PDBsum entry: 1oa9

Hydrolase

PDB id: 1oa9

Contents

Protein chain

208 a.a. *

Waters ×152

* Residue conservation analysis

PDB id:

1oa9

Name:

Hydrolase

Title:

Structure of melanocarpus albomyces endoglucanase

Structure:

Cellulase. Chain: a. Synonym: endoglucanase. Ec: 3.2.1.4

Source:

Melanocarpus albomyces. Organism_taxid: 204285

Resolution:

2.0Å R-factor: 0.215 R-free: 0.273

Authors:

M.Hirvonen,A.C.Papageorgiou

Key ref:

M.Hirvonen and A.C.Papageorgiou (2003). Crystal structure of a family 45 endoglucanase from Melanocarpus albomyces: mechanistic implications based on the free and cellobiose-bound forms. J Mol Biol, 329, 403-410. PubMed id: 12767825 DOI: 10.1016/S0022-2836(03)00467-4

Date:

04-Jan-03 Release date: 29-May-03

Protein chain

Q8J0K8  (Q8J0K8_MELAO) -  Cellulase (Precursor)

Seq: 235 a.a.

Struc: 208 a.a.

Enzyme reactions

• Enzyme class: E.C.3.2.1.4 - Cellulase.
• Reaction: Endohydrolysis of 1,4-beta-D-glucosidic linkages in cellulose, lichenin and cereal beta-D-glucans.

 Gene Ontology (GO) functional annotation 

• Biological process: metabolic process (2 terms)
• Biochemical function: hydrolase activity (3 terms)

DOI no: 10.1016/S0022-2836(03)00467-4

J Mol Biol 329:403-410 (2003)

PubMed id: 12767825

Crystal structure of a family 45 endoglucanase from Melanocarpus albomyces: mechanistic implications based on the free and cellobiose-bound forms.

M.Hirvonen, A.C.Papageorgiou.

ABSTRACT

Cellulose, a polysaccharide of beta-1,4-linked D-glucosyl units, is the major component of plant cell walls and one of the most abundant biopolymers in nature. Cellulases (cellobiohydrolases and endoglucanases) are enzymes that catalyse the hydrolysis of cellulose to smaller oligosaccharides, a process of paramount importance in biotechnology. The thermophilic fungus Melanocarpus albomyces produces a 20 kDa endoglucanase known as 20K-cellulase that has been found particularly useful in the textile industry. The crystal structures of free 20K-cellulase and its complex with cellobiose have been determined at 2.0 A resolution. The enzyme, classified into the glycoside hydrolase family 45, exhibits the characteristic six-stranded beta-barrel found before in Humicola insolens endoglucanase V structure. However, the active site in the 20K-cellulase shows a closing of approximately 2.5-3.5A while a mobile loop identified previously in Humicola insolens endoglucanase V and implicated in the catalytic mechanism is well-defined in 20K-cellulase. In addition, the crystal structure of the cellobiose complex shows a shift in the cellobiose position at the substrate-binding cleft. It is therefore proposed that these alterations may reflect differences in the binding mechanism and catalytic action of the enzyme.

Selected figure(s)

Figure 1.
Figure 1. Ribbon representation of maEG. The color changing is from blue (N terminus) to red (C terminus). Assignment of the secondary structure elements was based on the MOLAUTO suite in MOLSCRIPT.[25.] Figure drawn with BOBSCRIPT [26.] and Raster3D. [27.] Crystals of maEG were produced as described. [28.] Data to 2.0 Å were collected on station X11 at EMBL outstation in Hamburg (HASY-LAB, c/o DESY, Germany) from a single crystal at 100 K using 25% glycerol as cryoprotectant. Data were recorded on an 165 mm MARCCD detector. The crystal-to-detector distance was 200 mm, oscillation 0.5° and wavelength 0.8499 Å. Data were processed, scaled and merged in the tetragonal space group P4[3]2[1]2 using HKL.[29.] The intensities were subsequently converted to structure factor amplitudes with TRUNCATE from the CCP4 suite of programs. [30.] The R[sym] was 7.9% for 13,270 reflections with an overall completeness of 91.2% between 50.0-2.0 Å. Crystals of the maEG-cellobiose complex were grown in the presence of 20 mM cellobiose. Crystallisation conditions were the same as those for the uncomplexed maEG. The crystals were soaked overnight in a crystallisation solution containing 0.125 M freshly-prepared cellobiose prior to data collection. Data for the maEG-cellobiose complex to 2.0 Å were collected in-house on a MAR345 image plate detector mounted on a Rigaku Rotaflex rotating anode (Cu K[a], l=1.5418 Å) operating at 50 kV/100 mA, and equipped with Osmic mirrors. A single crystal soaked for vert, similar 15 seconds in a cryoprotectant solution containing 25% glycerol and flash-cooled in a nitrogen-gas cold stream was used. The crystal-to-detector distance was set to 170 mm, the exposure time was 15-20 minutes and the oscillation 1°. The structure of native maEG was determined by molecular replacement with the program AMoRe[31.] using the structure of EGV (PDB code 3eng; 1.9 Å) as the search model. Side-chains, water molecules and cellobiose were removed from the search model as well as the first three residues from the N-terminal and the last four residues from the C-terminal. Data from 8.0-3.0 Å were used in the rotation and translation search. After rigid-body optimisation, an unambiguous solution with correlation coefficient of 60.1% and R-factor of 44.4% was obtained. Examination of the crystal packing revealed no major clashes between individual molecules. The top solution from molecular replacement was initially subjected to rigid body refinement from 8.0 Å to 3.0 Å, followed by calculation of a (2|F[o]| -|F[c]|) map. Inspection of the initial map with O[14.] revealed good side-chain density for a number of residues. The structure was refined by simulated annealing using the maximum likelihood target and torsion-angle dynamics as implemented in CNS. [32.] The progress of refinement was followed by monitoring both the R[free] and R[cryst][33.] values. Engh and Huber geometric restraints [34.] were applied during refinement. The refinement was continued using alternate cycles of simulated annealing and model rebuilding followed by B-factor refinement. SigmaA-weighted electron density maps (|F[o]| -|F[c]| and 2|F[o]| -|F[c]|) were calculated after each cycle of refinement and visualised with O. Water molecules were added to the model towards the end of refinement by using the water_pick protocol in CNS. Water molecules with B-factors higher than 55 Å2 were excluded from subsequent rounds of refinement. The maEG-cellobiose complex was initially subjected to rigid body refinement (unit cell difference 0.9 Å along the c axis between the crystals of free and maEG-cellobiose complex). Calculation of an (|F[o]| -|F[c]|) electron density map at this stage revealed extra density at the predicted active site that could fit a cellobiose molecule. To avoid cross-validation errors, the same "free" subset of reflections was maintained as for the free maEG structure. Cellobiose was added to the model when the R[free] dropped below 30%. Solvent accessibility, inter- and intra-molecular contacts were calculated using the CCP4 suite.

Figure 2.
Figure 2. Stereo views displaying (A) C^a-traces of maEG (black) and EGV (blue). Every tenth residue in maEG is numbered. (B) C^a-traces of maEG (black), EGV-cellobiose (blue), maEG-cellobiose (green). Cellobiose in the EGV-cellobiose and maEG-cellobiose complexes is shown.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2003, 329, 403-410) copyright 2003.

Figures were selected by an automated process.