Mutations on aromatic residues of the active site to alter selectivity of the Sulfolobus solfataricus maltooligosyltrehalose synthase.
Mutations Y290F, Y367F, F405Y, and Y409F located near subsite +1 were constructed in maltooligosyltrehalose synthase (MTSase) to alter the selectivity of the enzyme. These mutations were designed to evaluate the effects of hydrophobic interactions and/or hydrogen bondings on transglycosylation and side hydrolysis reactions. The catalytic efficiencies of Y290F MTSase for hydrolysis and transglycosylation reactions were only 6.6 and 5.6%, respectively, of those of wildtype MTSase, whereas the catalytic efficiencies of Y367F MTSase were decreased by about half. F405Y MTSase had similar catalytic efficiencies for transglycosylation and a somewhat lower catalytic efficiency for hydrolysis. Y409F MTSase had somewhat lower catalytic efficiencies for the transglycosylation and a similar catalytic efficiency for hydrolysis. Y290F and Y367F MTSases had large changes in delta(deltaG), suggesting that there are hydrogen bonds between the substrate and residues Y290 and Y367 of wild-type MTSase. Compared with wild-type MTSase, F405Y MTSase had decreased ratios of hydrolysis to transglycosylation, whereas Y290F, Y367F, and Y409F MTSases had increased ratios. These results suggest that use of F405Y MTSase might result in a higher yield of trehalose production from starch when it replaces wild-type MTSase.
Fang
Tsuei-Yun
TY
Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan. tyfang@mail.ntou.edu.tw
Tseng
Wen-Chi
WC
Chung
Yao-Te
YT
Pan
Ching-Hsing
CH
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D006868:Hydrolysis; D009154:Mutation; D013379:Substrate Specificity / Q000235:genetics; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics; D014199:Trehalose / Q000378:metabolism
D014199:Trehalose; D005964:Glucosyltransferases; C099084:maltooligosyl trehalose synthase