The ‘pair of sugar tongs’ site on the non‐catalytic domain C of barley α‐amylase participates in substrate binding and activity

Abstract

Some starch‐degrading enzymes accommodate carbohydrates at sites situated at a certain distance from the active site. In the crystal structure of barley α‐amylase 1, oligosaccharide is thus bound to the ‘sugar tongs’ site. This site on the non‐catalytic domain C in the C‐terminal part of the molecule contains a key residue, Tyr380, which has numerous contacts with the oligosaccharide. The mutant enzymes Y380A and Y380M failed to bind to β‐cyclodextrin‐Sepharose, a starch‐mimic resin used for α‐amylase affinity purification. The Kd for β‐cyclodextrin binding to Y380A and Y380M was 1.4 mm compared to 0.20–0.25 mm for the wild‐type, S378P and S378T enzymes. The substitution in the S378P enzyme mimics Pro376 in the barley α‐amylase 2 isozyme, which in spite of its conserved Tyr378 did not bind oligosaccharide at the ‘sugar tongs’ in the structure. Crystal structures of both wild‐type and S378P enzymes, but not the Y380A enzyme, showed binding of the pseudotetrasaccharide acarbose at the ‘sugar tongs’ site. The ‘sugar tongs’ site also contributed importantly to the adsorption to starch granules, as Kd = 0.47 mg·mL−1 for the wild‐type enzyme increased to 5.9 mg·mL−1 for Y380A, which moreover catalyzed the release of soluble oligosaccharides from starch granules with only 10% of the wild‐type activity. β‐cyclodextrin both inhibited binding to and suppressed activity on starch granules for wild‐type and S378P enzymes, but did not affect these properties of Y380A, reflecting the functional role of Tyr380. In addition, the Y380A enzyme hydrolyzed amylose with reduced multiple attack, emphasizing that the ‘sugar tongs’ participates in multivalent binding of polysaccharide substrates.

An enzyme family reunion — similarities, differences and eccentricities in actions on α-glucans

“…Subsequently mutagenesis in AMY1 (Søgaard et al 1993) and crystallography of AMY2 (Nielsen et al, manuscript in preparation). Also in human salivary α-amylase four Bozonnet et al (2007). b Fukuda et al (2005); M6 = A42P AMY2.…”

“…6) that by mutational analysis was demonstrated to be important for oligosaccharide binding. Thus K D for the starch mimic β-CD was determined by surface plasmon resonance analysis to augment from 0.20 mM for wild-type to 1.4 mM for Y380A AMY1 (Table 1; Bozonnet et al 2007). The Y380A AMY1 mutant had a 13-fold reduced affinity and about 90% reduced catalytic efficiency towards starch granules as compared to the wild-type enzyme.…”

Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase

“…However, characterization of separate binding events at different binding sites (AS and SBSs) in these glycoside hydrolases is challenging. Surface plasmon resonance (SPR) experiments showed that substrate binding affinity changed upon sitedirected mutagenesis of the two SBSs in barley α-amylase [2,3]. Nuclear magnetic resonance (NMR)-monitored titration experiments demonstrated that binding of xylooligo-saccharides to the Bacillus circulans xylanase AS and SBS occurs independently, whereas binding of longer chains to both sites occurs cooperatively [4].…”

New insight in cereal starch degradation: identification and structural characterization of four α-amylases in bread wheat

“…In the high pI isozyme AMY2, despite the presence of the corresponding tyrosine (Tyr378), the serine was replaced by a proline (Pro376) that contributes to a more restrained loop and consequently may prevent the binding [10]. Of the four α-amylase isoforms from wheat, it is very probable that isoforms TaAMY2 and TaAMY3 can undertake this "sugar-tongs"-like surface binding activity, whereas for the isoforms TaAMY1 and TaAMY4 the binding would be hindered due to serine to proline replacement (Figure 3d,e), as observed in the high pI isozyme of barley α-amylase [50].…”

“…(b) Structural models of representatives of four wheat α-amylase families shown in a similar orientation as that of the barley α-amylase structure. All four models were obtained at the fold recognition server Phyre-2 [41] using the low pI α-amylase isozyme AMY1 (PDB code: 2QPU; [50]) as the best template in each case at the confidence equal to 100, sequence identity no lower than 64% and at least 91% alignment coverage. (c-e) Structural overlay of domain C emphasizing the 'pair of sugar tongs' site identified in the barley α-amylase AMY1 and corresponding residues in the models of four wheat α-amylases.…”