Cloned (Comment) | Organism |
---|---|
expression of wild-type and mutant enzymes in Bacillus subtilis strain DB104A, subcloning in Escherichia coli strain MC1061 | Thermoanaerobacterium thermosulfurigenes |
Crystallization (Comment) | Organism |
---|---|
purified recombinant mutant S77P CGTase in 10 mM sodium acetate, pH 5.5, large crystals from 17-20%-saturated ammonium sulfate at room temperature in 100 mM HEPES, pH 7.6, or 100 mM HEPES, pH 7.8, or 100 mM Tris/HCl, pH 8.0, like the wild-type enzyme, crystals are soaked in 25% v/v glycerol and directly flash-cooled, or soaked in 2% w/v acarbose, 4% w/v maltohexaose and 25% glycerol in 20% saturated ammonium sulfate for 25 min followed by flash-cooling, X-ray diffraction structure determination and analysis at 1.6 A resolution | Thermoanaerobacterium thermosulfurigenes |
Protein Variants | Comment | Organism |
---|---|---|
additional information | mutations in two residues, Ser-77 and Trp-239, on the outer region of the active site, lowers the hydrolytic activity up to 15fold with retention of cyclization activity | Thermoanaerobacterium thermosulfurigenes |
S77P | site-directed mutagenesis, mutant kinetics compared to the wild-type enzyme, molecular modelling of the location and effect of S77P mutation on the Tabium CGTase active-site conformation | Thermoanaerobacterium thermosulfurigenes |
W239L | site-directed mutagenesis, mutant kinetics compared to the wild-type enzyme, the mutation destroys a hydrogen-bonding interaction between the side chains of Asp209 and Trp239, compromising the stability of the mutant | Thermoanaerobacterium thermosulfurigenes |
W239R | site-directed mutagenesis, mutant kinetics compared to the wild-type enzyme, the mutation destroys a hydrogen-bonding interaction between the side chains of Asp209 and Trp239, compromising the stability of the mutant | Thermoanaerobacterium thermosulfurigenes |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | kinetics of the four reaction steps, overview | Thermoanaerobacterium thermosulfurigenes | |
0.15 | - |
4,6-O-ethylidene-4-nitrophenyl alpha-D-maltoheptaoside | pH 6.0, 60°C, wild-type enzyme | Thermoanaerobacterium thermosulfurigenes | |
0.2 | - |
4,6-O-ethylidene-4-nitrophenyl alpha-D-maltoheptaoside | pH 6.0, 60°C, mutant W239R | Thermoanaerobacterium thermosulfurigenes | |
0.24 | - |
4,6-O-ethylidene-4-nitrophenyl alpha-D-maltoheptaoside | pH 6.0, 60°C, mutant S77P | Thermoanaerobacterium thermosulfurigenes | |
0.27 | - |
4,6-O-ethylidene-4-nitrophenyl alpha-D-maltoheptaoside | pH 6.0, 60°C, mutant W239L | Thermoanaerobacterium thermosulfurigenes | |
2.4 | - |
maltose | pH 6.0, 60°C, mutant S77P | Thermoanaerobacterium thermosulfurigenes | |
3.3 | - |
maltose | pH 6.0, 60°C, wild-type enzyme | Thermoanaerobacterium thermosulfurigenes | |
4.1 | - |
maltose | pH 6.0, 60°C, mutant W239R | Thermoanaerobacterium thermosulfurigenes | |
4.2 | - |
maltose | pH 6.0, 60°C, mutant W239L | Thermoanaerobacterium thermosulfurigenes |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Thermoanaerobacterium thermosulfurigenes | - |
- |
- |
Thermoanaerobacterium thermosulfurigenes EM1 | - |
- |
- |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
alpha-D-glucopyranosyl-(1-4)-alpha-D-glucopyranosyl-(1-4)-alpha-D-glucopyranosyl-(1-4)-alpha-D-glucopyranosyl-(1-4)-alpha-D-glucopyranosyl-(1-4)-alpha-D-glucopyranosyl-(1-4)-alpha-D-glucopyranose = alpha-cyclodextrin + alpha-D-glucose | reaction in four steps via cyclization, disproportionation, hydrolysis, and coupling. The second step of the reaction involves the transfer of the covalently bound saccharide to an acceptor molecule | Thermoanaerobacterium thermosulfurigenes |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
4,6-O-ethylidene-4-nitrophenyl-alpha-D-maltoheptaoside + maltose | maltose as donor and acceptor, overview | Thermoanaerobacterium thermosulfurigenes | ? | - |
? | |
4,6-O-ethylidene-4-nitrophenyl-alpha-D-maltoheptaoside + maltose | maltose as donor and acceptor, overview | Thermoanaerobacterium thermosulfurigenes EM1 | ? | - |
? | |
additional information | the enzyme primarily catalyses the formation of cyclic alpha-1,4-linked cyclodextrins from starch. This enzyme also possesses unusually high hydrolytic activity as a side reaction, thought to be due to partial retention of ancestral enzyme function. Product formation, alpha-, beta-, and gamma-cyclodextrins, of wild-type and mutant enzymes, substrate-binding subsites of CGTase, and sugar binding structure, overview | Thermoanaerobacterium thermosulfurigenes | ? | - |
? | |
additional information | the enzyme primarily catalyses the formation of cyclic alpha-1,4-linked cyclodextrins from starch. This enzyme also possesses unusually high hydrolytic activity as a side reaction, thought to be due to partial retention of ancestral enzyme function. Product formation, alpha-, beta-, and gamma-cyclodextrins, of wild-type and mutant enzymes, substrate-binding subsites of CGTase, and sugar binding structure, overview | Thermoanaerobacterium thermosulfurigenes EM1 | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
CGTase | - |
Thermoanaerobacterium thermosulfurigenes |
More | the enzyme belongs to the superfamily of glycoside hydrolases | Thermoanaerobacterium thermosulfurigenes |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
60 | - |
assay at | Thermoanaerobacterium thermosulfurigenes |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
558 | - |
maltose | pH 6.0, 60°C, mutant S77P | Thermoanaerobacterium thermosulfurigenes | |
558 | - |
4,6-O-ethylidene-4-nitrophenyl alpha-D-maltoheptaoside | pH 6.0, 60°C, mutant S77P | Thermoanaerobacterium thermosulfurigenes | |
644 | - |
maltose | pH 6.0, 60°C, mutant W239R | Thermoanaerobacterium thermosulfurigenes | |
644 | - |
4,6-O-ethylidene-4-nitrophenyl alpha-D-maltoheptaoside | pH 6.0, 60°C, mutant W239R | Thermoanaerobacterium thermosulfurigenes | |
1082 | - |
maltose | pH 6.0, 60°C, mutant W239L | Thermoanaerobacterium thermosulfurigenes | |
1082 | - |
4,6-O-ethylidene-4-nitrophenyl alpha-D-maltoheptaoside | pH 6.0, 60°C, mutant W239L | Thermoanaerobacterium thermosulfurigenes | |
1244 | - |
maltose | pH 6.0, 60°C, wild-type enzyme | Thermoanaerobacterium thermosulfurigenes | |
1244 | - |
4,6-O-ethylidene-4-nitrophenyl alpha-D-maltoheptaoside | pH 6.0, 60°C, wild-type enzyme | Thermoanaerobacterium thermosulfurigenes |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6 | - |
assay at | Thermoanaerobacterium thermosulfurigenes |