Journal:Acta Cryst D:S2059798318014900

Structure of the AmyC GH13 alpha-amylase from Alicyclobacillus sp, reveals accommodation of starch branching points in the alpha-amylase family

Jon Agirre, Olga Moroz, Sebastian Meier, Jesper Brask, Astrid Munch, Tine Hoff, Carsten Andersen, Keith S. Wilsona and Gideon J. Davies ^[1]

Molecular Tour
The enzymatic degradation of starch has a myriad industrial applications. However, the branched nature of the polysaccharides that compose it poses problems, as branches have to be accommodated within an active centre best suited to linear polysaccharides. Alpha-amylases are glycoside hydrolases that break the α-1,4 bonds in starch and related glycans. The present work provides a rare insight into branch-point acceptance in these industrial catalysts.

The complex of α-amylase from Alicyclobacillus sp. 18711 (AliC) with acarbose was solved by molecular replacement, with two molecules of AliC in the asymmetric unit, at a resolution of 2.1 Å (6gxv). The fold, as expected, is a canonical three-domain arrangement with the A, B and C domains defined approximately as A, residues 4–104 and 210–397 (in deepskyblue), B, residues 105–209 (in yellow), and C, residues 398–484 (in white). A classical Ca²⁺–Na⁺–Ca²⁺ triad ^[2],^[3] is found at the A/B-domain interface. The structure of AliC was determined in the presence of the inhibitor acarbose (colored in green). As with many (retaining) α-amylase complexes, the acarbose is observed as a transglycosylated species, here a hexasaccharide which contains two of the acarviosin disaccharide motifs. The complex defines six subsites, -4 to +2, with the expected catalytic GH13 signature triad of Asp234 (nucleophile), Glu265 (acid/base) and Asp332 (interacting with O2/O3 of the -1 subsite sugar) all disposed for catalysis, here around the ²H₃ half-chair of the unsaturated cyclohexitol moiety. AliC must also be able to accommodate branching in the +2 subsite, which is consistent with the glucose moiety seen adjacent to O6 of the +2 sugar.

• Asp234 and Glu265 interactions.

A ‘branched-ligand’ AliC complex was obtained through co-crystallization, with crystals forming in a new space group. This form diffracted poorly and data could only be obtained to 2.95 Å resolution 6gya). Weak density in the -1 subsite, largely diffuse but greater than would be expected for discrete solvent, remained unmodelled. Density was clearer for a panose trisaccharide with an α-1,4-linked disaccharide in subsites +1 and +2 and, crucially, clear density for an α-1,6 branch accommodated in the +1 subsite, providing a structural context for the limit digest analysis of action on amylopectin starch. The binding of the branched oligosaccharide in subsites +1, +2 and +1' (oligosaccharide colored in green).

PDB references: Amylase in complex with acarbose, 6gxv; Amylase in complex with branched ligand, 6gya.

References

1. ↑ Agirre J, Moroz O, Meier S, Brask J, Munch A, Hoff T, Andersen C, Wilson KS, Davies GJ. The structure of the AliC GH13 alpha-amylase from Alicyclobacillus sp. reveals the accommodation of starch branching points in the alpha-amylase family. Acta Crystallogr D Struct Biol. 2019 Jan 1;75(Pt 1):1-7. doi:, 10.1107/S2059798318014900. Epub 2019 Jan 4. PMID:30644839 doi:http://dx.doi.org/10.1107/S2059798318014900
2. ↑ Machius M, Declerck N, Huber R, Wiegand G. Activation of Bacillus licheniformis alpha-amylase through a disorder-->order transition of the substrate-binding site mediated by a calcium-sodium-calcium metal triad. Structure. 1998 Mar 15;6(3):281-92. PMID:9551551
3. ↑ Brzozowski AM, Lawson DM, Turkenburg JP, Bisgaard-Frantzen H, Svendsen A, Borchert TV, Dauter Z, Wilson KS, Davies GJ. Structural analysis of a chimeric bacterial alpha-amylase. High-resolution analysis of native and ligand complexes. Biochemistry. 2000 Aug 8;39(31):9099-107. PMID:10924103