Characterization of the maltooligosyl trehalose synthase from the thermophilic archaeon Sulfolobus acidocaldarius.
We report the molecular characterization and the detailed study of the recombinant maltooligosyl trehalose synthase mechanism from the thermoacidophilic archaeon Sulfolobus acidocaldarius. The mts gene encoding a maltooligosyl trehalose synthase was overexpressed in Escherichia coli using the T7-expression system. The purified recombinant enzyme exhibited optimum activity at 75 degrees C and pH 5 with citrate-phosphate buffer and retained 60% of residual activity after 72 h of incubation at 80 degrees C. The recombinant enzyme was active on maltooligosaccharides such as maltotriose, maltotetraose, maltopentaose and maltoheptaose. Investigation of the enzyme action on maltooligosaccharides has brought much insight into the reaction mechanism. Results obtained from thin-layer chromatography suggested a possible mechanism of action for maltooligosyl trehalose synthase: the enzyme, after converting the alpha-1,4-glucosidic linkage to an alpha-1,1-glucosidic linkage at the reducing end of maltooligosaccharide glc(n) is able to release glucose and maltooligosaccharide glc(n-1) residues. And then, the intramolecular transglycosylation and the hydrolytic reaction continue, with the maltooligosaccharide glc(n-1) until the initial maltooligosaccharide is reduced to maltose. An hypothetical mechanism of maltooligosyl trehalose synthase acting on maltooligosaccharide is proposed.
Gueguen
Y
Y
IFREMER Centre de Brest, DRV/VP Laboratoire de Biotechnologie des Micro-organismes Hydrothermaux, Plouzané, France. ygueguen@ifremer.fr
Rolland
J L
JL
Schroeck
S
S
Flament
D
D
Defretin
S
S
Saniez
M H
MH
Dietrich
J
J
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002240:Carbohydrate Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D005964:Glucosyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D016945:Sulfolobus acidocaldarius / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D019843:Archaeal Proteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; C021705:maltooligosaccharides; D005964:Glucosyltransferases; C099084:maltooligosyl trehalose synthase