Literature summary for 2.4.1.19 extracted from

Kelly, R.M.; Leemhuis, H.; Rozeboom, H.J.; van Oosterwijk, N.; Dijkstra, B.W.; Dijkhuizen, L.
Elimination of competing hydrolysis and coupling side reactions of a cyclodextrin glucanotransferase by directed evolution (2008), Biochem. J., 413, 517-525.

Cloned(Commentary)

Cloned (Comment)	Organism
expression of wild-type and mutant enzymes in Bacillus subtilis strain DB104A, subcloning in Escherichia coli strain MC1061	Thermoanaerobacterium thermosulfurigenes

Crystallization (Commentary)

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

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

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 [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
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

Organism	UniProt	Comment	Textmining
Thermoanaerobacterium thermosulfurigenes	-	-	-
Thermoanaerobacterium thermosulfurigenes EM1	-	-	-

Reaction

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	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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

Synonyms	Comment	Organism
CGTase	-	Thermoanaerobacterium thermosulfurigenes
More	the enzyme belongs to the superfamily of glycoside hydrolases	Thermoanaerobacterium thermosulfurigenes

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
60	-	assay at	Thermoanaerobacterium thermosulfurigenes

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
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

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6	-	assay at	Thermoanaerobacterium thermosulfurigenes