Figure 1.
Production of trehalose from maltooligosaccharide via MTSase and MTHase.
In the first step, MTSase turns the α-1,4-linked terminal disaccharide of maltooligosaccharide into the α-1,1-linked terminal disaccharide. In the second step, MTHase cleaves the α-1,4-glucosidic linkage next to the α-1,1-linked terminal disaccharide of maltooligosyltrehalose and produces a trehalose.
Figure 2.
Production of trehalose from ligand G3T by MTHase.
Ligand G3T is a maltooligosyltrehalose consisting of five glucoses. The first four glucoses are α-1,4-linked while the terminal two ones are α-1,1-linked. The glucoses of G3T are numbered from G5 to T1. The catalytic triad residues identified in MTHase are D255, E286 and E380, respectively. The protein subsites are numbered from +3 to −2. The bond between T2 and G3 is cleaved during the hydrolysis reaction.
Figure 3.
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.
Figure 4.
The Lineweaver-Burk plots generated for both wild type and mutant MTHases.
In panel (a), the Lineweaver-Burk plots for the wild type and Y155A, Y155F, and H195A mutant MTHases were plotted. In panel (b), the Lineweaver-Burk plots for the wild type, D156A, R447A, and E450A mutant MTHases were plotted.
Figure 5.
The active site residues of MTHase and ligand G3T.
Ligand G3T is shown in stick while MTHase is shown in light blue ribbon. The key MTHase residues are also shown in stick. Oxygen atoms are colored in red, nitrogen atoms are colored in blue, and carbon atoms are colored in grey. The residues of catalytic triad are highlighted with yellow circles. The subsites of NTHase are numbered from +2 to −3.
Table 1.
The kinetic parameters measured for the generation° of trehalose from hydrolysis of G3T by both wild-type and mutant MTHases at 60°C in a 50 mM citrate-phosphate buffer at pH 5.
Figure 6.
The root-mean-square deviations (RMSDs) of MTHase backbone atoms (blue curve) and all the G3T (substrate) atoms (red curve) computed during the MD simulation.
Figure 7.
Free energy decomposition of wild type MTHase computed with the MM-GBSA method.
Table 2.
The free energy differences computed from the computational alanine scanning for the MTHase mutants.
Figure 8.
The transition state structure constructed using a truncated model system obtained from the optimized geometries with the 3-21G basis set and Hartree-Fock method.
Atoms O1, C1, and O2 represent respectively the oxygen atom of D255 and the carbon and oxygen atom of ligand G3T. Atom H1 is representing the hydrogen atom of E286.
Figure 9.
The energy profile of ab Initio quantum chemical calculation obtained using the truncated model system for ligand G3T in the presence (blue curve) or absence (red curve) of MTHase.
Figure 10.
The transition state structure of hydrolysis of G3T in the absence of MTHase or in water molecules.
Atoms O1, C1, and O2 represent respectively the oxygen atom of a water molecule and the carbon and oxygen atom of ligand G3T.