The crystal structure of the endoglucanase Cel10, a family 8 glycosyl hydrolase from Klebsiella pneumoniae

Abstract

Cellulases are produced by microorganisms that grow on cellulose biomass. Here, a cellulase, Cel10, was identified in a strain of Klebsiella pneumoniae isolated from Chinese bamboo rat gut. Analysis of substrate specificity showed that Cel10 is able to hydrolyze amorphous carboxymethyl cellulose (CMC) and crystalline forms of cellulose (Avicel and xylan) but is unable to hydrolyze p-nitrophenol β-D-glucopyranoside (p-NPG), proving that Cel10 is an endoglucanase. A phylogenetic tree analysis indicates that Cel10 belongs to the glycoside hydrolase 8 (GH8) subfamily. In order to further understanding of its substrate specificity, the structure of Cel10 was solved by molecular replacement and refined to 1.76 Å resolution. The overall fold is distinct from those of most other enzymes belonging to the GH8 subfamily. Although it forms the typical (α/α)₆-barrel motif fold, like Acetobacterxylinum CMCax, one helix is missing. Structural comparisons with Clostridium thermocellum CelA (CtCelA), the best characterized GH8 endoglucanase, revealed that sugar-recognition subsite -3 is completely missing in Cel10. The absence of this subsite correlates to a more open substrate-binding cleft on the cellooligosaccharide reducing-end side.

Keywords: Cel10; Klebsiella pneumoniae; carboxymethyl cellulase; cellulases; cellulose biosynthesis; crystal structure.

Figure 1

SDS–PAGE analysis of purified recombinant Cel10. Top: size-exclusion chromatography (SEC) chromatogram of Cel10 from the final purification column showing a notable peak. Bottom: SDS–PAGE gel of the peak fraction. The protein fractions were resolved on a gradient SDS–PAGE gel (15%) and stained using Coomassie Blue for visualization. Lane M contains molecular-weight markers (labelled in kDa).

Figure 2

Crystals were obtained in 0.1 M glycine–NaOH pH 9.0, 30% PEG 8K, 0.5 M potassium chloride by the sitting-drop vapour-diffusion method.

Figure 3

Phylogenetic tree showing the relationship between Cel10 and other hydrolase families. In the phylogenetic tree analysis, Cel10 was in the same cluster as members of different families and showed over 97% homology to Cel8A in GH8 (GenBank accession No. 440494657). These results demonstrate that Cel10 is a member of GH8. The phylogenetic tree was drawn using MEGA v.4.0. The amino-acid sequence of Cel10 was aligned with those from other different cellulase hydrolase families to generate a neighbour-joining phylogenetic tree. Bootstrap percentage values are indicated at branch points. Accession numbers are listed in the centre.

Figure 4

Structure of Cel10 (a) and structure superimposition of Cel10 (PDP entry 5gy3; blue) with CtCelA (PDB entry 1kwf; yellow) (b). Helix α11 in CtCelA (labelled in red) is missing and forms a flexible loop (labelled in green) in Ce110.

Figure 5

The structure and sequence alignment of Cel10 with CMCax (PDB entry 1wzz) and CtCelA (PDB entry 1kwf). Structure-based sequence alignment of enzymes belonging to GH8. Conserved catalytic residues are highlighted in yellow and the aromatic residues forming sugar-recognition subsites are shown in green. This figure was created using DaliLite (Holm & Rosenström, 2010 ▸).

Figure 6

Molecular surface-potential representation of CtCelA (left; PDB entry 1kwf) and Cel10 (right). A model of the substrate in PDB entry 1kwf is also shown in the cleft of Cel10. The electrostatic surface potentials were generated using PyMOL (http://www.pymol.org) in absolute mode. Areas coloured white, red and blue denote neutral, negative and positive potential, respectively.