Figure 1.
Schematic of the Molecular Architecture of the Enzymes Investigated in This Study
The mature forms of the C. japonicus esterases consist of a single catalytic module. The C. thermocellum enzyme containing a CE2 module (CtCel5C-CE2) consists of an N-terminal GH5 cellulase module (CtCel5C), a central type I dockerin module (Doc) that facilitates the integration of the enzyme into the cellulosome, and a C-terminal CE2 module (CtCE2). The CE2 modules of all the esterases contains an N-terminal domain of ∼160 residues that displays a jelly roll fold and a C-terminal domain that exhibits an α/β-hydrolase fold. The black lines are ∼15-residue P/T linker sequences. The residues in CjCE2C that carry an asterisk are predicted catalytic residues based on sequence alignments.
Table 1.
Catalytic Activity of CE2 Enzymes against 4-Nitrophenyl Acetate (4-NPAc)
Table 2.
Activity of CE2 Esterases against Acetylated Polysaccharides
Figure 2.
3-D Structures and Catalytic Constellation Geometries for Three CE2 Esterases
(A) 3-D structure of CjCE2B with the catalytic domain in green and the β-sheet domain in magenta. The catalytic Ser and His are shown in ball-and-stick representation.
(B) CjCE2A, drawn as above.
(C) CtCE2 as above and with cellopentaose in blue.
(D) The catalytic Ser-His-Asp triad of CjCE2A.
(E) The catalytic Ser-His dyad with the main-chain carbonyl interaction from Cys333 of CjCE2B.
(F) The catalytic dyad and main-chain carbonyl of CtCE2.
This figure was drawn with PyMOL (DeLano Scientific, http://pymol.sourceforge.net/).
Figure 3.
Binding of Cellooligosaccharides through the Esterase Active Centre of CtCE2
Observed electron density (maximum-likelihood weighted 2Fobs − Fcalc contoured at 1 σ) for cellohexaose (in which cellopentaose is ordered) bound to wild-type CtCE2. Ser-612 and His-791 form the catalytic dyad, with Trp-790 causing a change in position of Asp-789 and forming the binding-platform for the second glucose. Other interactions with aromatic residues discussed in the text are shown. The figure is in divergent (“wall-eyed”) stereo and was drawn with PyMOL.
Figure 4.
Binding of CE2 Esterases to Cellulose
(A) An AGE experiment in which the enzymes were subjected to nondenaturing gel electrophoresis in the absence (−) or presence (+) of 0.1 % (w/v) hydroxyethylcellulose. The lanes contained BSA (1), CtCE2 (2), CjCE2A (3), and CjCE2C (4). CjCE2B did not migrate on the nondenaturing gel.
(B) Shows a pull-down experiment using insoluble cellulose. The original protein samples (s) and bound protein eluted from cellulose with 10% SDS (b) were subjected to SDS-PAGE. The lanes contained CtCE2 (1), CjCE2A (2), CjCE2B (3), CjCE2C, (4), and BSA as a noninteracting control (5). Molecular weight markers are shown.
Figure 5.
Examples of Isothermal Titration Calorimetry of Wild-Type and Mutants of CtCE2
The proteins were titrated with cellohexaose in 50 mM sodium HEPES buffer, pH 7.0, at 25 °C. The protein concentration for each titration was 100 μM.
Table 3.
The Binding of CE2 Esterases to Cellohexaose and β-Glucan
Table 4.
ITC Analysis of CtCE2 Wild-Type and Mutants Binding to Cellooligosaccharides and β-Glucan
Figure 6.
Inhibition of Wild-Type CtCE2 by Cellohexaose
CtCE2 was assayed at 37 °C using either 4-nitrophenyl acetate (A) or acetylated glucomannan (B) as the substrate in the presence of different concentrations of cellohexaose (C6). The figure displays double reciprocal plots of the data.
Table 5.
Data Collection and Refinement Statistics