Role of the glutamate 332 residue in the transglycosylation activity of ThermusMaltogenic amylase.
A sequence alignment shows that residue 332 is conserved as glutamate in maltogenic amylases (MAases) and in other related enzymes such as cyclodextrinase and neopullulanase, while the corresponding position is conserved as histidine in alpha-amylases. We analyzed the role of Glu332 in the hydrolysis and the transglycosylation activity of Thermus MAase (ThMA) by site-directed mutagenesis. Replacing Glu332 with histidine reduced transglycosylation activity significantly, but enhanced hydrolysis activity on alpha-(1,3)-, alpha-(1,4)-, and alpha-(1,6)-glycosidic bonds relative to the wild-type (WT) enzyme. The mutant Glu332Asp had catalytic properties similar to those of the WT enzyme, but the mutant Glu332Gln resulted in significantly decreased transglycosylation activity. These results suggest that an acidic side chain at position 332 of MAase plays an important role in the formation and accumulation of transfer products by modulating the relative rates of hydrolysis and transglycosylation. From the structure, we propose that an acidic side chain at position 332, which is located in a pocket, is involved in aligning the acceptor molecule to compete with water molecules in the nucleophilic attack of the glycosyl-enzyme intermediate.
Kim
T J
TJ
Research Center for New Bio-Materials in Agriculture and Department of Food Science and Technology, Seoul National University, Suwon 441-744, Korea.
Park
C S
CS
Cho
H Y
HY
Cha
S S
SS
Kim
J S
JS
Lee
S B
SB
Moon
T W
TW
Kim
J W
JW
Oh
B H
BH
Park
K H
KH
D020909:Acarbose / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D018698:Glutamic Acid / Q000235:genetics* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D016695:Glycosyltransferases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008757:Methylglucosides / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D013379:Substrate Specificity
D001426:Bacterial Proteins; D008757:Methylglucosides; D018698:Glutamic Acid; C027020:methylglucoside; D016695:Glycosyltransferases; D006026:Glycoside Hydrolases; C120909:glucan 1,4-alpha-maltohydrolase; D020909:Acarbose