Substrate binding of a GH5 endoglucanase from the ruminal fungus Piromyces rhizinflata

Abstract

The endoglucanase EglA from Piromyces rhizinflata found in cattle stomach belongs to the GH5 family of glycoside hydrolases. The crystal structure of the catalytic domain of EglA shows the (β/α)(8)-barrel fold typical of GH5 enzymes. Adjacent to the active site of EglA, a loop containing a disulfide bond not found in other similar structures may participate in substrate binding. Because the active site was blocked by the N-terminal His tag of a neighbouring protein molecule in the crystal, enzyme-substrate complexes could not be obtained by soaking but were prepared by cocrystallization. The E154A mutant structure with a cellotriose bound to the -3, -2 and -1 subsites shows an extensive hydrogen-bonding network between the enzyme and the substrate, along with a stacking interaction between Trp44 and the -3 sugar. A possible dimer was observed in the crystal structure, but retention of activity in the E242A mutant suggested that the enzyme probably does not function as a dimer in solution. On the other hand, the first 100 amino acids encoded by the original cDNA fragment are very similar to those in the last third of the (β/α)(8)-barrel fold, indicating that EglA comprises at least two catalytic domains acting in tandem.

Figure 1

The unliganded crystal structure of rEglA. (a) The physical dimensions of the crystal in the centre of the picture were 0.5 × 0.2 × 0.2 mm. The E154A mutant–substrate complex crystals had a similar appearance. (b) The structure shows a typical (β/α)₈ fold. The protein model is rainbow-coloured from blue to red from the N-terminus to the C-terminus. The α-helices are labelled from 0 to 8 near their N-termini and the β-strands from I to VIII near their C-termini. The side chains of the catalytic glutamate and disulfide-forming cysteine residues are shown as stick models.

Figure 2

The bound cellotriose in the E154A mutant. (a) The mutant rEglA protein is shown as a molecular-surface model coloured from red to blue according to the electrostatic potential between −75 and 75 k _B T. The bound sugar is shown as a stick model and is superimposed on the F _o − F _c OMIT map contoured at the 5.0σ level. (b) A close-up view of the bound sugar and the electron density corresponding to the boxed region in (a) is shown with the surface rendered translucent to reveal the hidden part of the sugar. The nonreducing end (nre) and reducing end (re) of cellotriose are indicated. (c) The cellotriose molecule is shown as a bold stick model and the surrounding amino-acid residues are shown as thin sticks. The hydrogen bonds between the substrate and the enzyme are shown as dashed lines. Two mediating water molecules are shown as red spheres. The model is rotated clockwise by approximately 90° from those in (a) and (b).

Figure 3

His-tag interactions in the unliganded rEglA crystal. (a) Six enzyme molecules related by the P6₁ screw axis, representing one half of the unit-cell contents of the hexagonal P6₁22 crystal, are shown in different colours, along with a seventh molecule coloured as the first. The extended N-terminus of each enzyme molecule (e.g. the red molecule) penetrates into the active site of its neighbour (e.g. the yellow molecule). (b) The N-terminal His tag of one molecule is shown as purple sticks and the neighbouring molecule as a green surface model in a close-up view of the contacts between the two 6₁ symmetry-related rEglA molecules. The bound cellotriose from the E154A complex crystal after superposition of the mutant and the wild-type protein structures is shown as grey sticks.