Cloned (Comment) | Organism |
---|---|
overexpression of C-terminally His6-tagged wild-type and mutant enzymes in Pichia pastoris strain GS115 and secretion to the culture medium | Ostrinia furnacalis |
Crystallization (Comment) | Organism |
---|---|
purified recombinant His6-tagged wild-type enzyme and mutant E148Q unliganded, and wild-type enzyme and mutant E148A in complex with chitobiose and /or chitotriose, hanging drop vapour diffusion method, mixing of 0.001 ml of 10 mg/ml protein in 50 mM HEPES, pH 8.0, and 100 mM NaCl, with 0,001 ml of reservoir solution containing 100 mM HEPES, pH 7.5, 25% w/v PEG 3350 for the wild-type with soaking of crystals in ligand containing solution for the complex crystals, reservori solution containing 5 mM (GlcNAc)6 in 100 mM HEPES, pH 7.9, 23% w/v PEG 3350 and in 100 mM HEPES, pH 8.0, 21% w/v PEG 3350, respectively, for mutant E148A and E148Q, X-ray diffraction structure determination and analysis at 1.7 A and 2.2 A resolution | Ostrinia furnacalis |
Protein Variants | Comment | Organism |
---|---|---|
E148A | site-directed mutagenesis | Ostrinia furnacalis |
E148Q | site-directed mutagenesis | Ostrinia furnacalis |
F159A | site-directed mutagenesis | Ostrinia furnacalis |
F159A/F194A/W241A/Y290A | site-directed mutagenesis | Ostrinia furnacalis |
F159A/Y290A | site-directed mutagenesis | Ostrinia furnacalis |
F194A | site-directed mutagenesis | Ostrinia furnacalis |
F194A/W241A | site-directed mutagenesis | Ostrinia furnacalis |
W241A | site-directed mutagenesis | Ostrinia furnacalis |
Y290A | site-directed mutagenesis | Ostrinia furnacalis |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Ostrinia furnacalis | Q2V6H4 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged wild-type and mutant enzymes from Pichia pastoris strain GS115 culture supernatant by ammonium sulfate fractionation and nickel affinity chromatography | Ostrinia furnacalis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
chitin + H2O | the enzyme acts from non-reducing ends. Substrate binding structure, overview. The reducing sugar at subsite -1 is in an energetically unfavoured boat conformation, a state that possibly exists just before the completion of catalysis. A hydrophobic plane composed of four surface-exposed aromatic residues is adjacent to the entrance to the substrate-binding cleft | Ostrinia furnacalis | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
group I chitinase | - |
Ostrinia furnacalis |
OfChtI | - |
Ostrinia furnacalis |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme belongs to the glycosyl hydrolase family 18 (GH18) of chitinases | Ostrinia furnacalis |
additional information | catalytic domain structure with and without bound substrate, structure comparisons and structure-function relationship, overview | Ostrinia furnacalis |
physiological function | the enzyme is essential to moulting | Ostrinia furnacalis |