Literature summary for 5.4.99.16 extracted from

Wang, Y.L.; Chow, S.Y.; Lin, Y.T.; Hsieh, Y.C.; Lee, G.C.; Liaw, S.H.
Structures of trehalose synthase from Deinococcus radiodurans reveal that a closed conformation is involved in catalysis of the intramolecular isomerization (2014), Acta Crystallogr. Sect. D, 70, 3144-3154 .

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Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) from the pET-23a(+) vector	Deinococcus radiodurans

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant wild-type and N253A mutant enzymes in complex with inhibitor Tris, hanging drop vapour diffusion method, 15°C, for the wild-type enzyme, mixing of 0.002 ml of 30 mg/ml protein in 20 mM sodium phosphate, pH 7.4, with 0.002 ml of reservoir solution containing 9% PEG 4000, 0.2 M sodium acetate trihydrate, 0.3 M Tris-HCl, pH 8.5, 6-8 weeks, for the mutant enzyme, mixing of 0.002 ml of 60 mg/ml protein in 20 mM sodium phosphate, pH 7.4, with 0.002 ml of reservoir solution containing 11% PEG 4000, 0.2 M sodium acetate trihydrate, 0.3 M Tris-HCl, pH 8.5, and 5% glycerol, 2 weeks, X-ray diffracion structure determination and analysis at 2.21-2.70 A resolution	Deinococcus radiodurans

Crystallization (Comment)

Organism

purified recombinant wild-type and N253A mutant enzymes in complex with inhibitor Tris, hanging drop vapour diffusion method, 15°C, for the wild-type enzyme, mixing of 0.002 ml of 30 mg/ml protein in 20 mM sodium phosphate, pH 7.4, with 0.002 ml of reservoir solution containing 9% PEG 4000, 0.2 M sodium acetate trihydrate, 0.3 M Tris-HCl, pH 8.5, 6-8 weeks, for the mutant enzyme, mixing of 0.002 ml of 60 mg/ml protein in 20 mM sodium phosphate, pH 7.4, with 0.002 ml of reservoir solution containing 11% PEG 4000, 0.2 M sodium acetate trihydrate, 0.3 M Tris-HCl, pH 8.5, and 5% glycerol, 2 weeks, X-ray diffracion structure determination and analysis at 2.21-2.70 A resolution

Deinococcus radiodurans

Protein Variants

Protein Variants	Comment	Organism
N253A	site-directed mutagenesis, N253A structure shows a small pore created for water entry, the mutant shows a decrease in isomerase activity by 8-9fold and an increase in hydrolase activity by 1.5-1.8fold	Deinococcus radiodurans
R148A	site-directed mutagenesis, the mutant shows a decrease in isomerase activity by 8-9fold and an increase in hydrolase activity by 1.5-1.8fold	Deinococcus radiodurans

Inhibitors

Inhibitors	Comment	Organism	Structure
Tris	the enzyme-Tris complex may represent a substrate-induced closed conformation that facilitates intramolecular isomerization and minimize disaccharide hydrolysis	Deinococcus radiodurans

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Ca2+	the Ca2+ site is coordinated by the side chains of Asn105, Asp179 and Glu216 and the backbone O atoms of Tyr213 and Leu214. Many alpha-amylases contain a similar calcium site that involves the conserved asparagine and aspartate residues	Deinococcus radiodurans
Mg2+	the Mg2+ site consists of the side chains of Asp24, Asn26, Asp28 and Asp32 and the main-chain O atom of Lys30, which form part of the consensus signature DXNXDGXGD. Such a site is also found in some other GH13 member	Deinococcus radiodurans

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
maltose	Deinococcus radiodurans	-	alpha,alpha-trehalose	-	r

Organism

Organism	UniProt	Comment	Textmining
Deinococcus radiodurans	I3NX86	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography	Deinococcus radiodurans

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
maltose	-	Deinococcus radiodurans	alpha,alpha-trehalose	-	r
additional information	modeling of maltose into the active site, overview	Deinococcus radiodurans	?	-	?

Subunits

Subunits	Comment	Organism
dimer	DrTS consists of a catalytic (beta/alpha)8 barrel, subdomain B, a C-terminal beta-domain and two TS-unique subdomains, S7 and S8. The C-terminal domain and domain S8 contribute the majority of the dimeric interface, analytical ulatracentrifugation. Enzyme structure comparisons, detailed overview	Deinococcus radiodurans

Synonyms

Synonyms	Comment	Organism
DrTS	-	Deinococcus radiodurans
Trehalose synthase	-	Deinococcus radiodurans

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
20	-	assay at	Deinococcus radiodurans

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.4	-	assay at	Deinococcus radiodurans

General Information

General Information	Comment	Organism
malfunction	disruption of the interaction networks through the replacement of Arg148 and Asn253 with alanine results in a decrease in isomerase activity by 8-9fold and an increased hydrolase activity by 1.5-1.8fold. The N253A structure shows a small pore created for water entry. Active site structure and substrate-induced conformational changes, enzyme structure comparisons, detailed overview	Deinococcus radiodurans
additional information	residues Tyr213, Glu320 and Glu324 are essential within the +1 subsite for the enzyme activity. The interaction networks between subdomains B and S7 seal the active-site entrance	Deinococcus radiodurans