X-ray Structure of Novamyl, the Five-Domain “Maltogenic” α-Amylase from Bacillus stearothermophilus:  Maltose and Acarbose Complexes at 1.7 Å Resolution^,

Abstract

The three-dimensional structure of the Bacillus stearothermophilus "maltogenic" alpha-amylase, Novamyl, has been determined by X-ray crystallography at a resolution of 1.7 A. Unlike conventional alpha-amylases from glycoside hydrolase family 13, Novamyl exhibits the five-domain structure more usually associated with cyclodextrin glycosyltransferase. Complexes of the enzyme with both maltose and the inhibitor acarbose have been characterized. In the maltose complex, two molecules of maltose are found in the -1 to -2 and +2 to +3 subsites of the active site, with two more on the C and E domains. The C-domain maltose occupies a position identical to one previously observed in the Bacillus circulans CGTase structure [Lawson, C. L., et al. (1994) J. Mol. Biol. 236, 590-600], suggesting that the C-domain plays a genuine biological role in saccharide binding. In the acarbose-maltose complex, the tetrasaccharide inhibitor acarbose is found as an extended hexasaccharide species, bound in the -3 to +3 subsites. The transition state mimicking pseudosaccharide is bound in the -1 subsite of the enzyme in a 2H3 half-chair conformation, as expected. The active site of Novamyl lies in an open gully, fully consistent with its ability to perform internal cleavage via an endo as opposed to an exo activity.

Amylase action pattern on starch polymers

“…Some reports suggest that a maltogenic α-amylase from Bacillus stearothermophilus (BStA) acts as an exo-amylase (Bowles 1996;Kragh 2002). However, consistent with the characteristics of an endo-α-amylase, this enzyme does not require a non-reducing end, it does not invert the anomeric configuration (Christophersen et al 1998), its active site is located in an open cleft (Dauter et al 1999) and it can greatly reduce amylose molecular weight (MW) (Christophersen et al 1998;Leman et al 2005). Next to the three common domains (A, B and C), BStA possesses two additional domains, D and E, with the E domain involved in binding to native granular starch (Dauter et al 1999).…”

“…Indeed, putative starch interaction sites outside the active site have been identified for BStA (i.e. a starch binding domain E) (Dauter et al 1999) and PPA (i.e. two independent carbohydrate recognition sites) (Qian et al 1995).…”

Structure of the GTPase-binding Domain of Sec5 and Elucidation of its Ral Binding Site

“…4 and 5B, shown in green). The ␣-amylase IPT domain uses the same face to contact other domains within the same protein (30). NF-B and the other DNA-binding proteins use the same face both for contacting other IPT domains and for contacting other domains within the same protein (28, 29, 34 -40).…”

Carbohydrate-binding modules: fine-tuning polysaccharide recognition

“…Indeed, the structure and biochemistry of several family 20 CBMs, which bind to starch, have been analysed extensively (see [6][7][8][9][10][11][12][13] for examples). Furthermore, numerous crystal structures of starch-modifying enzymes have revealed malto-oligosaccharide-binding sites that are distinct from the substrate-binding cleft, indicating that these enzymes also contain starch-binding CBMs [14][15][16][17].…”