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

ABSTRACT: ?-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?Å 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.

SUBMITTER: Agirre J

PROVIDER: S-EPMC6333287 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Project description:The alpha-amylase from Bacillus sp. no. 195 (BAA) consists of two domains: one is the catalytic domain similar to alpha-amylases from animals and Streptomyces in the N-terminal region; the other is the functionally unknown domain composed of an approx. 90-residue direct repeat in the C-terminal region. The gene coding for BAA was expressed in Streptomyces lividans TK24. Three active forms of the gene products were found. The pH and thermal profiles of BAAs, and their catalytic activities for p-nitrophenyl maltopentaoside and soluble starch, showed almost the same behaviours. The largest, 69 kDa, form (BAA-alpha) was of the same molecular mass as that of the mature protein estimated from the nucleotide sequence, and had raw-starch-binding and -degrading abilities. The second largest, 60 kDa, form (BAA-beta), whose molecular mass was the same as that of the natural enzyme from Bacillus sp. no. 195, was generated by proteolytic processing between the two repeat sequences in the C-terminal region, and had lower activities for raw starch binding and degrading than those of BAA-alpha. The smallest, 50 kDa, form (BAA-gamma) contained only the N-terminal catalytic domain as a result of removal of the C-terminal repeat sequence, which led to loss of binding and degradation of insoluble starches. Thus the starch adsorption capacity and raw-starch-degrading activity of BAAs depends on the existence of the repeat sequence in the C-terminal region. BAA-alpha was specifically adsorbed on starch or dextran (alpha-1,4 or alpha-1,6 glucan), and specifically desorbed with maltose or beta-cyclodextrin. These observations indicated that the repeat sequence of the enzyme was functional in the starch-binding domain (SBD). We propose the designation of the homologues to the SBD of glucoamylase from Aspergillus niger as family I SBDs, the homologues to that of glucoamylase from Rhizopus oryzae as family II, and the homologues of this repeat sequence of BAA as family III.

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

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