X-ray structure of acarbose bound to amylomaltase from Thermus aquaticus . Implications for the synthesis of large cyclic glucans

Abstract

As a member of the a-amylase superfamily of enzymes, amylomaltase catalyzes either the transglycosylation from one a-1,4 glucan to another or an intramolecular cyclization. The latter reaction is typical for cyclodextrin glucanotransferases. In contrast to these enzymes, amylomaltase catalyzes the formation of cyclic glucans with a degree of polymerization larger than 22. To characterize the factors that determine the size of the synthesized cycloamyloses, we have analyzed the X-ray structure of amylomaltase from Thermus aquaticus in complex with the inhibitor acarbose, a maltotetraose derivative, at 1.9 A Ê resolution. Two acarbose molecules are bound to the enzyme, one in the active site groove at subsite 23 to 11 and a second one < 14 A Ê away from the nonreducing end of the acarbose bound to the catalytic site. The inhibitor bound to the catalytic site occupies subsites 23 to 11. Unlike the situation in other enzymes of the a-amylase family, the inhibitor is not processed and the inhibitory cyclitol ring of acarbose, which mimicks the half chair conformation of the transition state, does not bind to catalytic subsite 21. The minimum ring size of cycloamyloses produced by this enzyme is proposed to be determined by the distance of the specific substrate binding sites at the active site and near Tyr54 and by the size of the 460s loop. The 250s loop might be involved in binding of the substrate at the reducing end of the scissile bond.Keywords: acarbose; a-amylase family; (b, a) 8 barrel; glucanotransferase; protein crystallography.Besides cellulose, starch is one of the most abundant carbohydrate polymers in nature. Starch consists of two compounds, the linear amylose with a-1,4 linked glucoses and the branched amylopectin with a-1,4 and a-1,6 connected repeating units. Due to its important role in energy storage and uptake many enzymes are known to act on starch. All starch hydrolases and related enzymes have been classified together with the other O-glycosyl hydrolases into a group of glycoside hydrolase families, which covers more than 80 families [1,2]. Amylomaltase belongs to the a-amylase family (family 13), which includes about 20 different enzymes. These proteins catalyze the transformation of a-1,4 and a-1,6 glucosidic linkages with retention of the anomeric center [3±6]. Well studied enzymes of this family are the a-amylases and the cyclodextrin glucanotransferases (CGTases). a-Amylases catalyze the hydrolysis of the a-1,4 glycosidic bonds of amylose, whereas CGTases catalyze a reversible transfer of a-1,4 glucans. By intramolecular cyclization these enzymes synthesize small cycloamyloses, termed cyclodextrins. The final cycloamylose products of CGTase activity consist mainly of 6±8 glucose units. However, CGTase initially produces large cycloamyloses, which are subsequently transformed into the smaller cyclodextrins [7].The doughnut-shaped cyclodextrins can form complexes with guest molecules in their central hydrophobic cavity and have found many applications in the food, pharmaceutical and cosmetic...

Tyrosine 105 and Threonine 212 at Outermost Substrate Binding Subsites –6 and +4 Control Substrate Specificity, Oligosaccharide Cleavage Patterns, and Multiple Binding Modes of Barley α-Amylase 1

“…Repeated A, SH, and DH generate left helices, which C or E disrupt (46). The present S1, S2, and S3 solutions share disaccharide conformations with those compiled for oligosaccharides bound in different GH-H crystal structures (28). S2 AMY1 adopts mainly the more stable DH with a single break (C: 118, Ϫ106) between subsites ϩ2 and ϩ3.…”

“…The structures validate modeled substrate complexes and subsite maps (8,12,24,25) by highlighting (i) aromatic stacking and hydrogen bonds between carbohydrate and protein (9,10,21,24,26,27), (ii) conformational features of the bound carbohydrate (8,21,28), (iii) conserved geometry of the catalytic site (10,14,15,21,22,29,30), and (iv) substrate binding motifs in ␤ 3 ␣ loops of the catalytic (␤/␣) 8 barrel (15). The macromolecular substrate starch most probably also interacts with distinct areas outside the cleft as suggested by oligosaccharide occupation at so-called surface or secondary sites in several structures from GH-H (10,14,28,31). This additional substrate binding is only proven, however, for starch binding domains of family 20 (afmb.…”

“…The protein engineering strategy used took advantage of: (i) the modeled AMY2/maltodecaose (25), (ii) insight in earlier AMY1 mutants of substrate binding residues (4, 30, 39 -41), and (iii) numerous crystal structures of ␣-amylases from Aspergillus oryzae (21), Aspergillus niger (50), B. licheniformis (51), Bacillus subtilis (11), Pseudoalteromonas haloplanktis (29), AMY2 (10), porcine (9), and human pancreas (8), and other GH-H members; e.g. CGTase (16,22,52), TVAII ␣-amylase (53), and amylomaltase (28). The enzymatic activities on starch, a short-chain amylose DP17, and various oligosaccharides of the eight mutants of AMY1 residues Tyr 105 and Thr 212 at subsites Ϫ6 and ϩ4, including the AMY2 mimic [T212Y]AMY1, were interpreted using structural models established by AMY1/maltododecaose docking.…”

Selectivity of the surface binding site (SBS) on barley starch synthase I

“…1) and forms numerous interactions with other residues in HvSSI (Cuesta- Seijo et al 2013). This architecture is common for SBSs that bind α-1,4 linked d-glucose chains and mutation of the aromatic residues that the α-glucan curls around has resulted in decreases of activity in all cases (Przylas et al 2000;Robert et al 2005;Sevcík et al 2006;Bozonnet et al 2007;Nielsen et al 2009;Díaz et al 2011). Thus the F358A mutant in HvSSI had four fold lower activity on soluble starch and six-and 12-fold lower activity on oyster and rabbit liver glycogen, respectively, compared to HvSSI wild-type.…”

Reaction Mechanism Based on X-ray Crystallography at Atomic Resolution of Endopolygalacutronase I from Fungus <i>Stereum purpureum</i>

“…Thus, the ring structure of GalpA in the 1 subsite was built based on the distorted conformation of the valienamine pseudosaccharide moiety in acarbose, a transition state analogue inhibitor of glucoamylase. 23) The resulting model of the di galacturonate substrate shows the following characteristics in the 1 subsite: 1) The pyranose ring of the GalpA residue has been distorted into the 4 H3 half chair conformation. In that half chair conformation, the C2, C1, O5 and C5 atoms are co planar, a very important characteristic of the postulated oxocarbenium ion like transition state in the glycosyl hydrolase reaction.…”