Sophie Bozonnet

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.

Two Secondary Carbohydrate Binding Sites on the Surface of Barley α-Amylase 1 Have Distinct Functions and Display Synergy in Hydrolysis of Starch Granules

Some polysaccharide processing enzymes possess secondary carbohydrate binding sites situated on the surface far from the active site. In barley alpha-amylase 1 (AMY1), two such sites, SBS1 and SBS2, are found on the catalytic (beta/alpha)(8)-barrel and the noncatalytic C-terminal domain, respectively. Site-directed mutagenesis of Trp(278) and Trp(279), stacking onto adjacent ligand glucosyl residues at SBS1, and of Tyr(380) and His(395), making numerous ligand contacts at SBS2, suggested that SBS1 and SBS2 act synergistically in degradation of starch granules. While SBS1 makes the major contribution to binding and hydrolysis of starch granules, SBS2 exhibits a higher affinity for the starch mimic beta-cyclodextrin. Compared to that of wild-type AMY1, the K(d) of starch granule binding by the SBS1 W278A, W279A, and W278A/W279A mutants thus increased 15-35 times; furthermore, the k(cat)/K(m) of W278A/W279A was 2%, whereas both affinity and activity for Y380A at SBS2 were 10% of the wild-type values. Dual site double and triple SBS1/SBS2 substitutions eliminated binding to starch granules, and the k(cat)/K(m) of W278A/W279A/Y380A AMY1 was only 0.4% of the wild-type value. Surface plasmon resonance analysis of mutants showed that beta-cyclodextrin binds to SBS2 and SBS1 with K(d,1) and K(d,2) values of 0.07 and 1.40 mM, respectively. A model that accounts for the observed synergy in starch hydrolysis, where SBS1 and SBS2 bind ordered and free alpha-glucan chains, respectively, thus targeting the enzyme to single alpha-glucan chains accessible for hydrolysis, is proposed. SBS1 and SBS2 also influence the kinetics of hydrolysis for amylose and maltooligosaccharides, the degree of multiple attack on amylose, and subsite binding energies.

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.

Multi-site substrate binding and interplay in barley α-amylase 1

Certain starch hydrolases possess secondary carbohydrate binding sites outside of the active site, suggesting that multi‐site substrate interactions are functionally significant. In barley α‐amylase both Tyr380, situated on a remote non‐catalytic domain, and Tyr105 in subsite −6 of the active site cleft are principal carbohydrate binding residues. The dual active site/secondary site mutants Y105A/Y380A and Y105A/Y380M show that each of Tyr380 and Tyr105 is important, albeit not essential for binding, degradation, and multiple attack on polysaccharides, while Tyr105 predominates in oligosaccharide hydrolysis. Additional delicate structure/function relationships of the secondary site are uncovered using Y380A/H395A, Y380A, and H395A AMY1 mutants.

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

Abstractα-Glucans in general, including starch, glycogen and their derived oligosaccharides are processed by a host of more or less closely related enzymes that represent wide diversity in structure, mechanism, specificity and biological role. Sophisticated three-dimensional structures continue to emerge hand-in-hand with the gaining of novel insight in modes of action. We are witnessing the “test of time” blending with remaining questions and new relationships for these enzymes. Information from both within and outside of ALAMY_3 Symposium will provide examples on what the family contains and outline some future directions. In 2007 a quantum leap crowned the structural biology by the glucansucrase crystal structure. This initiates the disclosure of the mystery on the organisation of the multidomain structure and the “robotics mechanism” of this group of enzymes. The central issue on architecture and domain interplay in multidomain enzymes is also relevant in connection with the recent focus on carbohydrate-binding domains as well as on surface binding sites and their long underrated potential. Other questions include, how different or similar are glycoside hydrolase families 13 and 31 and is the lid finally lifted off the disguise of the starch lyase, also belonging to family 31? Is family 57 holding back secret specificities? Will the different families be sporting new “eccentric” functions, are there new families out there, and why are crystal structures of “simple” enzymes still missing? Indeed new understanding and discovery of biological roles continuously emphasize value of the collections of enzyme models, sequences, and evolutionary trees which will also be enabling advancement in design for useful and novel applications.

Roles of multiple surface sites, long substrate binding clefts, and carbohydrate binding modules in the action of amylolytic enzymes on polysaccharide substrates

Germinating barley seeds contain multiple forms of α-amylase, which are subject to both differential gene expression and differential degradation as part of the repertoire of starch-degrading enzymes. The α-amylases are endo-acting and possess a long substrate binding cleft with a characteristic subsite binding energy profile around the catalytic site. Furthermore, several amylolytic enzymes that facilitate attack on the natural substrate, i.e. the endosperm starch granules, have secondary sugar binding sites either situated on the surface of the protein domain or structural unit that contains the catalytic site or belonging to a separate starch binding domain. The role of surface sites in the function of barley α-amylase 1 has been investigated by using mutational analysis in conjunction with carbohydrate binding analyses and crystallography. The ability to bind starch depends on the surface sites and varies for starch granules of different genotypes and botanical origin. The surface sites, moreover, are candidates for being involved in degradation of polysaccharides by a multiple attack mechanism. Future studies of the molecular nature of the multivalent enzyme-substrate interactions will address surface sites in both barley α-amylase 1 and in the related isozyme 2.

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.