X-ray structure of Novamyl, the five-domain "maltogenic" alpha-amylase from Bacillus stearothermophilus: Maltose and acarbose complexes at 1.7 angstrom resolution

TY - JOUR

T1 - X-ray structure of Novamyl, the five-domain "maltogenic" alpha-amylase from Bacillus stearothermophilus: Maltose and acarbose complexes at 1.7 angstrom resolution

AU - Dauter, Z

AU - Dauter, M

AU - Brzozowski, A M

AU - Christensen, S

AU - Borchert, T V

AU - Beier, L

AU - Wilson, K S

AU - Davies, G J

PY - 1999/6/29

Y1 - 1999/6/29

N2 - 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 Angstrom. 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 H-2(3) 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.

AB - 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 Angstrom. 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 H-2(3) 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.

KW - SEQUENCE-BASED CLASSIFICATION

KW - GLYCOSYL HYDROLASES

KW - CYCLODEXTRIN GLYCOSYLTRANSFERASE

KW - MOLECULAR REPLACEMENT

KW - CATALYTIC MECHANISM

KW - INHIBITOR ACARBOSE

KW - CRYSTAL-STRUCTURE

KW - REFINEMENT

KW - ASPERGILLUS

KW - SIMILARITIES

M3 - Article

SN - 0006-2960

VL - 38

SP - 8385

EP - 8392

JO - Biochemistry

JF - Biochemistry

IS - 26

ER -