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Interaction between trehalose and MTHase from Sulfolobus solfataricus studied by theoretical computation and site-directed mutagenesis.

Fu CW, Wang YP, Fang TY, Lin TH - PLoS ONE (2013)

Bottom Line: It was found that residues Y155, D156, and W218 of subsites -2 and -3 of the enzyme might play an important role in interacting with the ligand.The theoretically constructed structure of the enzyme-ligand complex was further validated through an ab initio quantum chemical calculation using the Gaussian09 package.The activation energy computed from this latter study was very similar to those reported in literatures for the same type of hydrolysis reactions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan.

ABSTRACT
Maltooligosyltrehalose trehalohydrolase (MTHase) catalyzes the release of trehalose by cleaving the α-1,4-glucosidic linkage next to the α-1,1-linked terminal disaccharide of maltooligosyltrehalose. Computer simulation using the hydrogen bond analysis, free energy decomposition, and computational alanine scanning were employed to investigate the interaction between maltooligosyltrehalose and the enzyme. The same residues that were chosen for theoretical investigation were also studied by site-directed mutagenesis and enzyme kinetic analysis. The importance of residues determined either experimentally or computed theoretically were in good accord with each other. It was found that residues Y155, D156, and W218 of subsites -2 and -3 of the enzyme might play an important role in interacting with the ligand. The theoretically constructed structure of the enzyme-ligand complex was further validated through an ab initio quantum chemical calculation using the Gaussian09 package. The activation energy computed from this latter study was very similar to those reported in literatures for the same type of hydrolysis reactions.

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SDS-polyacrylamide gel electrophoresis of the purified wild type and mutant MTHases.The purified wild type and mutant MTHases were analyzed by a 12% minigel and stained with Coomassic Brilliant Blue R-250. Lane M: the molecular weight standards; lanes 1−7: 2.2 µg of wild-type, Y155A, Y155F, D156A, H195A, R447A, and E450A MTHases, respectively.
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pone-0068565-g003: SDS-polyacrylamide gel electrophoresis of the purified wild type and mutant MTHases.The purified wild type and mutant MTHases were analyzed by a 12% minigel and stained with Coomassic Brilliant Blue R-250. Lane M: the molecular weight standards; lanes 1−7: 2.2 µg of wild-type, Y155A, Y155F, D156A, H195A, R447A, and E450A MTHases, respectively.

Mentions: The purities of wild type and mutant MTHases prepared were good as shown by the SDS-PAGE analysis (Figure 3). In fact, the purities of all the purified wild type and mutant MTHases estimated using GelAnalyzer 2010 [51] were greater than 97%. The residual activities of purified wild type and mutant MTHases measured were 87.4 ± 0.3 (wild type), 91.8±2.4 (Y155A), 111.7±0.4 (Y155F), 110.4±0.8 (D156A), 84.6±0.9 (H195A), 100.1±2.2 (R447A), and 104.9±1.4 (E450A) %, respectively, when they were all incubated at 80°C for two hours. This implied that the mutant enzymes were as thermostable as the wild type one.


Interaction between trehalose and MTHase from Sulfolobus solfataricus studied by theoretical computation and site-directed mutagenesis.

Fu CW, Wang YP, Fang TY, Lin TH - PLoS ONE (2013)

SDS-polyacrylamide gel electrophoresis of the purified wild type and mutant MTHases.The purified wild type and mutant MTHases were analyzed by a 12% minigel and stained with Coomassic Brilliant Blue R-250. Lane M: the molecular weight standards; lanes 1−7: 2.2 µg of wild-type, Y155A, Y155F, D156A, H195A, R447A, and E450A MTHases, respectively.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3716775&req=5

pone-0068565-g003: SDS-polyacrylamide gel electrophoresis of the purified wild type and mutant MTHases.The purified wild type and mutant MTHases were analyzed by a 12% minigel and stained with Coomassic Brilliant Blue R-250. Lane M: the molecular weight standards; lanes 1−7: 2.2 µg of wild-type, Y155A, Y155F, D156A, H195A, R447A, and E450A MTHases, respectively.
Mentions: The purities of wild type and mutant MTHases prepared were good as shown by the SDS-PAGE analysis (Figure 3). In fact, the purities of all the purified wild type and mutant MTHases estimated using GelAnalyzer 2010 [51] were greater than 97%. The residual activities of purified wild type and mutant MTHases measured were 87.4 ± 0.3 (wild type), 91.8±2.4 (Y155A), 111.7±0.4 (Y155F), 110.4±0.8 (D156A), 84.6±0.9 (H195A), 100.1±2.2 (R447A), and 104.9±1.4 (E450A) %, respectively, when they were all incubated at 80°C for two hours. This implied that the mutant enzymes were as thermostable as the wild type one.

Bottom Line: It was found that residues Y155, D156, and W218 of subsites -2 and -3 of the enzyme might play an important role in interacting with the ligand.The theoretically constructed structure of the enzyme-ligand complex was further validated through an ab initio quantum chemical calculation using the Gaussian09 package.The activation energy computed from this latter study was very similar to those reported in literatures for the same type of hydrolysis reactions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan.

ABSTRACT
Maltooligosyltrehalose trehalohydrolase (MTHase) catalyzes the release of trehalose by cleaving the α-1,4-glucosidic linkage next to the α-1,1-linked terminal disaccharide of maltooligosyltrehalose. Computer simulation using the hydrogen bond analysis, free energy decomposition, and computational alanine scanning were employed to investigate the interaction between maltooligosyltrehalose and the enzyme. The same residues that were chosen for theoretical investigation were also studied by site-directed mutagenesis and enzyme kinetic analysis. The importance of residues determined either experimentally or computed theoretically were in good accord with each other. It was found that residues Y155, D156, and W218 of subsites -2 and -3 of the enzyme might play an important role in interacting with the ligand. The theoretically constructed structure of the enzyme-ligand complex was further validated through an ab initio quantum chemical calculation using the Gaussian09 package. The activation energy computed from this latter study was very similar to those reported in literatures for the same type of hydrolysis reactions.

Show MeSH