyme modulation mechanism and demonstrates how specificity can be achieved by integrating non-specific catalytic and binding modules.
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