Literature summary for 5.1.3.17 extracted from

Qin, Y.; Ke, J.; Gu, X.; Fang, J.; Wang, W.; Cong, Q.; Li, J.; Tan, J.; Brunzelle, J.S.; Zhang, C.; Jiang, Y.; Melcher, K.; Li, J.P.; Xu, H.E.; Ding, K.
Structural and functional study of D-glucuronyl C5-epimerase (2015), J. Biol. Chem., 290, 4620-4630 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene glce, recombinant expression of enzyme mutant comprising residues Arg50-Asn585 as N-terminally His6-SUMO-tagged enzyme in Escherichia coli strain BL21(DE3), expression of N-terminally His6-SUMO-tagged SeMet-substituted Glce protein in Escherichia coli strain B834, expression of diverse enzyme point mutants	Danio rerio

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified enzyme in apo-form and in complex with heparin hexasaccharide, both in a stable dimer conformation, hanging drop vapour diffusion method, mixing of 0.001 ml of 15-20 mg/ml protein in in 20 mM Tris, pH 8.0, 200 mM ammonium acetate, 1 mM dithiothreitol, and 1 mM EDTA, with 0.001 ml of 16% w/v PEG 3350, 0.1 M sodium citrate tribasic dihydrate, pH 5.6, and 2% v/v Tacsimate, pH 5.0 for the unlabeled enzyme and 16% w/v PEG 3350, 0.06 M citric acid, and 0.04 M Bis-Tris propane, pH 4.1, for the SeMet-labeled enzyme, a concentration of 5.0 mg/ml is used for the enzyme complex with heparin oligosaccharide at a molar ratio of 1:5, 20°C, 3 days, X-ray diffraction structure determination and analysis at 1.9-2.2 A resolution	Danio rerio

Crystallization (Comment)

Organism

purified enzyme in apo-form and in complex with heparin hexasaccharide, both in a stable dimer conformation, hanging drop vapour diffusion method, mixing of 0.001 ml of 15-20 mg/ml protein in in 20 mM Tris, pH 8.0, 200 mM ammonium acetate, 1 mM dithiothreitol, and 1 mM EDTA, with 0.001 ml of 16% w/v PEG 3350, 0.1 M sodium citrate tribasic dihydrate, pH 5.6, and 2% v/v Tacsimate, pH 5.0 for the unlabeled enzyme and 16% w/v PEG 3350, 0.06 M citric acid, and 0.04 M Bis-Tris propane, pH 4.1, for the SeMet-labeled enzyme, a concentration of 5.0 mg/ml is used for the enzyme complex with heparin oligosaccharide at a molar ratio of 1:5, 20°C, 3 days, X-ray diffraction structure determination and analysis at 1.9-2.2 A resolution

Danio rerio

Protein Variants

Protein Variants	Comment	Organism
H584A	site-directed mutagenesis, the mutant shows 80% reduced activity compared to wild-type	Danio rerio
N585A	site-directed mutagenesis, the mutant shows 90% reduced activity compared to wild-type	Danio rerio
R154A	site-directed mutagenesis, the mutant shows 60% reduced activity compared to wild-type	Danio rerio
R156A	site-directed mutagenesis, the mutant shows over 90% reduced activity compared to wild-type	Danio rerio
R396A	site-directed mutagenesis, the mutant shows 90% reduced activity compared to wild-type	Danio rerio
R531A	site-directed mutagenesis, the mutant shows about 90% reduced activity compared to wild-type	Danio rerio
R543A	site-directed mutagenesis, the mutant shows over 90% reduced activity compared to wild-type	Danio rerio
Y149F	site-directed mutagenesis, the mutant shows over 90% reduced activity compared to wild-type	Danio rerio
Y177F	site-directed mutagenesis, the mutant shows 40% reduced activity compared to wild-type	Danio rerio
Y179F	site-directed mutagenesis, the mutant shows slightly reduced activity compared to wild-type	Danio rerio
Y468A	site-directed mutagenesis, the mutant shows over 90% reduced activity compared to wild-type	Danio rerio
Y468F	site-directed mutagenesis, the mutant shows 75% reduced activity compared to wild-type	Danio rerio
Y482F	site-directed mutagenesis, the mutant shows 20% reduced activity compared to wild-type	Danio rerio
Y528A	site-directed mutagenesis, the mutant shows over 90% reduced activity compared to wild-type	Danio rerio
Y528F	site-directed mutagenesis, the mutant shows over 90% reduced activity compared to wild-type	Danio rerio
Y546A	site-directed mutagenesis, the mutant shows over 90% reduced activity compared to wild-type	Danio rerio
Y546F	site-directed mutagenesis, the mutant shows over 90% reduced activity compared to wild-type	Danio rerio

Inhibitors

Inhibitors	Comment	Organism	Structure
heparin hexasaccharide	mechanism of product inhibition of the enzyme: 2-O- and 6-O-sulfation of heparan sulfate keeps the C5 carbon of L-iduronic acid away from the active-site tyrosine residues, binding structure, detailed overview	Danio rerio

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
125000	-	recombinant enzyme, gel filtration	Danio rerio

Organism

Organism	UniProt	Comment	Textmining
Danio rerio	F1QR43	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His6-SUMO-tagged enzyme mutants from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, dialysis, tag cleavage, another step of nickel affinity chromatography, and gel filtration of the eluate	Danio rerio

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
additional information	heparin hexasaccharide is product of Glce following O-sulfation, structure of the Glce dimer in complex with heparin hexasaccharide, detailed overview	Danio rerio	?	-	?

Subunits

Subunits	Comment	Organism
homodimer	2 * 60600, recombinant enzyme, SDS-PAGE	Danio rerio
More	the enzyme forms a dimer with two catalytic sites, each at a positively charged cleft in C-terminal alpha-helical domains binding one negatively charged hexasaccharide	Danio rerio

Synonyms

Synonyms	Comment	Organism
D-glucuronyl C5-epimerase	-	Danio rerio
Glce	-	Danio rerio

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Danio rerio

General Information

General Information	Comment	Organism
metabolism	D-glucuronyl C5-epimerase is a crucial modifying enzyme in the heparan sulfate biosynthesis pathway	Danio rerio
additional information	enzyme structure-function relationship, active site structure and function, overview. Three tyrosine residues, Tyr468, Tyr528, and Tyr546, in the active site are crucial for the enzymatic activity	Danio rerio
physiological function	heparan sulfate (HS) is a glycosaminoglycan present on the cell surface and in the extracellular matrix, which interacts with diverse signal molecules and is essential for many physiological processes including embryonic development, cell growth, inflammation, and blood coagulation. D-Glucuronyl C5-epimerase (Glce) is a crucial enzyme in HS synthesis, converting D-glucuronic acid to L-iduronic acid to increase HS flexibility. This modification of HS is important for protein ligand recognition	Danio rerio