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 | 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 | Comment | Organism | Structure |
---|---|---|---|
arabinohydroxamate | - |
Halalkalibacterium halodurans | |
D-arabinarate | - |
Halalkalibacterium halodurans |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
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 | Comment | Organism | Structure |
---|---|---|---|
Zn2+ | URI contains up to 1 equivalent | Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | A0A140N3B4 | gene uxaC | - |
Halalkalibacterium halodurans | - |
- |
- |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
D-Glucuronate = D-fructuronate | reaction mechanism, overview | Halalkalibacterium halodurans | |
D-Glucuronate = D-fructuronate | reaction mechanism, overview | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | Comment | Organism |
---|---|---|
More | structure modelling, overview | Halalkalibacterium halodurans |
More | structure modelling using the crystal structure of URI from Bacillus halodurans, overview | Escherichia coli |
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 Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Escherichia coli |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
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 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 | Comment | Organism | Structure |
---|---|---|---|
NADH | required | Halalkalibacterium halodurans | |
NADH | required | Escherichia coli |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
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 |