α-amylase (α-1,4-glucan-4-glucanohydrolase; EC 18.104.22.168) catalyses the hydrolysis of the α-1,4-d
-glucosidic linkages in starch and related oligosaccharides and polysaccharides. The enzyme has been classified as a member of glycoside hydrolase (GH) family 13 (Henrissat, 1991
), which is the largest of the 109 GH families currently identified. It shows 100% sequence identity to its A. oryzae
homologue, for which a 3.0 Å resolution crystal structure was reported as long ago as 1984 (space group P
, with unit-cell parameters a
= 91.9, b
= 133.3, c
= 94.3 Å, β = 102.7° and three molecules per asymmetric unit; PDB code 2taa
; Matsuura et al.
). Since the A. oryzae
enzyme was isolated from ‘Takadistase Sankyo’ it was named TAKA-amylase. Being the first structurally characterized GH family 13 enzyme, the TAKA-amylase structure has often been used as the representative for the entire family (Kuriki & Imanaka, 1999
). Later, two P
crystal structures were published. The first one was of the native enzyme at 2.1 Å resolution, with unit-cell parameters a
= 50.9, b
= 67.2, c
= 132.7 Å and one molecule per asymmetric unit (PDB code 6taa
; Swift et al.
). The second was a 2.0 Å resolution structure of a complex of TAKA-amylase with the inhibitor acarbose, with unit-cell parameters a
= 50.8, b
= 67.1, c
= 131.6 Å and one molecule per asymmetric unit (PDB code 7taa
; Brzozowski & Davies, 1997
). Two other crystal forms have been reported for this enzyme, but their coordinates have not been deposited in the RCSB Protein Data Bank (Berman et al.
): a tetragonal form (space group P
2; unit-cell parameters a
= 63.8, c
= 231 Å; Akabori et al.
; Matsuura et al.
) and another monoclinic form (space group P
, unit-cell parameters a
= 75.0, b
= 104.3, c
= 67.4 Å, β = 104.5°; Swift et al.
The three-dimensional structures of TAKA-amylase revealed three domains: domain A (residues 1–121 and 177–380), which is the N-terminal catalytic (β/α)8
-barrel domain, domain B (residues 122–176), which is inserted between the third β-strand and the third α-helix of domain A, and domain C (residues 384–478), which is folded into an eight-stranded β-sandwich domain at the C-terminus of the enyzme. The active site is positioned at the bottom of a substrate-binding groove at the C-terminal ends of β-strands β4, β5 and β7 of the catalytic domain. Seven substrate-binding subsites (numbered from −4 to +3) were proposed for TAKA-amylase on the basis of kinetic studies performed in the early 1970s (Nitta et al.
). Six of them were structurally characterized in a complex of TAKA-amylase with an acarbose-derived hexasaccharide (Brzozowski & Davies, 1997
). The non-reducing end of the carbohydrate chain was bound at the −3 subsite, with the sugar residues extending into subsite +3 (Fig. 1
). Cleavage of the α-1,4-glycosidic bond in GH family 13 occurs between subsites −1 and +1 via
a double-displacement mechanism involving a covalent glucosyl-enzyme intermediate at subsite −1 and with retention of the α-anomeric configuration of the sugar upon hydrolysis (Koshland, 1953
; Uitdehaag et al.
). Three acidic amino acids participate in the hydrolysis reaction: Glu230 acts as catalytic acid/base and Asp206 is the nucleophile, while Asp297 is involved in stabilization of the oxocarbenium ion-like transition state (Matsuura et al.
; Uitdehaag et al.
). We report here the purification, crystallization and structure determination of a hitherto undescribed monoclinic crystal form of the A. niger
α-amylase in complex with maltose, the shortest chain-length substrate of this enzyme (Nitta et al.
), at 1.8 Å resolution. Furthermore, a 1.6 Å new orthorhombic crystal form is presented (P
2). The two molecules A
in the asymmetric unit of the monoclinic unit cell bind four and two maltose molecules, respectively. In both proteins maltose molecules occupy subsites −1 and −2 as well as +1 and +2 in the active-site cleft. In addition, in molecule A
two more maltose molecules are bound. One occupies the until now unobserved subsites +4 and +5 in the active-site groove. The other binds to two distant binding sites d1 and d2, also previously unobserved. These latter two binding sites are located in a loop connecting the A and C domains and could function to bind the polysaccharide chain extending from the active site. Furthermore, alternative modes of sugar binding at subsites +1 and +2 are observed when comparing the maltose–α-amylase with the acarbose–TAKA-amylase complexes (Brzozowski & Davies, 1997
). This plasticity of the active-site groove in the proximity to the catalytic centre might be important both for the formation of the productive substrate–enzyme complex as well as for the release of the product from the +1 to +n
Figure 1 Maltose versus acarbose binding in A. niger α-amylase. (a) Schematic presentation of the acarbose-derived hexasaccharide bound in the TAKA-amylase structure (PDB code 7taa; Brzozowski & Davies, 1997 ) with the inhibitor acarbose (more ...)