Structure determination and refinement of the Humicola insolens endoglucanase V at 1.5 Å Resolution

Abstract

The structure of the catalytic core of the endoglucanase V (EGV) from Humicola insolens has been determined by the method of multiple isomorphous replacement at 1.5 A, resolution. The final model, refined with X-PLOR and PROLSQ, has a crystallographic R factor of 0.163 (Rfree = 0.240) with deviations from stereochemical target values of 0.012 ,~ and 0.037 ° for bonds and angles, respectively. The model was further refined with SHELXL, including anisotropic modelling of the protein-atom temperature factors, to give a final model with an R factor of 0.105 and an Rfre~ of 0.154. The initial isomorphous replacement electron-density map was poor and uninterpretable but was improved by the use of synchrotron data collected at a wavelength chosen so as to optimize the f" contribution of the anomalous scattering from the heavy atoms. The structure of H. insolens EGV consists of a six-stranded/3-barrel domain, similar to that found in a family of plant defence proteins, linked by a number of disulfide-bonded loop regions. A long open groove runs across the surface of the enzyme either side of which lie the catalytic aspartate residues. The 9/~ separation of the catalytic carboxylate groups is consistent with the observation that EGV catalyzes the hydrolysis of the cellulose ,0(144) links with inversion of configuration at the anomeric CI atom. This structure is the first representative from the glycosyl hydrolase family 45.

Characterization of an Endoglucanase Belonging to a New Subfamily of Glycoside Hydrolase Family 45 of the BasidiomycetePhanerochaete chrysosporium

“…A BLAST search of the amino acid sequence of PcCel45A showed that the protein may be classified into the GH family 45, which includes many small EGs (8,9,11,36), and this conclusion is supported by the result of hydrophobic cluster analysis (performed by B. Henrissat, personal communication). Since only a low sequence similarity was found with other known GH family 45 EGs, the protein represents a new subdivision of this family.…”

Model cellulose films exposed to H. insolens glucoside hydrolase family 45 endo-cellulase—the effect of the carbohydrate-binding module

“…Finally, they were rinsed in Milli-Q water and heattreated at 150 • C for 60 min. The films prepared in this way [33], here shown in complex with cellohexaose, and a carbohydrate-binding module (B) [34], here represented by the carbohydrate-binding module (CBM) of Cel7A (Trichoderma reesei), belonging to the same classification family. The CBM is connected to the C-terminal of the catalytic core.…”

“…The native enzyme consists of two parts, a catalytic core containing 213 amino acids ( Fig. 1A [33]) and a C-terminal carbohydrate-binding module (CBM) containing 38 amino acids ( Fig. 1B [34]), held together by a linkage containing 33 amino acids [29].…”

Cellulases: ambiguous nonhomologous enzymes in a genomic perspective

“…(images are taken from the RCSB PDB (www.pdb.org). The following labels correspond to PDB accession numbers: ‘Hexokinase’ - 1ig8[43]; ‘GH5, GH44, GH51’ - 1e5j[44]; ‘GH6’ - 2boe[45]; ‘GH7, GH12’ - 2jen[46]; ‘GH8, GH9, GH48’ - 1ia6[47]; ‘GH23’ – 2xqo[33]; ‘GH45’ - 4eng[48]; ‘GH61’ - 2vtc[49]; ‘GH74’ - 2cn2[50]. …”