Identification of the essential catalytic residues and selectivity-related residues of maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092.
Maltooligosyltrehalose trehalohydrolase (MTHase) catalyzes the release of trehalose by cleaving the alpha-1,4-glucosidic linkage next to the alpha-1,1-linked terminal disaccharide of maltooligosyltrehalose. Mutations at residues D255, E286, and D380 were constructed to identify the essential catalytic residues of MTHase, while mutations at residues W218, A259, Y328, F355, and R356 were constructed to identify selectivity-related residues of the enzyme. The specific activities of the purified D255A, E286A, and D380A MTHases were only 0.15, 0.09 and 0.01%, respectively, of that of wild-type MTHase, suggesting that these three residues are essential catalytic residues. Compared with wild-type MTHase, A259S, Y328F, F355Y, and R356K MTHases had increased selectivity ratios, which were defined as the ratios of the catalytic efficiencies for glucose formation to those for trehalose formation in the hydrolysis of maltooligosaccharides and maltooligosyltrehaloses, respectively, while W218A and W218F MTHases had decreased selectivity ratios. When starch digestion was carried out at 75 degrees C and wild-type and mutant MTHases were, respectively, used with isoamylase and maltooligosyltrehalose synthase (MTSase), the ratios of initial rates of glucose formation to those of trehalose formation were inversely correlated to the peak trehalose yields.
Fang
Tsuei-Yun
TY
Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan. tyfang@mail.ntou.edu.tw
Tseng
Wen-Chi
WC
Shih
Tong-Yuan
TY
Wang
Mei-Ying
MY
D001665:Binding Sites; D002384:Catalysis; D005947:Glucose / Q000378:metabolism; D005959:Glucosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013213:Starch / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology*; D014199:Trehalose / Q000378:metabolism
D013213:Starch; D014199:Trehalose; D005959:Glucosidases; C099156:maltooligosyl trehalose trehalohydrolase; D005947:Glucose