Literature summary for 5.4.99.11 extracted from

Xu, Z.; Li, S.; Li, J.; Li, Y.; Feng, X.; Wang, R.; Xu, H.; Zhou, J.
The structural basis of Erwinia rhapontici isomaltulose synthase (2013), PLoS ONE, 8, e74788.

Search for more literature for 5.4.99.11

Cloned(Commentary)

Cloned (Comment)	Organism
expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Erwinia rhapontici

Crystallization (Commentary)

Crystallization (Comment)	Organism
wild-type NX-5 and enzyme mutant complexes E295Q-sucrose and D241A-glucose, sitting drop vapor diffusion method, mixing of 0.001 ml of 10 mg/ml protein solution with 0.001 ml of reservoir solution containing 0.1 M trisodium citrate, pH 5.6, 0.2 M ammonium acetate, and 30% PEG 4000, and for sucrose-complexed mutant enzyme crystal 20 mM sucrose, 0.1 M bicine, pH 9.0, and 30% PEG 6000. The D241A-glucose complex and R335H/R336T/K337I/D338P-glucose complex are co-crystallized in the presence of 20 mM sucrose, 0.1 M sodium cacodylate, pH 6.5, and 29% PEG 8000, 20°C, 3-5 days, X-ray diffraction structure determination and analysis at 1.70 A, 1.70 a, and 2.00 A resolutions, respectively	Erwinia rhapontici

Crystallization (Comment)

Organism

wild-type NX-5 and enzyme mutant complexes E295Q-sucrose and D241A-glucose, sitting drop vapor diffusion method, mixing of 0.001 ml of 10 mg/ml protein solution with 0.001 ml of reservoir solution containing 0.1 M trisodium citrate, pH 5.6, 0.2 M ammonium acetate, and 30% PEG 4000, and for sucrose-complexed mutant enzyme crystal 20 mM sucrose, 0.1 M bicine, pH 9.0, and 30% PEG 6000. The D241A-glucose complex and R335H/R336T/K337I/D338P-glucose complex are co-crystallized in the presence of 20 mM sucrose, 0.1 M sodium cacodylate, pH 6.5, and 29% PEG 8000, 20°C, 3-5 days, X-ray diffraction structure determination and analysis at 1.70 A, 1.70 a, and 2.00 A resolutions, respectively

Erwinia rhapontici

Protein Variants

Protein Variants	Comment	Organism
D241A	site-directed mutagenesis, glucose binding structure in comparison to the wild-type enzyme by crystal structure analysis	Erwinia rhapontici
E295Q	site-directed mutagenesis, sucrose binding structure in comparison to the wild-type enzyme by crystal structure analysis	Erwinia rhapontici
F297A	site-directed mutagenesis, the mutant shows an altered product ratio of trehalulose and isomaltulose compared to the wild-type enzyme	Erwinia rhapontici
F321A	site-directed mutagenesis, the mutant shows an altered product ratio of trehalulose and isomaltulose compared to the wild-type enzyme	Erwinia rhapontici
additional information	mutations of the loop region of enzyme NX-5 result in significant changes of the product ratio between isomaltulose and trehalulose	Erwinia rhapontici
R325D	site-directed mutagenesis, the mutant shows an altered product ratio of trehalulose and isomaltulose compared to the wild-type enzyme	Erwinia rhapontici
R328D	site-directed mutagenesis, the mutant shows an altered product ratio of trehalulose and isomaltulose compared to the wild-type enzyme	Erwinia rhapontici
R335H/R336T/K337I/D338P	site-directed mutagenesis, glucose binding structure in comparison to the wild-type enzyme by crystal structure analysis	Erwinia rhapontici

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
additional information	Erwinia rhapontici	the enzyme catalyzes the isomerization of sucrose (alpha-D-glucosylpyranosyl-1,2-beta-D-fructofuranose) to isomaltulose (alpha-D-glucosylpyranosyl-1,6-D-fructofuranose) as the main product, and trehalulose (alpha-D-glucosylpyranosyl-1,1-D-fructofuranose) as minor product, and produces glucose and fructose in residual amounts because of sucrose hydrolysis	?	-	?
additional information	Erwinia rhapontici NX-5	the enzyme catalyzes the isomerization of sucrose (alpha-D-glucosylpyranosyl-1,2-beta-D-fructofuranose) to isomaltulose (alpha-D-glucosylpyranosyl-1,6-D-fructofuranose) as the main product, and trehalulose (alpha-D-glucosylpyranosyl-1,1-D-fructofuranose) as minor product, and produces glucose and fructose in residual amounts because of sucrose hydrolysis	?	-	?
sucrose	Erwinia rhapontici	product specificity of enzyme NX-5 towards isomaltulose and the role of the loop330-339 in NX-5 catalysis	1-O-alpha-D-glucopyranosyl-D-fructofuranose + alpha-D-glucosylpyranosyl-1,6-D-fructofuranose	-	?
sucrose	Erwinia rhapontici NX-5	product specificity of enzyme NX-5 towards isomaltulose and the role of the loop330-339 in NX-5 catalysis	1-O-alpha-D-glucopyranosyl-D-fructofuranose + alpha-D-glucosylpyranosyl-1,6-D-fructofuranose	-	?

Organism

Organism	UniProt	Comment	Textmining
Erwinia rhapontici	-	-	-
Erwinia rhapontici NX-5	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatograhy and gel filtration	Erwinia rhapontici

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
additional information	the enzyme catalyzes the isomerization of sucrose (alpha-D-glucosylpyranosyl-1,2-beta-D-fructofuranose) to isomaltulose (alpha-D-glucosylpyranosyl-1,6-D-fructofuranose) as the main product, and trehalulose (alpha-D-glucosylpyranosyl-1,1-D-fructofuranose) as minor product, and produces glucose and fructose in residual amounts because of sucrose hydrolysis	Erwinia rhapontici	?	-	?
additional information	the enzyme catalyzes the isomerization of sucrose (alpha-D-glucosylpyranosyl-1,2-beta-D-fructofuranose) to isomaltulose (alpha-D-glucosylpyranosyl-1,6-D-fructofuranose) as the main product, and trehalulose (alpha-D-glucosylpyranosyl-1,1-D-fructofuranose) as minor product, and produces glucose and fructose in residual amounts because of sucrose hydrolysis	Erwinia rhapontici NX-5	?	-	?
sucrose	product specificity of enzyme NX-5 towards isomaltulose and the role of the loop330-339 in NX-5 catalysis	Erwinia rhapontici	1-O-alpha-D-glucopyranosyl-D-fructofuranose + alpha-D-glucosylpyranosyl-1,6-D-fructofuranose	-	?
sucrose	product specificity of enzyme NX-5 towards isomaltulose and the role of the loop330-339 in NX-5 catalysis	Erwinia rhapontici	1-O-alpha-D-glucopyranosyl-D-fructofuranose + alpha-D-glucosylpyranosyl-1,6-D-fructofuranose	main product isomaltulose, i.e. alpha-D-glucopyranosyl-1,6-D-fructofuranose or palatinose, and by-product trehalulose, i.e. alpha-D-glucopyranosyl-1,1-D-fructofuranose are two structural isomers of sucrose	?
sucrose	product specificity of enzyme NX-5 towards isomaltulose and the role of the loop330-339 in NX-5 catalysis	Erwinia rhapontici NX-5	1-O-alpha-D-glucopyranosyl-D-fructofuranose + alpha-D-glucosylpyranosyl-1,6-D-fructofuranose	-	?
sucrose	product specificity of enzyme NX-5 towards isomaltulose and the role of the loop330-339 in NX-5 catalysis	Erwinia rhapontici NX-5	1-O-alpha-D-glucopyranosyl-D-fructofuranose + alpha-D-glucosylpyranosyl-1,6-D-fructofuranose	main product isomaltulose, i.e. alpha-D-glucopyranosyl-1,6-D-fructofuranose or palatinose, and by-product trehalulose, i.e. alpha-D-glucopyranosyl-1,1-D-fructofuranose are two structural isomers of sucrose	?

Subunits

Subunits	Comment	Organism
More	enzyme structural analysis, overview	Erwinia rhapontici

Synonyms

Synonyms	Comment	Organism
NX-5	-	Erwinia rhapontici
SIase	-	Erwinia rhapontici
sucrose isomerase	-	Erwinia rhapontici

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Erwinia rhapontici

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6	-	assay at	Erwinia rhapontici

General Information

General Information	Comment	Organism
additional information	structural basis for the product specificity of NX-5, docking of fructofuranose and fructopyranose into the active site of the enzyme mutant D241A-glucose complex, molecular dynamics simulations, molecular mechanism controlling sucrose isomer formation, overview. Phe297 and Phe321 in enzyme NX-5 form an aromatic clamp near the entrance of the active pocket. Residues Asp102, His145, Arg239, His368, Asp369, Glu428, and Arg456 are responsible for sucrose binding and residues Asp241 (the nucleophile) and Glu295 (the acid catalyst) are crucial for sucrose hydrolysis. The active site pocket is stabilized by the salt bridge interactions between Arg239-Asp241, Asp102-Arg456 and Asp369-Arg456. Residues Arg325 and Arg328 are located in the325RLDRD329 motif and play roles in the product specificity	Erwinia rhapontici