α−amylases are glycoside hydrolases that break the α−1,4 bonds in starch and related glycans. The degradation of starch is rendered difficult by varying degrees of α−1,6 branch points and their possible accommodation within the active centre of α−amylase enzymes. Given the myriad industrial uses for starch and thus also for α−amylase-catalysed starch degradation and modification, there is considerable interest in how different α−amylases might accommodate these branches thus impacting on the potential limit dextrins and societal applications. Here, we sought to probe the branch-point accommodation of the Alicyclobacillus sp. CAZy family GH13 α−amylase, prompted by our observation of a molecule of glucose in the position that may represent a branch point in an acarbose complex solved at 2.1 Å resolution. Limit digest analysis, by 2D NMR, using both pullulan (a regular linear polysaccharide of α−1,4, α−1,4, α−1,6 repeating trisaccharides) and amylopectin starch showed how the Alicyclobacillus sp enzyme could accept α−1,6 branches in, at least, -2, +1 and +2 subsites consistent with 3-D structures with glucosyl moieties in +1 and +2 subsites and the solvent exposure of the -2 6-hydroxyl group. Together the work provides a rare insight into branch point acceptance in these industrial catalysts.
|Journal||Acta crystallographica Section D: Structural biology|
|Number of pages||7|
|Publication status||Published - 2019|
- AliC GH13 α-analyse
- Starch brancing points
- Glycoside hydrolases
- Carbohydrate-active enzymes