Crystal structure of a raw-starch-degrading bacterial α-amylase belonging to subfamily 37 of the glycoside hydrolase family GH13

Abstract

Subfamily 37 of the glycoside hydrolase family GH13 was recently established on the basis of the discovery of a novel α-amylase, designated AmyP, from a marine metagenomic library. AmyP exhibits raw-starch-degrading activity and consists of an N-terminal catalytic domain and a C-terminal starch-binding domain. To understand this newest subfamily, we determined the crystal structure of the catalytic domain of AmyP, named AmyPΔSBD, complexed with maltose, and the crystal structure of the E221Q mutant AmyPΔSBD complexed with maltotriose. Glu221 is one of the three conserved catalytic residues, and AmyP is inactivated by the E221Q mutation. Domain B of AmyPΔSBD forms a loop that protrudes from domain A, stabilizes the conformation of the active site and increases the thermostability of the enzyme. A new calcium ion is situated adjacent to the -3 subsite binding loop and may be responsible for the increased thermostability of the enzyme after the addition of calcium. Moreover, Tyr36 participates in both stacking and hydrogen bonding interactions with the sugar motif at subsite -3. This work provides the first insights into the structure of α-amylases belonging to subfamily 37 of GH13 and may contribute to the rational design of α-amylase mutants with enhanced performance in biotechnological applications.

Functional Characterization of Recombinant Raw Starch Degrading α-Amylase from Roseateles terrae HL11 and Its Application on Cassava Pulp Saccharification

“…Several factors are related to raw starch granule digestibility, for example, surface area, number of pores on the granule, crystalline organization, degree of crystallinity, shape and granule size, which affects the binding of enzymes via the starch-binding domain [52,53,[61][62][63][64]. Among three starches from different botanical sources, HL11Amy exhibited the highest specific activity and kinetic parameters (V max , k cat and k cat /K m ) toward raw rice starch, followed by raw potato starch and raw cassava starch.…”

Resistant starch, microbiome, and precision modulation

“… 80 This remarkable difference may simply be due to rice starch’s higher intrinsic digestibility, 26 but may also be explained by specificity imparted by its starch-binding domain, CBM69. 81 At present, there is no obvious pattern by which the copy number of GH13’s or CBM family domains can predict whether a bacterial strain can degrade starch or RS ( Table 2 ).…”

Mangrove soil as a source for novel xylanase and amylase as determined by cultivation-dependent and cultivation-independent methods

“…This method favors the discovery of enzymes from uncultivated microorganisms without performing a cultivation step [15]. The metagenomics libraries have permitted the discovery of xylanases [16] and α-amylases [17].…”