Cloned (Comment) | Organism |
---|---|
expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Pyrococcus horikoshii |
Crystallization (Comment) | Organism |
---|---|
purified enzyme mutants in complex with either the substrate or product ligands, hanging drop vapour diffusion method, 20 mg/ml protein in 50 mM Tris/HCl, pH 8.0, mixing of 0.0015 ml of both protein and reservoir solution, the latter contaning 1.5 M ammonium phosphate, and 0.1 M MES, pH 6.5, 22°C, 3 days, X-ray diffraction structure determination and analysis at 1.65-2.01 A resolution | Pyrococcus horikoshii |
Protein Variants | Comment | Organism |
---|---|---|
E201A | site-directed mutagenesis, crystal structure determination with bound ligands | Pyrococcus horikoshii |
E342A | site-directed mutagenesis, crystal structure determination with bound ligands | Pyrococcus horikoshii |
Y299F | site-directed mutagenesis, crystal structure determination with bound ligands, the mutant shows reduced activity compare to the wild-type enzyme, and a rare enzyme-substrate complex structure | Pyrococcus horikoshii |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
membrane | - |
Pyrococcus horikoshii | 16020 | - |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Zn2+ | zinc ions tightly bound between the two catalytic glutamate residues, which present an obstacle for the entrance of ligand in the active site | Pyrococcus horikoshii |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pyrococcus horikoshii | O58925 | - |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | with substrate cellulose, the substrate position is fixed by the alignment of one cellobiose unit between the two aromatic amino acid residues at subsites +1 and +2. During the enzyme reaction, the glucose structure of cellulose substrates is distorted at subsite -1, and the beta-1,4-glucoside bond between glucose moieties is twisted between subsites -1 and +1. Subsite -2 specifically recognizes the glucose residue, but recognition by subsites +1 and +2 is loose during the enzyme reaction. This type of recognition is important for creation of the distorted boat form of the substrate at subsite -1 | Pyrococcus horikoshii | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
beta-1,4-endoglucanase | - |
Pyrococcus horikoshii |
endocellulase | - |
Pyrococcus horikoshii |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
85 | - |
assay at | Pyrococcus horikoshii |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
5.5 | - |
assay at | Pyrococcus horikoshii |