Literature summary for 2.4.1.245 extracted from

Woo, E.J.; Ryu, S.I.; Song, H.N.; Jung, T.Y.; Yeon, S.M.; Lee, H.A.; Park, B.C.; Park, K.H.; Lee, S.B.
Structural insights on the new mechanism of trehalose synthesis by trehalose synthase TreT from Pyrococcus horikoshii (2010), J. Mol. Biol., 404, 247-259.

Search for more literature for 2.4.1.245

Cloned(Commentary)

Cloned (Comment)	Organism
gene treT, DNA and amino acid sequence determination and analysis, sequence comparison	Pyrococcus horikoshii

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified enzyme free or as TreT-UDP binary complex, 10 mg/ml native enzyme from PEG 3350 25%, 0.2 M MgCl2, and 0.1 M sodium HEPES, 18°C, the selenomethionine-substituted protein crystal grow from 21% methoxy PEG 2000, 0.18 M ammonium sulfate, and 0.1 M sodium acetate, pH 4.6, for the UDP-glucose complex crystal, 5 mM UDPG is added to 10 mg/ml E326A protein for 1 h prior to setup of the crystallization using the same conditions as for the native crystal, X-ray diffraction structure determination and analysis at 2.3-3.0 A resolution, single-wavelength anomalous dispersion phasing	Pyrococcus horikoshii

Crystallization (Comment)

Organism

purified enzyme free or as TreT-UDP binary complex, 10 mg/ml native enzyme from PEG 3350 25%, 0.2 M MgCl2, and 0.1 M sodium HEPES, 18°C, the selenomethionine-substituted protein crystal grow from 21% methoxy PEG 2000, 0.18 M ammonium sulfate, and 0.1 M sodium acetate, pH 4.6, for the UDP-glucose complex crystal, 5 mM UDPG is added to 10 mg/ml E326A protein for 1 h prior to setup of the crystallization using the same conditions as for the native crystal, X-ray diffraction structure determination and analysis at 2.3-3.0 A resolution, single-wavelength anomalous dispersion phasing

Pyrococcus horikoshii

Protein Variants

Protein Variants	Comment	Organism
D134E	the mutant shows 10% reduced activity in the forward reaction, and 50% reduced activity in the reverse reaction	Pyrococcus horikoshii
D134R	inactive mutant	Pyrococcus horikoshii
D274R/D275A	inactive mutant	Pyrococcus horikoshii
E326A	inactive mutant	Pyrococcus horikoshii
F85R	the mutant shows 95% reduced activity in the forward reaction, and no activity in the reverse reaction	Pyrococcus horikoshii
F85Y	the mutant shows 10% reduced activity in the forward reaction, and 40% reduced activity in the reverse reaction	Pyrococcus horikoshii
H155D	inactive in the forward reaction, 80% reduced activity in the reverse reaction compared to wild-type	Pyrococcus horikoshii
H92A	the mutant shows wild-type activity in the forward reaction, and 90% reduced activity in the reverse reaction	Pyrococcus horikoshii
K209R	the mutant shows wild-type activity	Pyrococcus horikoshii
Q96A	the mutant shows 10% reduced activity in the forward reaction, and 90% reduced activity in the reverse reaction	Pyrococcus horikoshii
R239A	inactive in the reverse reaction, 10% reduced activity in the forward reaction compared to wild-type	Pyrococcus horikoshii
T49H	the mutant shows 90% reduced activity in the forward reaction, and 98% reduced activity in the reverse reaction	Pyrococcus horikoshii
T49R	inactive mutant	Pyrococcus horikoshii
V309L	the mutant shows wild-type activity in the forward reaction, and 25% reduced activity in the reverse reaction	Pyrococcus horikoshii

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ADP-alpha-D-glucose + D-glucose	Pyrococcus horikoshii	-	ADP + alpha,alpha-trehalose	-	r

Organism

Organism	UniProt	Comment	Textmining
Pyrococcus horikoshii	O58762	gene treT	-

Reaction

Reaction	Comment	Organism	Reaction ID
NDP-alpha-D-glucose + D-glucose = alpha,alpha-trehalose + NDP	reaction mechanism, overview. The acceptor binding site of TreT shows a wide and commodious groove and lacks the long flexible loop that plays a gating role in ligand binding in trehalose phosphate synthase, TPS, EC 2.4.1.15. A wide space at the fissure between two domains and the relative shift of the N-domain in one of the crystal forms suggest that an interactive conformational change between two domains would occur, allowing a more compact architecture for catalysis	Pyrococcus horikoshii	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ADP-alpha-D-glucose + D-glucose	-	Pyrococcus horikoshii	ADP + alpha,alpha-trehalose	-	r
GDP-glucose + D-glucose	-	Pyrococcus horikoshii	alpha,alpha-trehalose + GDP	-	r
additional information	molecular basis of the synthetic mechanism of trehalose, or the nucleotide sugar in the reverse reaction of TreT. TreT can utilize ADP-glucose and GDP-glucose as well as UDP-glucose to synthesize trehalose, the nucleotide sugar molecule is recognized by the nucleotide-sensing Gly-Gly-Leu motif that is located at the domain interface in many GT-B family members, and TreT contains this motif at residues 52-55, the acceptor molecule binds predominantly to the N-terminal domain	Pyrococcus horikoshii	?	-	?
UDP-glucose + D-glucose	-	Pyrococcus horikoshii	alpha,alpha-trehalose + UDP	-	r

Synonyms

Synonyms	Comment	Organism
trehalose glycosyltransferring synthase	-	Pyrococcus horikoshii
Trehalose synthase	-	Pyrococcus horikoshii
TreT	-	Pyrococcus horikoshii

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Pyrococcus horikoshii

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6	-	assay at	Pyrococcus horikoshii

General Information

General Information	Comment	Organism
additional information	the acceptor binding site of TreT shows a wide and commodious groove and lacks the long flexible loop that plays a gating role in ligand binding in trehalose phosphate synthase, TPS, active site, and donor and acceptor binding pocket structure, overview. A wide space at the fissure between two domains and the relative shift of the N-domain in one of the crystal forms suggest that an interactive conformational change between two domains would occur, allowing a more compact architecture for catalysis	Pyrococcus horikoshii