Literature summary for 5.1.3.31 extracted from

Yoshida, H.; Yoshihara, A.; Ishii, T.; Izumori, K.; Kamitori, S.
X-ray structures of the Pseudomonas cichorii D-tagatose 3-epimerase mutant form C66S recognizing deoxy sugars as substrates (2016), Appl. Microbiol. Biotechnol., 100, 10403-10415 .

Application

Application	Comment	Organism
synthesis	enzyme PcDTE is used industrially to produce D-psicose from the more abundant sugar D-fructose	Pseudomonas cichorii

Cloned(Commentary)

Cloned (Comment)	Organism
gene dte, recombinant expression of C-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain JM109	Pseudomonas cichorii

Crystallization (Commentary)

Crystallization (Comment)	Organism
recombinant enzyme mutant PcDTE_C66S in complexes with four deoxy rare sugars, 6-deoxy L-psicose, 1-deoxy 3-keto D-galactitol, 1-deoxy D-tagatose, and 1-deoxy L-tagatose, and with L-erythrulose (a sugar without groups at the 5- and 6-positions), hanging drop vapor diffusion method, mixing of 0.002 ml of 6-7 mg/ml protein in 5 mM Tris-HCl, pH 8.0, with 0.002 ml of reservoir solution containing 6.0-11.0 % w/v PEG 4000 and 100 mM CH3COONa, pH 4.6, and equilibration against 0.45 ml of reservoir solution, microgravity, X-ray diffraction structure determination and analysis at 1.59-2.3 A resolution, molecular replacement using crystal structure, PDB ID 1QUL, as a search model	Pseudomonas cichorii

Crystallization (Comment)

Organism

recombinant enzyme mutant PcDTE_C66S in complexes with four deoxy rare sugars, 6-deoxy L-psicose, 1-deoxy 3-keto D-galactitol, 1-deoxy D-tagatose, and 1-deoxy L-tagatose, and with L-erythrulose (a sugar without groups at the 5- and 6-positions), hanging drop vapor diffusion method, mixing of 0.002 ml of 6-7 mg/ml protein in 5 mM Tris-HCl, pH 8.0, with 0.002 ml of reservoir solution containing 6.0-11.0 % w/v PEG 4000 and 100 mM CH3COONa, pH 4.6, and equilibration against 0.45 ml of reservoir solution, microgravity, X-ray diffraction structure determination and analysis at 1.59-2.3 A resolution, molecular replacement using crystal structure, PDB ID 1QUL, as a search model

Pseudomonas cichorii

Protein Variants

Protein Variants	Comment	Organism
C66S	site-directed mutagenesis, the enzyme mutant recognizes deoxy sugars as substrates. In PcDTE_C66S/deoxy sugar complex structures, bound ligand molecules in both the linear and ring forms are detected in the hydrophobic groove, while bound ligand molecules in the catalytic site are in the linear form	Pseudomonas cichorii

Metals/Ions

Metals/Ions	Comment	Organism
Co2+	activates, best metal ion	Pseudomonas cichorii
Mn2+	activates	Pseudomonas cichorii
additional information	metal enzyme, divalent cations are required for activity	Pseudomonas cichorii

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
D-fructose	Pseudomonas cichorii	-	D-psicose	-	r
D-tagatose	Pseudomonas cichorii	-	D-sorbose	-	r

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas cichorii	O50580	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant C-terminally His-tagged wild-type and mutant enzymes from Escherichia coli strain JM109 by nickel affinity chromatography and ultrafiltration	Pseudomonas cichorii

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
0.127	-	recombinant enzyme mutant C66S, substrate 1-deoxy D-sorbose, pH 9.0, 60°C	Pseudomonas cichorii
0.15	-	recombinant enzyme mutant C66S, substrate 1-deoxy L-sorbose, pH 9.0, 60°C	Pseudomonas cichorii
7.6	-	recombinant enzyme mutant C66S, substrate 1-deoxy 3-keto D-allitol, pH 9.0, 60°C	Pseudomonas cichorii
16.9	-	recombinant enzyme mutant C66S, substrate 6-deoxy L-fructose, pH 9.0, 60°C	Pseudomonas cichorii
108	-	recombinant enzyme mutant C66S, substrate D-tagatose, pH 9.0, 60°C	Pseudomonas cichorii

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
1-deoxy 3-keto D-galactitol	7% activity compared to the activity with D-tagatose	Pseudomonas cichorii	1-deoxy 3-keto D-allitol	-	r
1-deoxy D-tagatose	0.12% activity compared to the activity with D-tagatose	Pseudomonas cichorii	1-deoxy D-sorbose	-	r
1-deoxy L-tagatose	0.14% activity compared to the activity with D-tagatose	Pseudomonas cichorii	1-deoxy L-sorbose	-	r
6-deoxy L-psicose	15.7% activity compared to the activity with D-tagatose	Pseudomonas cichorii	6-deoxy L-fructose	-	r
D-fructose	-	Pseudomonas cichorii	D-psicose	-	r
D-tagatose	-	Pseudomonas cichorii	D-sorbose	-	r
additional information	D-tagatose 3-epimerase (PcDTE) has a broad substrate specificity, it efficiently catalyzes the epimerization of not only D-tagatose to D-sorbose but also D-fructose to D-psicose (D-allulose) and also recognizes the deoxy sugars as substrates. Substrate recognition by the enzyme at the 1-, 2-, and 3-positions is responsible for enzymatic activity and substrate-enzyme interactions at the 4-, 5-, and 6-positions are not essential for the catalytic reaction of the enzyme leading to the broad substrate specificity of PcDTE. 1-Deoxy sugars may bind to the catalytic site in the inhibitor-binding mode. Ligand-binding structure at the catalytic site, overview. Binding structures of 6-deoxy L-psicose, 1-deoxy-3-oxo-D-galactitol, 1-deoxy-D-tagatose, 1-deoxy L-tagatose, L-erythrulose, D-talitol, and glycerol	Pseudomonas cichorii	?	-	?

Synonyms

Synonyms	Comment	Organism
DTE	-	Pseudomonas cichorii
PcDTE	-	Pseudomonas cichorii

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
60	-	assay at	Pseudomonas cichorii

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
9	-	assay at	Pseudomonas cichorii

General Information

General Information	Comment	Organism
evolution	the enzyme belongs to the D-tagatose 3-epimerase (D-TE) family	Pseudomonas cichorii
additional information	the hydrophobic groove that acts as an accessible surface for substrate binding is formed through the dimerization of PcDTE. The sugar-ring opening of a substrate may occur in the hydrophobic groove and also that the narrow channel of the passageway to the catalytic site allows a substrate in the linear form to pass through. Ligand-binding structure at the catalytic site, overview	Pseudomonas cichorii
physiological function	D-tagatose 3-epimerase (DTE) catalyzes epimerization between D-tagatose and D-sorbose. DTE from Pseudomonas cichorii (PcDTE) has a broad substrate specificity and efficiently catalyzes the epimerization of not only D-tagatose to D-sorbose but also D-fructose to D-psicose (D-allulose)	Pseudomonas cichorii