3WX5: Crystal Structure Of Metagenome-derived Glycoside Hydrolase Family 12 Endoglucanase

Ten genes encoding novel cellulases with putative signal peptides at the N-terminus, termed pre-LC-CelA-J, were isolated from a fosmid library of a leaf-branch compost metagenome by functional screening using agar plates containing carboxymethyl cellulose and trypan blue. All the cellulases except pre-LC-CelG have a 14-29 residue long flexible linker (FL) between the signal peptide and the catalytic domain. LC-CelA without a signal peptide (residues 20-261), which shows 76% amino acid sequence identity to Cel12A from Rhodothermus marinus (RmCel12A), was overproduced in Escherichia coli, purified and characterized. LC-CelA exhibited its highest activity across a broad pH range (pH 5-9) and at 90 degrees C, indicating that LC-CelA is a highly thermostable cellulase, like RmCel12A. The crystal structure of LC-CelA was determined at 1.85 A resolution and is nearly identical to that of RmCel12A determined in a form without the FL. Both proteins contain two disulfide bonds. LC-CelA has a 16-residue FL (residues 20-35), most of which is not visible in the electron density map, probably due to structural disorder. However, Glu34 and Pro35 form hydrogen bonds with the central region of the protein. DeltaFL-LC-CelA (residues 36-261) and E34A-LC-CelA with a single Glu34 --> Ala mutation were therefore constructed and characterized. DeltaFL-LC-CelA and E34A-LC-CelA had lower melting temperatures (T m) than LC-CelA by 14.7 and 12.0 degrees C respectively. The T m of LC-CelA was also decreased by 28.0 degrees C in the presence of dithiothreitol. These results suggest that Glu34-mediated hydrogen bonds and the two disulfide bonds contribute to the stabilization of LC-CelA.
PDB ID: 3WX5Download
MMDB ID: 124618
PDB Deposition Date: 2014/7/17
Updated in MMDB: 2014/11
Experimental Method:
x-ray diffraction
Resolution: 1.85  Å
Source Organism:
Similar Structures:
Biological Unit for 3WX5: monomeric; determined by author and by software (PISA)
Molecular Components in 3WX5
Label Count Molecule
Protein (1 molecule)
Molecule annotation
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Citing MMDB