Tertiary structure and characterization of a glycoside hydrolase family 44 endoglucanase from Clostridium acetobutylicum

Abstract

A gene encoding a glycoside hydrolase family 44 (GH44) protein from Clostridium acetobutylicum ATCC 824 was synthesized and transformed into Escherichia coli. The previously uncharacterized protein was expressed with a C-terminal His tag and purified by nickel-nitrilotriacetic acid affinity chromatography. Crystallization and X-ray diffraction to a 2.2-A resolution revealed a triose phosphate isomerase (TIM) barrel-like structure with additional Greek key and beta-sandwich folds, similar to other GH44 crystal structures. The enzyme hydrolyzes cellotetraose and larger cellooligosaccharides, yielding an unbalanced product distribution, including some glucose. It attacks carboxymethylcellulose and xylan at approximately the same rates. Its activity on carboxymethylcellulose is much higher than that of the isolated C. acetobutylicum cellulosome. It also extensively converts lichenan to oligosaccharides of intermediate size and attacks Avicel to a limited extent. The enzyme has an optimal temperature in a 10-min assay of 55 degrees C and an optimal pH of 5.0.

FIG. 1.

Structural organization of genes coding for GH44 CDs, excluding GH44 members with only a signal peptide and a CD. The gene encoding O. terrae's GH44 member produces a 920-residue protein whose domain structure is unclassified. The sequence was searched against the Pfam library, and a low E-value, 1.22 × 10⁻²², was found for a PKD (polycystic kidney disease)-type domain. Ig, immunoglobulin.

FIG. 2.

Phylogenetic tree of GH44 CDs.

FIG. 3.

Crystal structure of C. acetobutylicum ATCC 824 EG shown with a 90° rotation. The TIM barrel, a β-sandwich domain, and ψ-loop domains are shown. The catalytic acid/base, Glu180, and catalytic nucleophile, Glu352, are shown as sticks, as are amino acids involved in ligand binding. A chloride ion, orange, is located next to the catalytic acid in the TIM barrel. A cartoon diagram of the ψ-loop with respect to the rest of the protein is highlighted in the orange box. It contains a β-strand located between two antiparallel β-strands and hydrogen bonded to them. A calcium ion, magenta, appears to stabilize this domain.

FIG. 4.

Structural alignments of C. acetobutylicum ATCC 824 EG with C. thermocellum EG or CelM2. (A) Alignment of cartoon representations of C. acetobutylicum EG and C. thermocellum EG. Ligand binding residues of C. thermocellum EG and their C. acetobutylicum EG analogs are shown as sticks, as are the catalytic residues. The chloride and calcium ions of both enzymes are orange and magenta spheres, respectively. Minimal structural differences are observed, with those additional secondary structure elements present in one enzyme but not the other shown in blue (C. thermocellum EG) or green (C. acetobutylicum EG). (B) Cartoon alignment of C. acetobutylicum EG and CelM2. Secondary structural features present in C. acetobutylicum EG but not in CelM2 are shown in green, and those found in CelM2 but not in C. acetobutylicum EG are shown in yellow. Key ligand binding amino acids and catalytic residues are shown as sticks and are colored like their secondary structure. The chloride and calcium ions of both enzymes are orange and magenta spheres, respectively. The ψ-loop of C. acetobutylicum EG and small domain of CelM2 that replaces it are shown in an orange circle. The blue ovals highlight both additional α-helices present in C. acetobutylicum EG, as well as its ligand binding residues (Arg41, Tyr65, and Trp324). (C) The alignment in panel B is rotated to show how the small domain of CelM2 extends the upper face of the ligand binding cleft relative to the ψ-loop of C. acetobutylicum EG. (D) A surface representation of the cartoon alignment in panel B, with C. acetobutylicum EG shown in blue and CelM2 shown in red. As in panel B, key differences are shown in yellow or green. Catalytic residues are shown in orange.

FIG. 5.

Thin-layer chromatography of hydrolysis products when enzyme was incubated with 10 g/liter cellooligosaccharides or 10 g/liter various polysaccharides in 0.1 M NaOAc buffer, pH 5.0, at 25°C for 16 h. (A) Left-hand lane: glucose, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose standards (from top to bottom). Lanes 2 to 11: cellobiose control (without enzyme), cellobiose incubation (with enzyme), cellotriose control, cellotriose incubation, cellotetraose control, cellotetraose incubation, cellopentaose control, cellopentaose incubation, cellohexaose control, and cellohexaose incubation, respectively. (B) Left- and right-hand lanes: glucose, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose standards (from top to bottom). Lanes 2 to 11: CMC control (without enzyme), CMC incubation, lichenan control, lichenan incubation, birch wood xylan control, birch wood xylan incubation, larch wood xylan control, larch wood xylan incubation, Avicel control and Avicel incubation, respectively.