Idnhdmerf Venhdkprf Leshdtsrl Vskhdnqtl Idvhdgmtl

Figure 4 Amino acid sequence alignment of the four conserved regions in members of the «-amylase family. TAA: «-amylase, Aspergillus oryzae (Taka-amylase A) (33); CGT: CGTase, Bacillus circulans strain 251 (31); CD: cyclodextrinase, Klebsiella oxytoca (72); PUL: pullulanase, Klebsiella aerogenes (73); ISO: isoamylase, Pseudomonas amyloderamosa (74). The residues are numbered according to the CGTase from B. circulans strain 251. An asterisk indicates amino acid identity; a dot indicates amino acid similarity.

«-amylases (33) and CGTases (31, 34-37) are quite similar. «-Amylases generally consist of three structural domains, A, B, and C, while CGTases show a similar domain organization with two additional domains, D and E (Fig. 5). Domain A contains a highly symmetrical fold of eight parallel ^-strands arranged in a barrel encircled by eight «-helices. This so-called (6/«)8-or TIM-barrel catalytic domain of 300-400 residues is present in all enzymes of the «-amylase family (38). The (^/«)8 barrel was first found in the structure of chicken muscle triose phosphate isomerase (39), but it has been shown to be widespread in functionally diverse enzymes (40). Several prolines and glycines flanking loops connecting the strands and «-helices have been found to be highly conserved in these enzymes (41). The catalytic and substrate-binding residues conserved in the «-amylase family are located in loops at the C-terminal of y8-strands in domain A. The loop between ^-strand 3 and «-helix 3 of the catalytic domain is rather large and is regarded as a separate structural domain. This B-domain consists of 44-133 amino acid residues and contributes to substrate binding. The C-domain is ~ 100 amino acids long and has an antiparallel ^-sand-wich fold. Domain C of the CGTase from B. circulans strain 251 contains one of the maltose-binding sites (MBS) observed in the structure derived from maltose-dependent crystals (see below) (31). This MBS is involved in raw starch binding (42); the C-domain thus contributes in substrate binding. This C-domain may determine bond specificity, since in enzymes hydrolyz-ing or forming «(1,6) bonds (e.g., pullulanase, isoamy-lase, branching enzyme) the A-domain is followed by a different domain (Fig. 5) (43). The D-domain is ~ 90 amino acids in size and is almost exclusively found in CGTases; it carries an immunoglobulin fold but its function remains unknown. The E-domain is more widespread in starch degrading enzymes. In enzymes of the «-amylase family it is present as the C-terminal domain; in glucoamylases (family 15 of glycoside hydrolases) it is attached to the C- or N-terminus of the catalytic domain via a glycosylated linker (Fig. 5). The E-domain consists of ~ 110 amino acids and is responsible for the adsorption of the enzyme onto granular starch (see below).

All CGTases studied not only catalyze the three transglycosylation reactions but also display hydrolytic activity (Fig. 2). Incubation of CGTase with starch thus may not only yield cyclodextrins but also linear products. In some cases this has resulted in misidentification of CGTase enzymes. The Thermoanaerobacterium thermosulfurigenes EM1 CGTase was initially identified as an «-amylase because of its relatively high hydrolytic activity (44). Further studies showed that it possesses a clear cycli-zation activity, as in other CGTases; amino acid alignments with other CGTases also show a high overall sequence similarity, with all five domains present (25). The CGTase converts starch not only into cyclo-dextrins (35%) but also into linear sugars (11%) (45). In contrast, the CGTase from B. circulans strain 251 has minor hydrolytic activity; this enzyme exclusively forms cyclodextrins (46).

Recently the 3D structure of the Bacillus stearother-mophilus "maltogenic" «-amylase Novamyl has been determined (47). Unlike the conventional «-amylases of family 13, Novamyl exhibits the five-domain structure associated with CGTase enzymes (Fig. 5, G2A). Its overall sequence similarity with CGTase is high, and the 3D structures are very similar. Most interestingly, Novamyl contains a 5 amino acid residue insertion in the B-domain, partially enclosing the active site at the -3 subsite. The absence of this insertion in CGTases allows for additional subsites, with up to -7 characterized crystallographically (32). Novamyl thus is either an «-amylase or a CGTase with unusual properties. Studies of evolutionary relationships within the «-amylase family have provided evidence that

Figure 5 Domain level organization of starch degrading enzymes. CGT: CGTase, Bacillus circulans; G2A: maltogenic a-amylase, Bacillus stearothermophilus; G4A: maltotetraose forming a-amylase, Pseudomonas stutzerl; TAA: a-amylase, Aspergillus oryzae (Taka-amylase A); CD: cyclodextrinase, Klebsiella oxytoca; ISO: isoamylase, Pseudomonas amyloder-amosa; PUL: pullulanase, Klebsiella aerogenes: GA: glucoa-mylase (family 15 of glycoside hydrolases) from Aspergillus niger. (From Ref. 43.)

Figure 5 Domain level organization of starch degrading enzymes. CGT: CGTase, Bacillus circulans; G2A: maltogenic a-amylase, Bacillus stearothermophilus; G4A: maltotetraose forming a-amylase, Pseudomonas stutzerl; TAA: a-amylase, Aspergillus oryzae (Taka-amylase A); CD: cyclodextrinase, Klebsiella oxytoca; ISO: isoamylase, Pseudomonas amyloder-amosa; PUL: pullulanase, Klebsiella aerogenes: GA: glucoa-mylase (family 15 of glycoside hydrolases) from Aspergillus niger. (From Ref. 43.)

CGTase enzymes evolved from a-amylases (48). Novamyl may thus be an example of an enzyme at an intermediary stage in between "true" a-amylases and "true" CGTases. It appears more likely, however, that Novamyl is a "true" CGTase with a few essential mutations modifying product specificity.

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