Three-way Stabilization of the Covalent Intermediate in Amylomaltase, an α-Amylase-like Transglycosylase

Abstract

Amylomaltases are glycosyl hydrolases belonging to glycoside hydrolase family 77 that are capable of the synthesis of large cyclic glucans and the disproportionation of oligosaccharides. Using protein crystallography, we have generated a flip book movie of the amylomaltase catalytic cycle in atomic detail. The structures include a covalent glycosyl enzyme intermediate and a covalent intermediate in complex with an analogue of a cosubstrate and show how the structures of both enzyme and substrate respond to the changes required by the catalytic cycle as it proceeds. Notably, the catalytic nucleophile changes conformation dramatically during the reaction. Also, Gln-256 on the 250s loop is involved in orienting the substrate in the ؉1 site. The absence of a suitable base in the covalent intermediate structure explains the low hydrolysis activity.Amylomaltases are 4-␣-glucanotransferase enzymes (1-4) that are structurally and mechanistically related to ␣-amylases (family 13 of the glycoside hydrolases or GH13) (5-7) despite their classification in a separate glycoside hydrolase family (glycoside hydrolase family 77). However, amylomaltases almost exclusively catalyze transglycosylation reactions, whereas ␣-amylase-like enzymes mostly catalyze hydrolysis. As a result, the amylomaltases perform so-called disproportionation reactions, the net effect of which is that two amylose chains of certain lengths react in such a way that one of the chains (the "donor") becomes shorter and the other one (the "acceptor") longer as shown in Reaction 1.Instead of using two different sugar chains for this reaction, the enzyme can also take the non-reducing end of the donor substrate as the acceptor substrate, which results in the formation of a cyclic glucan product of at least 16 glucose units (8 -11). These properties, amylose disproportionation and the synthesis of large cyclic glucans, make these enzymes interesting biocatalysts for the synthesis of valuable fine chemicals and pharmaceutically important materials (12)(13)(14). Over the years, several incisive studies have contributed to our current understanding of catalysis by ␣-amylase-type enzymes. Importantly, a structure of a covalent reaction intermediate, obtained by Uitdehaag et al. (15), provided a detailed look into the active site, showing among other things how the (mutated) acid/base catalyst is poised to activate a water molecule for attack on the C 1 carbon atom. Since then, two other examples of covalent glycosyl enzyme intermediate structures of ␣-amylase-type enzymes have been published (16,17). These structures provided insight into the hydrolysis mechanism, i.e. in the way in which a covalent intermediate is attacked by a water molecule. Three conserved carboxylic acid residues play a central role in the catalytic mechanism (15, 18 -20). The first carboxylic acid residue acts as an acid/base catalyst that protonates the oxygen atom of the scissile glycosidic bond. Simultaneously, the C 1 carbon atom is attacked by the second carboxylate, the nucleophile, which...

Structural Insight into the Bifunctional Mechanism of the Glycogen-debranching Enzyme TreX from the Archaeon Sulfolobus solfataricus

“…The glucose moiety at the reducing end of the acarbose ligand is thought to be cleaved, resulting in the covalent intermediate bound to Asp 363 . The inhibitory acarviosine moiety binds to subsites Ϫ2 to Ϫ3, showing a similar binding mode to the acarbose intermediate observed in the amylomaltase structure (18,19 491 , is positioned in subsite Ϫ4 and is involved in a stacking interaction with the substrate. Given the typical left-handed helical configuration of the substrate, helix ␣4 may provide a platform for the stable binding of the longer substrate, explaining the substrate specificity of TreX.…”

“…In the structure of Thermotoga maritima 4-␣-glucanotransferase, a loop in the N-terminal domain from the same molecule forms a clamp over the active site that captures the sugar rings of the substrate at the acceptor-binding site (26). In the amylomaltase structure, the corresponding loop (aa 246 -258) is involved in acceptor binding (18), whereas the lid (aa 627-630) protruding from the helix bundle deeply penetrates the active site upon binding of the substrate in the 4-␣-glucanotransferase structure (27). In cyclodextrin glucanotransferase, tyrosine or phenylalanine corresponding to the same region also protrudes, whereas various mutants of that region affect cyclization, demonstrating its involvement in transfer activity (28).…”

A Novel Transition-state Analogue for Lysozyme, 4-O-β-Tri-N-acetylchitotriosyl Moranoline, Provided Evidence Supporting the Covalent Glycosyl-enzyme Intermediate*

“…However, the situation has been changed after the report of a crystal structure of HEWL covalently bound to C1 carbon of the Ϫ1 sugar, which exhibits a chair conformation with the C1 carbon in sp 3 hybridization (12). Nowadays, the catalytic mechanism through the covalently bound intermediate is more widely accepted by enzyme researchers (33)(34)(35). However, the crystal structure capturing the covalent glycosyl-HEWL intermediate was obtained only by use of the inactive mutant HEWL(E35Q) and 2-acetamido-2-deoxy-␤-D-glucopyranosyl-(134)-2-deoxy-2-fluoro-␤-D-glucopyranosyl fluoride (NAG2FGlcF).…”

Enzymatic Synthesis of Acarviosyl-maltooligosaccharides Using Disproportionating Enzyme 1

“…This is the first example of an acarbosemodifying enzyme in GH family 77. Other GH family 77 enzymes, Thermus aquaticus amylomaltase 23) and Thermus thermophilus amylomaltase, 24) may possess such a property to modify acarbose. Their crystal structures, of complex form bound with acarbose, indicate that the acarviosyl unit of acarbose occupies not the active site, but subsites À3 and À2.…”