The structure of the AliC GH13-amylase from Alicyclobacillus sp. reveals the accommodation of starch branching points in the -amylase family

alpha-Amylases are glycoside hydrolases that break the -1,4 bonds in starch and related glycans. The degradation of starch is rendered difficult by the presence of varying degrees of -1,6 branch points and their possible accommodation within the active centre of -amylase enzymes. Given the myriad industrial uses for starch and thus also for -amylase-catalysed starch degradation and modification, there is considerable interest in how different -amylases might accommodate these branches, thus impacting on the potential processing of highly branched post-hydrolysis remnants (known as limit dextrins) and societal applications. Here, it was sought to probe the branch-point accommodation of the Alicyclobacillus sp. CAZy family GH13 -amylase AliC, prompted by the observation of a molecule of glucose in a position that may represent a branch point in an acarbose complex solved at 2.1 angstrom resolution. Limit digest analysis by two-dimensional NMR using both pullulan (a regular linear polysaccharide of -1,4, -1,4, -1,6 repeating trisaccharides) and amylopectin starch showed how the Alicyclobacillus sp. enzyme could accept -1,6 branches in at least the -2, +1 and +2 subsites, consistent with the three-dimensional structures with glucosyl moieties in the +1 and +2 subsites and the solvent-exposure of the -2 subsite 6-hydroxyl group. Together, the work provides a rare insight into branch-point acceptance in these industrial catalysts.