Literature summary for 3.5.1.116 extracted from

Shin, I.; Han, K.; Rhee, S.
Structural insights into the substrate specificity of (S)-ureidoglycolate amidohydrolase and its comparison with allantoate amidohydrolase (2014), J. Mol. Biol., 426, 3028-3040.

Search for more literature for 3.5.1.116

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of seleno-L-methionine-substituted, N-terminally His-tagged enzyme in Escherichia coli strain BL21(DE3)	Arabidopsis thaliana

Crystallization (Commentary)

Crystallization (Comment)	Organism
wild-type enzyme and mutant E183A complexed with substrate, reaction intermediate, and product, sitting drop vapor diffusion method, mixing of 10 mg/ml protein in 50 mM Tris, pH 7.4, 1 mM MnCl2, and 5 mM DTT, with crystallization buffer of 0.1 M 4-morpholineethanesulfonic acid, pH 6.5, and 12% w/v PEG 20000, at 22°C, crystals of the AtUAH binary complex with substrate or reaction intermediate by co-crystallization of mutant E183A with a (S)-ureidoglycolate, from a crystallization solution containing 0.2 M calcium acetate hydrate, 20% w/v PEG 3350, 1 mM MnCl2, and 5 or 2 mM, respectively, (S)-ureidoglycolate, X-ray diffraction structure determination and analysis at 1.45-2.20 A resolution	Arabidopsis thaliana

Crystallization (Comment)

Organism

wild-type enzyme and mutant E183A complexed with substrate, reaction intermediate, and product, sitting drop vapor diffusion method, mixing of 10 mg/ml protein in 50 mM Tris, pH 7.4, 1 mM MnCl2, and 5 mM DTT, with crystallization buffer of 0.1 M 4-morpholineethanesulfonic acid, pH 6.5, and 12% w/v PEG 20000, at 22°C, crystals of the AtUAH binary complex with substrate or reaction intermediate by co-crystallization of mutant E183A with a (S)-ureidoglycolate, from a crystallization solution containing 0.2 M calcium acetate hydrate, 20% w/v PEG 3350, 1 mM MnCl2, and 5 or 2 mM, respectively, (S)-ureidoglycolate, X-ray diffraction structure determination and analysis at 1.45-2.20 A resolution

Arabidopsis thaliana

Protein Variants

Protein Variants	Comment	Organism
D149A	site-directed mutagenesis, inactive mutant	Arabidopsis thaliana
D149N	site-directed mutagenesis, inactive mutant	Arabidopsis thaliana
D351A	site-directed mutagenesis, inactive mutant	Arabidopsis thaliana
E183A	site-directed mutagenesis, inactive mutant	Arabidopsis thaliana
E183A	site-directed mutagenesis, inactive mutant, crystal structure analysis	Arabidopsis thaliana
E183D	site-directed mutagenesis, inactive mutant	Arabidopsis thaliana
E183Q	site-directed mutagenesis, inactive mutant	Arabidopsis thaliana
E184A	site-directed mutagenesis, inactive mutant	Arabidopsis thaliana
H138A	site-directed mutagenesis, inactive mutant	Arabidopsis thaliana
H254A	site-directed mutagenesis, inactive mutant	Arabidopsis thaliana
H290A	site-directed mutagenesis, inactive mutant	Arabidopsis thaliana
H290N	site-directed mutagenesis, inactive mutant	Arabidopsis thaliana
H290Q	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Arabidopsis thaliana
H424N	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Arabidopsis thaliana
H448A	site-directed mutagenesis, inactive mutant	Arabidopsis thaliana
additional information	conferring an allantoate amidinohydrolase-like activity on the enzyme by redesigning the size of the (S)-ureidoglycolate amidohydrolase active site and modifying the substrate specifiicty is not successfull by simply relieving the possible steric hindrance of Tyr423 to the incoming pro-S ureido group in allantoate.The AtUAH Y423G mutant is inactive with allantoate. Substrate specificity in the (S)-ureidoglycolate amidohydrolase is a function of interactions more complex than those conferred by a single active-site residue	Arabidopsis thaliana
N340A	site-directed mutagenesis, almost inactive mutant	Arabidopsis thaliana
N340D	site-directed mutagenesis, inactive mutant	Arabidopsis thaliana
Q257A	site-directed mutagenesis, inactive mutant	Arabidopsis thaliana
Q257E	site-directed mutagenesis, inactive mutant	Arabidopsis thaliana
Q257N	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Arabidopsis thaliana
R353A	site-directed mutagenesis, inactive mutant	Arabidopsis thaliana
R353K	site-directed mutagenesis, inactive mutant	Arabidopsis thaliana
Y423A	site-directed mutagenesis, inactive mutant	Arabidopsis thaliana
Y423F	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Arabidopsis thaliana

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	steady-state kinetic analysis of wild-type and mutant enzymes	Arabidopsis thaliana
0.0113	-	(S)-ureidoglycolate	wild-type enzyme, pH 8.5, 30°C	Arabidopsis thaliana
0.021	-	(S)-ureidoglycolate	mutant Y423F, pH 8.5, 30°C	Arabidopsis thaliana
0.033	-	(S)-ureidoglycolate	mutant H424N, pH 8.5, 30°C	Arabidopsis thaliana
700	-	(S)-ureidoglycolate	mutant Q257N, pH 8.5, 30°C	Arabidopsis thaliana
840	-	(S)-ureidoglycolate	mutant H290Q, pH 8.5, 30°C	Arabidopsis thaliana

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
endoplasmic reticulum	sequences of endoplasmic reticulum-targeting signals comprise residues 1-25	Arabidopsis thaliana	5783	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mn2+	Mn2+ is required for maximum catalytic activity. The bimetal reaction center of the enzyme is located at the pocket enclosed by the catalytic domain, dimerization domain, and other structural elements from a different subunit, structure, overview	Arabidopsis thaliana

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
(S)-ureidoglycolate + H2O	Arabidopsis thaliana	-	glyoxylate + 2 NH3 + CO2	-	?

Organism

Organism	UniProt	Comment	Textmining
Arabidopsis thaliana	Q8VXY9	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant seleno-L-methionine-substituted, N-terminally His-tagged enzyme from Escherichia coli strain BL21(DE3) by immobilized metal affinity achromatography, tag cleavage by TEV protease, followed by dialysis and gel filtration	Arabidopsis thaliana

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
(S)-ureidoglycolate + H2O	-	Arabidopsis thaliana	glyoxylate + 2 NH3 + CO2	-	?
(S)-ureidoglycolate + H2O	via the reaction intermediate (S)-hydroxyglycine, enzyme binding structure of substrate, intermediate, and product, overview	Arabidopsis thaliana	glyoxylate + 2 NH3 + CO2	-	?
additional information	substrate specificity, overview	Arabidopsis thaliana	?	-	?

Synonyms

Synonyms	Comment	Organism
(S)-ureidoglycolate amidohydrolase	-	Arabidopsis thaliana
UAH	-	Arabidopsis thaliana

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Arabidopsis thaliana

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.05	-	(S)-ureidoglycolate	mutant Q257N, pH 8.5, 30°C	Arabidopsis thaliana
0.23	-	(S)-ureidoglycolate	mutant H424N, pH 8.5, 30°C	Arabidopsis thaliana
0.37	-	(S)-ureidoglycolate	mutant H290Q, pH 8.5, 30°C	Arabidopsis thaliana
0.59	-	(S)-ureidoglycolate	mutant Y423F, pH 8.5, 30°C	Arabidopsis thaliana
1.43	-	(S)-ureidoglycolate	wild-type enzyme, pH 8.5, 30°C	Arabidopsis thaliana

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8.5	-	assay at	Arabidopsis thaliana

General Information

General Information	Comment	Organism
metabolism	the enzyme is involved in the plant ureide pathway, overview	Arabidopsis thaliana
additional information	amino acid and structure comparisons, e.g. to allantoate amidinohydrolase, enzyme monomer structure modeling, overview. Monomeric AtUAH (Asn54 to Asp476) is composed of 13 alpha-helices, 12 beta-strands, and 2 short 310-helices. It folds largely into two structural domains: a catalytic domain (residues 54-275 and 392-476) and a dimerization domain (residues 276-391) that is inserted between beta6 and alpha11 of the catalytic domain. The two structural domains are connected by a so-called hinge region (residues 273-275 and 392-394), structure-function analysis, overview. The catalytic domain exhibits an alpha/beta/alpha-folded architecture. Substrate specificity in the (S)-ureidoglycolate amidohydrolase is a function of interactions more complex than those conferred by a single active-site residue	Arabidopsis thaliana

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.08	-	(S)-ureidoglycolate	mutant Q257N, pH 8.5, 30°C	Arabidopsis thaliana
0.43	-	(S)-ureidoglycolate	mutant H290Q, pH 8.5, 30°C	Arabidopsis thaliana
7	-	(S)-ureidoglycolate	mutant H424N, pH 8.5, 30°C	Arabidopsis thaliana
28	-	(S)-ureidoglycolate	mutant Y423F, pH 8.5, 30°C	Arabidopsis thaliana
126.5	-	(S)-ureidoglycolate	wild-type enzyme, pH 8.5, 30°C	Arabidopsis thaliana