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Literature summary extracted from

  • Nguyen, T.T.; Fedorov, A.A.; Williams, L.; Fedorov, E.V.; Li, Y.; Xu, C.; Almo, S.C.; Raushel, F.M.
    The mechanism of the reaction catalyzed by uronate isomerase illustrates how an isomerase may have evolved from a hydrolase within the amidohydrolase superfamily (2009), Biochemistry, 48, 8879-8890.
    View publication on PubMedView publication on EuropePMC

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
5.3.1.12 enzyme Bh0493 in complex with substrate D-glucuronate, D-fructuronate, or two inhibitory mimics of the cis-enediol intermediate, hanging drop method at room temperature, B16 mg/ml h0493 in 10 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM methionine, 10% glycerol, 1.0 mM DTT, 0.2 mM ZnCl2, and the corresponding substrate or inhibitor at 40 mM, precipitation solutions are 20% PEG 3350 and 0.2 M sodium citrate, pH 6.0, or 25% PEG 3350, 0.1 M Tris, pH 8.5, and 0.2 M NaCl, X-ray diffraction structure determination and analysis at 1.9-2.2 A resolution Halalkalibacterium halodurans

Protein Variants

EC Number Protein Variants Comment Organism
5.3.1.12 D238N site-directed mutagenesis, the mutant shows altered kinetics and zinc content compared to the wild-type enzyme Escherichia coli
5.3.1.12 D412A site-directed mutagenesis, the mutant shows altered kinetics and zinc content compared to the wild-type enzyme Escherichia coli
5.3.1.12 D412N site-directed mutagenesis, the mutant shows altered kinetics and zinc content compared to the wild-type enzyme Escherichia coli
5.3.1.12 H297A site-directed mutagenesis, the mutant shows altered kinetics and zinc content compared to the wild-type enzyme Escherichia coli
5.3.1.12 H297N site-directed mutagenesis, the mutant shows altered kinetics and zinc content compared to the wild-type enzyme Escherichia coli
5.3.1.12 H33A site-directed mutagenesis, the mutant shows altered kinetics and zinc content compared to the wild-type enzyme Escherichia coli
5.3.1.12 H33N site-directed mutagenesis, the mutant shows altered kinetics and zinc content compared to the wild-type enzyme Escherichia coli
5.3.1.12 H35A site-directed mutagenesis, the mutant shows altered kinetics and zinc content compared to the wild-type enzyme Escherichia coli
5.3.1.12 H35N site-directed mutagenesis, the mutant shows altered kinetics and zinc content compared to the wild-type enzyme Escherichia coli
5.3.1.12 H59A site-directed mutagenesis, the mutant shows altered kinetics and zinc content compared to the wild-type enzyme Escherichia coli
5.3.1.12 H59N site-directed mutagenesis, the mutant shows altered kinetics and zinc content compared to the wild-type enzyme Escherichia coli
5.3.1.12 additional information construction of mutants in association with the active site structure of URI from Bacillus halodurans Escherichia coli
5.3.1.12 R186K site-directed mutagenesis, the mutant shows altered kinetics and zinc content compared to the wild-type enzyme Escherichia coli
5.3.1.12 R186M site-directed mutagenesis, the mutant shows altered kinetics and zinc content compared to the wild-type enzyme Escherichia coli
5.3.1.12 R302K site-directed mutagenesis, the mutant shows altered kinetics and zinc content compared to the wild-type enzyme Escherichia coli
5.3.1.12 R302M site-directed mutagenesis, the mutant shows altered kinetics and zinc content compared to the wild-type enzyme Escherichia coli
5.3.1.12 R414K site-directed mutagenesis, the mutant shows altered kinetics and zinc content compared to the wild-type enzyme Escherichia coli
5.3.1.12 R414M site-directed mutagenesis, the mutant shows altered kinetics and zinc content compared to the wild-type enzyme Escherichia coli
5.3.1.12 W381A site-directed mutagenesis, the mutant shows altered kinetics and zinc content compared to the wild-type enzyme Escherichia coli
5.3.1.12 W381F site-directed mutagenesis, the mutant shows altered kinetics and zinc content compared to the wild-type enzyme Escherichia coli
5.3.1.12 Y60A site-directed mutagenesis, the mutant shows altered kinetics and zinc content compared to the wild-type enzyme Escherichia coli
5.3.1.12 Y60F site-directed mutagenesis, the mutant shows altered kinetics and zinc content compared to the wild-type enzyme Escherichia coli

Inhibitors

EC Number Inhibitors Comment Organism Structure
5.3.1.12 arabinohydroxamate
-
Halalkalibacterium halodurans
5.3.1.12 D-arabinarate
-
Halalkalibacterium halodurans

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
5.3.1.12 additional information
-
additional information primary isotope effects on the kinetic constants with D-glucuronate and the effects of changes in solvent viscosity are consistent with product release being the rate-limiting step Escherichia coli
5.3.1.12 0.05
-
D-glucuronate pH 8.0, 25°C, mutant H33N Escherichia coli
5.3.1.12 0.16
-
D-glucuronate pH 8.0, 25°C, mutant Y60F Escherichia coli
5.3.1.12 0.2
-
D-glucuronate pH 8.0, 25°C, mutant H33A Escherichia coli
5.3.1.12 0.4
-
D-glucuronate pH 8.0, 25°C, mutant D412A Escherichia coli
5.3.1.12 0.5
-
D-glucuronate pH 8.0, 25°C, wild-type enzyme Escherichia coli
5.3.1.12 0.7
-
D-glucuronate pH 8.0, 25°C, mutant H59A Escherichia coli
5.3.1.12 0.7
-
D-glucuronate pH 8.0, 25°C, mutant H59N Escherichia coli
5.3.1.12 0.82
-
D-glucuronate pH 8.0, 25°C, mutant R414K Escherichia coli
5.3.1.12 1
-
D-glucuronate pH 8.0, 25°C, mutant D412N Escherichia coli
5.3.1.12 1.3
-
D-glucuronate pH 8.0, 25°C, mutant D238N Escherichia coli
5.3.1.12 1.4
-
D-glucuronate pH 8.0, 25°C, mutant R414M Escherichia coli
5.3.1.12 1.7
-
D-glucuronate pH 8.0, 25°C, mutant W381F Escherichia coli
5.3.1.12 2.5
-
D-glucuronate pH 8.0, 25°C, mutant R302K Escherichia coli
5.3.1.12 2.6
-
D-glucuronate pH 8.0, 25°C, mutant R186K Escherichia coli
5.3.1.12 9.4
-
D-glucuronate pH 8.0, 25°C, mutant H35N Escherichia coli
5.3.1.12 10.21
-
D-glucuronate pH 8.0, 25°C, mutant Y60A Escherichia coli
5.3.1.12 21
-
D-glucuronate pH 8.0, 25°C, mutant W381A Escherichia coli
5.3.1.12 38
-
D-glucuronate pH 8.0, 25°C, mutant R186M Escherichia coli
5.3.1.12 39
-
D-glucuronate pH 8.0, 25°C, mutant H35A Escherichia coli
5.3.1.12 56
-
D-glucuronate pH 8.0, 25°C, mutant H297N Escherichia coli
5.3.1.12 200
-
D-glucuronate pH 8.0, 25°C, mutant R302M Escherichia coli
5.3.1.12 220
-
D-glucuronate pH 8.0, 25°C, mutant H297A Escherichia coli

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
5.3.1.12 Zn2+ URI contains up to 1 equivalent Escherichia coli

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
5.3.1.12 D-Galacturonate Halalkalibacterium halodurans
-
D-Tagaturonate
-
r
5.3.1.12 D-Galacturonate Escherichia coli
-
D-Tagaturonate
-
r
5.3.1.12 D-Glucuronate Halalkalibacterium halodurans
-
D-Fructuronate
-
r
5.3.1.12 D-Glucuronate Escherichia coli
-
D-Fructuronate
-
r

Organism

EC Number Organism UniProt Comment Textmining
5.3.1.12 Escherichia coli A0A140N3B4 gene uxaC
-
5.3.1.12 Halalkalibacterium halodurans
-
-
-

Reaction

EC Number Reaction Comment Organism Reaction ID
5.3.1.12 D-Glucuronate = D-fructuronate reaction mechanism, overview Halalkalibacterium halodurans
5.3.1.12 D-Glucuronate = D-fructuronate reaction mechanism, overview Escherichia coli

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
5.3.1.12 D-Galacturonate
-
Halalkalibacterium halodurans D-Tagaturonate
-
r
5.3.1.12 D-Galacturonate
-
Escherichia coli D-Tagaturonate
-
r
5.3.1.12 D-Glucuronate
-
Halalkalibacterium halodurans D-Fructuronate
-
r
5.3.1.12 D-Glucuronate
-
Escherichia coli D-Fructuronate
-
r
5.3.1.12 D-Glucuronate the mononuclear metal center in the active site is ligated to the C6 carboxylate and the C5 hydroxyl group of the substrate, this hydroxyl group is also hydrogen-bonded to Asp355. The C2 and C3 hydroxyl groups of the substrate are hydrogen bonded to Arg357 and the carbonyl group at C1 is hydrogen bonded to Tyr50 Halalkalibacterium halodurans D-Fructuronate
-
r
5.3.1.12 additional information active site structure and molecular reaction mechanism, proton transfer from C2 of D-glucuronate to C1 that is initiated by the combined actions of Asp-355 from the end of ?-strand 8 and the C-5 hydroxyl of the substrate that is bound to the metal ion. Formation of the proposed cis-enediol intermediate is further facilitated by the shuttling of the proton between the C2 and C1 oxygens by the conserved Tyr50 and/or Arg355 Halalkalibacterium halodurans ?
-
?
5.3.1.12 additional information chemical mechanism and active site structure, mutational analysis, overview Escherichia coli ?
-
?

Subunits

EC Number Subunits Comment Organism
5.3.1.12 More structure modelling, overview Halalkalibacterium halodurans
5.3.1.12 More structure modelling using the crystal structure of URI from Bacillus halodurans, overview Escherichia coli

Synonyms

EC Number Synonyms Comment Organism
5.3.1.12 Bh0493
-
Halalkalibacterium halodurans
5.3.1.12 More URI is a member of the amidohydrolase superfamily, AHS Halalkalibacterium halodurans
5.3.1.12 More URI is a member of the amidohydrolase superfamily, AHS Escherichia coli
5.3.1.12 URI
-
Halalkalibacterium halodurans
5.3.1.12 URI
-
Escherichia coli
5.3.1.12 Uronate isomerase
-
Halalkalibacterium halodurans
5.3.1.12 Uronate isomerase
-
Escherichia coli
5.3.1.12 UxaC
-
Escherichia coli

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
5.3.1.12 25
-
assay at Escherichia coli

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
5.3.1.12 0.6
-
D-glucuronate pH 8.0, 25°C, mutant D412N Escherichia coli
5.3.1.12 0.6
-
D-glucuronate pH 8.0, 25°C, mutant H33A Escherichia coli
5.3.1.12 0.6
-
D-glucuronate pH 8.0, 25°C, mutant H59A Escherichia coli
5.3.1.12 0.7
-
D-glucuronate pH 8.0, 25°C, mutant H35A Escherichia coli
5.3.1.12 0.7
-
D-glucuronate pH 8.0, 25°C, mutant R414M Escherichia coli
5.3.1.12 2.1
-
D-glucuronate pH 8.0, 25°C, mutant H33N Escherichia coli
5.3.1.12 4
-
D-glucuronate pH 8.0, 25°C, mutant H35N Escherichia coli
5.3.1.12 4.7
-
D-glucuronate pH 8.0, 25°C, mutant R186M Escherichia coli
5.3.1.12 5.8
-
D-glucuronate pH 8.0, 25°C, mutant R414K Escherichia coli
5.3.1.12 9
-
D-glucuronate pH 8.0, 25°C, mutant D412A Escherichia coli
5.3.1.12 10
-
D-glucuronate pH 8.0, 25°C, mutant H297A Escherichia coli
5.3.1.12 13.9
-
D-glucuronate pH 8.0, 25°C, mutant Y60A Escherichia coli
5.3.1.12 15
-
D-glucuronate pH 8.0, 25°C, mutant H59N Escherichia coli
5.3.1.12 16
-
D-glucuronate pH 8.0, 25°C, mutant W381F Escherichia coli
5.3.1.12 21.7
-
D-glucuronate pH 8.0, 25°C, mutant Y60F Escherichia coli
5.3.1.12 30
-
D-glucuronate pH 8.0, 25°C, mutant H297N Escherichia coli
5.3.1.12 54
-
D-glucuronate pH 8.0, 25°C, mutant R186K Escherichia coli
5.3.1.12 60
-
D-glucuronate pH 8.0, 25°C, mutant D238N Escherichia coli
5.3.1.12 160
-
D-glucuronate pH 8.0, 25°C, mutant R302K Escherichia coli
5.3.1.12 180
-
D-glucuronate pH 8.0, 25°C, mutant R302M Escherichia coli
5.3.1.12 196
-
D-glucuronate pH 8.0, 25°C, wild-type enzyme Escherichia coli
5.3.1.12 250
-
D-glucuronate pH 8.0, 25°C, mutant W381A Escherichia coli

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
5.3.1.12 additional information
-
the pH-rate profiles for kcat and kcat/Km for URI from Escherichia coli are bellshaped and indicate that one group must be unprotonated and another residue must be protonated for catalytic activity Escherichia coli
5.3.1.12 8
-
assay at Escherichia coli

Cofactor

EC Number Cofactor Comment Organism Structure
5.3.1.12 NADH required Halalkalibacterium halodurans
5.3.1.12 NADH required Escherichia coli

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
5.3.1.12 0.018
-
D-glucuronate pH 8.0, 25°C, mutant H35A Escherichia coli
5.3.1.12 0.021
-
D-glucuronate pH 8.0, 25°C, mutant D412A Escherichia coli
5.3.1.12 0.043
-
D-glucuronate pH 8.0, 25°C, mutant H297A Escherichia coli
5.3.1.12 0.054
-
D-glucuronate pH 8.0, 25°C, mutant R414M Escherichia coli
5.3.1.12 0.06
-
D-glucuronate pH 8.0, 25°C, mutant D412N Escherichia coli
5.3.1.12 0.088
-
D-glucuronate pH 8.0, 25°C, mutant R302M Escherichia coli
5.3.1.12 0.13
-
D-glucuronate pH 8.0, 25°C, mutant R186M Escherichia coli
5.3.1.12 0.43
-
D-glucuronate pH 8.0, 25°C, mutant H35N Escherichia coli
5.3.1.12 0.5
-
D-glucuronate pH 8.0, 25°C, mutant H297N Escherichia coli
5.3.1.12 0.83
-
D-glucuronate pH 8.0, 25°C, mutant H59A Escherichia coli
5.3.1.12 3
-
D-glucuronate pH 8.0, 25°C, mutant H33A Escherichia coli
5.3.1.12 7.1
-
D-glucuronate pH 8.0, 25°C, mutant R414K Escherichia coli
5.3.1.12 9.5
-
D-glucuronate pH 8.0, 25°C, mutant W381F Escherichia coli
5.3.1.12 12
-
D-glucuronate pH 8.0, 25°C, mutant W381A Escherichia coli
5.3.1.12 21
-
D-glucuronate pH 8.0, 25°C, mutant H59N Escherichia coli
5.3.1.12 21
-
D-glucuronate pH 8.0, 25°C, mutant R186K Escherichia coli
5.3.1.12 46
-
D-glucuronate pH 8.0, 25°C, mutant D238N Escherichia coli
5.3.1.12 47
-
D-glucuronate pH 8.0, 25°C, mutant H33N Escherichia coli
5.3.1.12 63
-
D-glucuronate pH 8.0, 25°C, mutant R302K Escherichia coli
5.3.1.12 66
-
D-glucuronate pH 8.0, 25°C, mutant Y60A Escherichia coli
5.3.1.12 140
-
D-glucuronate pH 8.0, 25°C, mutant Y60F Escherichia coli
5.3.1.12 400
-
D-glucuronate pH 8.0, 25°C, wild-type enzyme Escherichia coli