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Literature summary 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.
    View publication on PubMed

Cloned(Commentary)

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

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.5.1.116 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
3.5.3.4 in complex with allantoate, sitting drop vapor diffusion method, using 0.1 M sodium citrate (pH 5.6), 20% (w/v) polyethylene glycol 4000, 20%(v/v) isopropanol, 1 mMMnCl2, and 5 mM allantoate Escherichia coli

Protein Variants

EC Number Protein Variants Comment Organism
3.5.1.116 D149A site-directed mutagenesis, inactive mutant Arabidopsis thaliana
3.5.1.116 D149N site-directed mutagenesis, inactive mutant Arabidopsis thaliana
3.5.1.116 D351A site-directed mutagenesis, inactive mutant Arabidopsis thaliana
3.5.1.116 E183A site-directed mutagenesis, inactive mutant Arabidopsis thaliana
3.5.1.116 E183A site-directed mutagenesis, inactive mutant, crystal structure analysis Arabidopsis thaliana
3.5.1.116 E183D site-directed mutagenesis, inactive mutant Arabidopsis thaliana
3.5.1.116 E183Q site-directed mutagenesis, inactive mutant Arabidopsis thaliana
3.5.1.116 E184A site-directed mutagenesis, inactive mutant Arabidopsis thaliana
3.5.1.116 H138A site-directed mutagenesis, inactive mutant Arabidopsis thaliana
3.5.1.116 H254A site-directed mutagenesis, inactive mutant Arabidopsis thaliana
3.5.1.116 H290A site-directed mutagenesis, inactive mutant Arabidopsis thaliana
3.5.1.116 H290N site-directed mutagenesis, inactive mutant Arabidopsis thaliana
3.5.1.116 H290Q site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Arabidopsis thaliana
3.5.1.116 H424N site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Arabidopsis thaliana
3.5.1.116 H448A site-directed mutagenesis, inactive mutant Arabidopsis thaliana
3.5.1.116 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
3.5.1.116 N340A site-directed mutagenesis, almost inactive mutant Arabidopsis thaliana
3.5.1.116 N340D site-directed mutagenesis, inactive mutant Arabidopsis thaliana
3.5.1.116 Q257A site-directed mutagenesis, inactive mutant Arabidopsis thaliana
3.5.1.116 Q257E site-directed mutagenesis, inactive mutant Arabidopsis thaliana
3.5.1.116 Q257N site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Arabidopsis thaliana
3.5.1.116 R353A site-directed mutagenesis, inactive mutant Arabidopsis thaliana
3.5.1.116 R353K site-directed mutagenesis, inactive mutant Arabidopsis thaliana
3.5.1.116 Y423A site-directed mutagenesis, inactive mutant Arabidopsis thaliana
3.5.1.116 Y423F site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Arabidopsis thaliana
3.5.3.4 E126A inactive Escherichia coli

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.5.1.116 additional information
-
 additional information steady-state kinetic analysis of wild-type and mutant enzymes Arabidopsis thaliana
3.5.1.116 0.0113
-
 (S)-ureidoglycolate wild-type enzyme, pH 8.5, 30°C Arabidopsis thaliana
3.5.1.116 0.021
-
 (S)-ureidoglycolate mutant Y423F, pH 8.5, 30°C Arabidopsis thaliana
3.5.1.116 0.033
-
 (S)-ureidoglycolate mutant H424N, pH 8.5, 30°C Arabidopsis thaliana
3.5.1.116 700
-
 (S)-ureidoglycolate mutant Q257N, pH 8.5, 30°C Arabidopsis thaliana
3.5.1.116 840
-
 (S)-ureidoglycolate mutant H290Q, pH 8.5, 30°C Arabidopsis thaliana

Localization

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

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.5.1.116 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
3.5.3.4 Co2+ required for maximum activity Escherichia coli
3.5.3.4 Mn2+ required for maximum activity Escherichia coli
3.5.3.4 Ni2+ required for maximum activity Escherichia coli

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.5.1.116 (S)-ureidoglycolate + H2O Arabidopsis thaliana
-
 glyoxylate + 2 NH3 + CO2
-
 ?
3.5.3.4 allantoate + H2O Escherichia coli
-
 (S)-ureidoglycolate + urea
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.5.1.116 Arabidopsis thaliana Q8VXY9
-
-
3.5.3.4 Escherichia coli P77425
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.5.1.116 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)

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

Synonyms

EC Number Synonyms Comment Organism
3.5.1.116 (S)-ureidoglycolate amidohydrolase
-
 Arabidopsis thaliana
3.5.1.116 UAH
-
 Arabidopsis thaliana
3.5.3.4 AAH
-
 Escherichia coli
3.5.3.4 allantoate amidohydrolase
-
 Escherichia coli

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.5.1.116 30
-
 assay at Arabidopsis thaliana

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.5.1.116 0.05
-
 (S)-ureidoglycolate mutant Q257N, pH 8.5, 30°C Arabidopsis thaliana
3.5.1.116 0.23
-
 (S)-ureidoglycolate mutant H424N, pH 8.5, 30°C Arabidopsis thaliana
3.5.1.116 0.37
-
 (S)-ureidoglycolate mutant H290Q, pH 8.5, 30°C Arabidopsis thaliana
3.5.1.116 0.59
-
 (S)-ureidoglycolate mutant Y423F, pH 8.5, 30°C Arabidopsis thaliana
3.5.1.116 1.43
-
 (S)-ureidoglycolate wild-type enzyme, pH 8.5, 30°C Arabidopsis thaliana

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.5.1.116 8.5
-
 assay at Arabidopsis thaliana

General Information

EC Number General Information Comment Organism
3.5.1.116 metabolism the enzyme is involved in the plant ureide pathway, overview Arabidopsis thaliana
3.5.1.116 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]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.5.1.116 0.08
-
 (S)-ureidoglycolate mutant Q257N, pH 8.5, 30°C Arabidopsis thaliana
3.5.1.116 0.43
-
 (S)-ureidoglycolate mutant H290Q, pH 8.5, 30°C Arabidopsis thaliana
3.5.1.116 7
-
 (S)-ureidoglycolate mutant H424N, pH 8.5, 30°C Arabidopsis thaliana
3.5.1.116 28
-
 (S)-ureidoglycolate mutant Y423F, pH 8.5, 30°C Arabidopsis thaliana
3.5.1.116 126.5
-
 (S)-ureidoglycolate wild-type enzyme, pH 8.5, 30°C Arabidopsis thaliana