Literature summary for 2.4.1.25 extracted from

Barends, T.R.; Bultema, J.B.; Kaper, T.; van der Maarel, M.J.; Dijkhuizen, L.; Dijkstra, B.W.
Three-way stabilization of the covalent intermediate in amylomaltase, an alpha-amylase-like transglycosylase (2007), J. Biol. Chem., 282, 17242-17249.

Cloned(Commentary)

Cloned (Comment)	Organism
expression of the His-tagged enzyme in Escherichia coli strain BL21(DE3)	Thermus thermophilus

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant enzyme, hanging drop vapour diffusion method, 0.003 ml of protein solution containing 10 mM MES-NaOH, pH 6.5, with 1 mM dithiothreitol is mixed with 0.001-0.003 ml of reservoir solution containing 0.4-0.8 M sodium malonate, pH 5.6, and 1 mM dithiothreitol, equilibration against reservoir solution at room temperature, 1 week, for enzyme-acarbose complexing the crystals are soaked in 0.5 ml of 0.8 M sodium malonate, pH 5.6, with 5 mg/ml acarbose and with or without and 4-deoxyglucose, for 30 min, X-ray diffraction structure determination and analysis at 1.9-2.5 A resolution	Thermus thermophilus

Crystallization (Comment)

Organism

purified recombinant enzyme, hanging drop vapour diffusion method, 0.003 ml of protein solution containing 10 mM MES-NaOH, pH 6.5, with 1 mM dithiothreitol is mixed with 0.001-0.003 ml of reservoir solution containing 0.4-0.8 M sodium malonate, pH 5.6, and 1 mM dithiothreitol, equilibration against reservoir solution at room temperature, 1 week, for enzyme-acarbose complexing the crystals are soaked in 0.5 ml of 0.8 M sodium malonate, pH 5.6, with 5 mg/ml acarbose and with or without and 4-deoxyglucose, for 30 min, X-ray diffraction structure determination and analysis at 1.9-2.5 A resolution

Thermus thermophilus

Protein Variants

Protein Variants	Comment	Organism
E758Q	the mutant shows highly reduced activity compared to the wild-type enzyme	Thermus thermophilus
additional information	the deletion mutant DELTAN130 is unable to use glycogen but has high disproportionating activity with maltodextrins	Thermus thermophilus

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	initial rate kintics	Thermus thermophilus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
additional information	Thermus thermophilus	amylomaltases are capable of the synthesis of large cyclic glucans and the disproportionation of oligosaccharides	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Thermus thermophilus	O87172	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by heat treatment and nickel affinity chromatography, removal of the His-tag through cleavage with bovine thrombin	Thermus thermophilus

Reaction

Reaction	Comment	Organism	Reaction ID
Transfers a segment of a (1->4)-alpha-D-glucan to a new position in an acceptor, which may be glucose or a (1->4)-alpha-D-glucan	reaction mechanism and catalytic cycle, the catalytic nucleophile changes conformation dramatically during the reaction, Gln256 on the 250s loop is involved in orienting the substrate in the +1 site. The absence of a suitable base in the covalent intermediate structure explains the low hydrolysis activity, overview	Thermus thermophilus	a

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
0.0027	-	mutant E758Q	Thermus thermophilus
5.19	-	wild-type enzyme	Thermus thermophilus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
additional information	amylomaltases are capable of the synthesis of large cyclic glucans and the disproportionation of oligosaccharides	Thermus thermophilus	?	-	?
additional information	glucose transfer/production using maltose and glycogen. Wild-type DPE2 can bind to starch and glycogen has very little, if any, ability to dissociate DPE2 from the starch pellet	Thermus thermophilus	?	-	?

Synonyms

Synonyms	Comment	Organism
alpha-amylase-like transglycosylase	-	Thermus thermophilus
amylomaltase	-	Thermus thermophilus
additional information	amylomaltases are glycosyl hydrolases belonging to glycoside hydrolase family 77	Thermus thermophilus