Birte Kramhøft

The 'pair of sugar tongs' site on the non-catalytic domain C of barley alpha-amylase participates in substrate binding and activity

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.

Mapping of barley α-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

Subsite affinity maps of long substrate binding clefts in barley α‐amylases, obtained using a series of maltooligosaccharides of degree of polymerization of 3–12, revealed unfavorable binding energies at the internal subsites −3 and −5 and at subsites −8 and +3/+4 defining these subsites as binding barriers. Barley α‐amylase 1 mutants Y105A and T212Y at subsite −6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high‐affinity subsites (−2 and +2) and barriers. The double mutant Y105A‐T212Y displayed a hybrid subsite affinity profile, converting barriers to binding areas. These findings highlight the dynamic binding energy distribution and the versatility of long maltooligosaccharide derivatives in mapping extended binding clefts in α‐amylases.

Degradation of the starch components amylopectin and amylose by barley α-amylase 1: role of surface binding site 2.

Barley α-amylase isozyme 1 (AMY1, EC 3.2.1.1) contains two surface binding sites, SBS1 and SBS2, involved in the degradation of starch granules. The distinct role of SBS1 and SBS2 remains to be fully understood. Mutational analysis of Tyr-380 situated at SBS2 previously revealed that Tyr-380 is required for binding of the amylose helix mimic, β-cyclodextrin. Also, mutant enzymes altered at position 380 displayed reduced binding to starch granules. Similarly, binding of wild type AMY1 to starch granules was suppressed in the presence of β-cyclodextrin. We investigated the role of SBS2 by comparing kinetic properties of the wild type AMY1 and the Y380A mutant enzyme in hydrolysis of amylopectin, amylose and β-limit dextrin, and the inhibition by β-cyclodextrin. Progress curves of the release of reducing ends revealed a bi-exponential hydrolysis of amylopectin and β-limit dextrin, whereas hydrolysis of amylose progressed mono-exponentially. β-Cyclodextrin, however, inhibited only one of the two reaction rates of amylopectin and β-limit dextrin hydrolysis, whereas hydrolysis of amylose was unaffected. The Y380A enzyme showed no detectable inhibition by β-cyclodextrin but displayed similar kinetics to the inhibited wild type AMY1. These results point to SBS2 as an important binding site in amylopectin depolymerization.

Engineering of Barley α-Amylase

Abstract Protein engineering of barley α-amylase addressed the roles of Ca2+ in activity and inhibition by barley α-amylase/subtilisin inhibitor (BASI), multiple attach in polysaccharide hydrolysis, secondary starch binding sites, and BASI hot spots in AMY2 recognition. AMY1/AMY2 isozyme chimeras faciliatated assignment of function to specific regions of the structure. An AMY1 fusion with starch binding domain and AMY1 mutants in the substrate binding cleft gave degree of multiple attack of 0.9–3.3, compared to 1.9 for wild-type. About 40% of the secondary attacks, succeeding the initial endo-attack, produced DP5-10 maltooligosaccharides in similar proportion for all enzyme variants, whereas shorter products, comprising about 25%, varied depending on the mutation. Secondary binding sites were important in both multiple attack and starch granule hydrolysis. Surface plasmon resonance and inhibition analyses indicated the importance of fully hydrated Ca2+ at the AMY2/BASI interface to strengthen the complex. Engineering of intermolecular contacts in BASI modulated the affinity for AMY2 and the target enzyme specificity.

Interactions of barley α-amylase isozymes with Ca2 + , substrates and proteinaceous inhibitors

α-Amylases are endo-acting retaining enzymes of glycoside hydrolase family 13 with a catalytic (β/α)8-domain containing an inserted loop referred to as domain B and a C-terminal anti-parallel β-sheet termed domain C. New insights integrate the roles of Ca2 + , different substrates, and proteinaceous inhibitors for α-amylases. Isozyme specific effects of Ca2 +  on the 80% sequence identical barley α-amylases AMY1 and AMY2 are not obvious from the two crystal structures, containing three superimposable Ca2 +  with identical ligands. A fully hydrated fourth Ca2 +  at the interface of the AMY2/barley α-amylase/subtilisin inhibitor (BASI) complex interacts with catalytic groups in AMY2, and Ca2 +  occupies an identical position in AMY1 with thiomaltotetraose bound at two surface sites. EDTA-treatment, DSC, and activity assays indicate that AMY1 has the highest affinity for Ca2 + . Subsite mapping has revealed that AMY1 has ten functional subsites which can be modified by means protein engineering to modulate the substrate specificity. Other mutational analyses show that surface carbohydrate binding sites are critical for interaction with polysaccharides. The conserved Tyr380 in the newly discovered ‘sugar tongs’ site in domain C of AMY1 is thus critical for binding to starch granules. Furthermore, mutations of binding sites mostly reduced the degree of multiple attack in amylose hydrolysis. AMY1 has higher substrate affinity than AMY2, but isozyme chimeras with AMY2 domain C and other regions from AMY1 have higher substrate affinity than both parent isozymes. The latest revelations addressing various structural and functional aspects that govern the mode of action of barley α-amylases are reported in this review.