Literature summary for 5.3.1.12 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.

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

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

Inhibitors

Inhibitors	Comment	Organism	Structure
arabinohydroxamate	-	Halalkalibacterium halodurans
D-arabinarate	-	Halalkalibacterium halodurans

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
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
0.05	-	D-glucuronate	pH 8.0, 25°C, mutant H33N	Escherichia coli
0.16	-	D-glucuronate	pH 8.0, 25°C, mutant Y60F	Escherichia coli
0.2	-	D-glucuronate	pH 8.0, 25°C, mutant H33A	Escherichia coli
0.4	-	D-glucuronate	pH 8.0, 25°C, mutant D412A	Escherichia coli
0.5	-	D-glucuronate	pH 8.0, 25°C, wild-type enzyme	Escherichia coli
0.7	-	D-glucuronate	pH 8.0, 25°C, mutant H59A	Escherichia coli
0.7	-	D-glucuronate	pH 8.0, 25°C, mutant H59N	Escherichia coli
0.82	-	D-glucuronate	pH 8.0, 25°C, mutant R414K	Escherichia coli
1	-	D-glucuronate	pH 8.0, 25°C, mutant D412N	Escherichia coli
1.3	-	D-glucuronate	pH 8.0, 25°C, mutant D238N	Escherichia coli
1.4	-	D-glucuronate	pH 8.0, 25°C, mutant R414M	Escherichia coli
1.7	-	D-glucuronate	pH 8.0, 25°C, mutant W381F	Escherichia coli
2.5	-	D-glucuronate	pH 8.0, 25°C, mutant R302K	Escherichia coli
2.6	-	D-glucuronate	pH 8.0, 25°C, mutant R186K	Escherichia coli
9.4	-	D-glucuronate	pH 8.0, 25°C, mutant H35N	Escherichia coli
10.21	-	D-glucuronate	pH 8.0, 25°C, mutant Y60A	Escherichia coli
21	-	D-glucuronate	pH 8.0, 25°C, mutant W381A	Escherichia coli
38	-	D-glucuronate	pH 8.0, 25°C, mutant R186M	Escherichia coli
39	-	D-glucuronate	pH 8.0, 25°C, mutant H35A	Escherichia coli
56	-	D-glucuronate	pH 8.0, 25°C, mutant H297N	Escherichia coli
200	-	D-glucuronate	pH 8.0, 25°C, mutant R302M	Escherichia coli
220	-	D-glucuronate	pH 8.0, 25°C, mutant H297A	Escherichia coli

Metals/Ions

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

Natural Substrates/ Products (Substrates)

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

Organism

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

Reaction

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

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
D-Galacturonate	-	Halalkalibacterium halodurans	D-Tagaturonate	-	r
D-Galacturonate	-	Escherichia coli	D-Tagaturonate	-	r
D-Glucuronate	-	Halalkalibacterium halodurans	D-Fructuronate	-	r
D-Glucuronate	-	Escherichia coli	D-Fructuronate	-	r
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
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	?	-	?
additional information	chemical mechanism and active site structure, mutational analysis, overview	Escherichia coli	?	-	?

Subunits

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

Synonyms

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

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
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
8	-	assay at	Escherichia coli

Cofactor

Cofactor	Comment	Organism	Structure
NADH	required	Halalkalibacterium halodurans
NADH	required	Escherichia coli

kcat/KM [mM/s]

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