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Literature summary for 4.1.1.23 extracted from

  • Desai, B.J.; Wood, B.M.; Fedorov, A.A.; Fedorov, E.V.; Goryanova, B.; Amyes, T.L.; Richard, J.P.; Almo, S.C.; Gerlt, J.A.
    Conformational changes in orotidine 5-monophosphate decarboxylase: a structure-based explanation for how the 5-phosphate group activates the enzyme (2012), Biochemistry, 51, 8665-8678.
    View publication on PubMedView publication on EuropePMC

Activating Compound

Activating Compound Comment Organism Structure
orotidine 5'-phosphate the 5'-phosphate group of the substrate activates the enzyme 240000000fold. The binding of orotidine 5'-monophosphate is accompanied by a conformational change of the enzyme from an open, inactive conformation to a closed, active conformation. As the substrate traverses the reaction coordinate to form the stabilized vinyl carbanion/carbene intermediate, interactions that destabilize the carboxylate group of the substrate and stabilize the intermediate are enforced. The activation is equivalently described by an intrinsic binding energy of 11.4 kcal/mol. The residues that directly contact the 5'-phosphate group, participate in a hydrophobic cluster near the base of the active site loop that sequesters the bound substrate from the solvent, or that form hydrogen bonding interactions across the interface between the mobile and fixed half-barrel domains of the (beta/alpha)8-barrel structure. The data support a model in which the intrinsic binding energy provided by the 5'-phosphate group is used to allow interactions both near the N-terminus of the active site loop and across the domain interface that stabilize the complexes of the enzyme in the active closed conformation with the substrate or with the substrate intermediate with the destabilized carboxylate group relative to the complex of the enzyme in the open inactive conformation with the substrate Methanothermobacter thermautotrophicus

Crystallization (Commentary)

Crystallization (Comment) Organism
eight different crystal forms are grown by the sitting drop method at room temperature for single mutant enzymes liganded with 1-(5-phospho-beta-ribofuranosyl)barbituric acid: Q185A/[1-(5-phospho-beta-D-ribofuranosyl)barbituric acid], R203A/[1-(5-phospho-beta-ribofuranosyl)barbituric acid], T159V/[1-(5-phospho-beta-D-ribofuranosyl)barbituric acid], T159A/[1-(5-phospho-beta-D-ribofuranosyl)barbituric acid], T159S/[1-(5-phospho-beta-D-ribofuranosyl)barbituric acid], R160A/[1-(5-phospho-beta-D-ribofuranosyl)barbituric acid], Y206F/[1-(5-phospho-beta-ribofuranosyl)barbituric acid], K82A/[1-(5-phospho-beta-D-ribofuranosyl)barbituric acid] Methanothermobacter thermautotrophicus
mutants Q185A, R203A, T159V, T159A, T159S, R160A, Y206F, K82A, and double- and triple mutants, to 1.3-1.6 A resolution Methanothermobacter thermautotrophicus

Protein Variants

Protein Variants Comment Organism
K82A crystallization data Methanothermobacter thermautotrophicus
Q185A 220fold decrease in catalytic efficiency Methanothermobacter thermautotrophicus
Q185A/R203A loss of activity Methanothermobacter thermautotrophicus
R160A 43fold decrease in catalytic efficiency Methanothermobacter thermautotrophicus
R160A/R203A loss of activity Methanothermobacter thermautotrophicus
R160A/V182A 1700fold decrease in catalytic efficiency Methanothermobacter thermautotrophicus
R160A/Y206F crystallization data Methanothermobacter thermautotrophicus
R160A/Y206F 340fold decrease in catalytic efficiency Methanothermobacter thermautotrophicus
R203A 1900fold decrease in catalytic efficiency Methanothermobacter thermautotrophicus
R203A/R160A crystallization data Methanothermobacter thermautotrophicus
R203A/T159V crystallization data Methanothermobacter thermautotrophicus
R203A/V182A crystallization data Methanothermobacter thermautotrophicus
T159A crystallization data Methanothermobacter thermautotrophicus
T159S crystallization data Methanothermobacter thermautotrophicus
T159V 41fold decrease in catalytic efficiency Methanothermobacter thermautotrophicus
T159V/R203A loss of activity Methanothermobacter thermautotrophicus
T159V/V182A 3600fold decrease in catalytic efficiency Methanothermobacter thermautotrophicus
T159V/V182A/Y206F 20000fold decrease in catalytic efficiency Methanothermobacter thermautotrophicus
T159V/Y206F 780fold decrease in catalytic efficiency Methanothermobacter thermautotrophicus
V182A 22fold decrease in catalytic efficiency Methanothermobacter thermautotrophicus
V182A/R203A loss of activity Methanothermobacter thermautotrophicus
V182A/Y206F 190fold decrease in catalytic efficiency Methanothermobacter thermautotrophicus
Y206F 4fold decrease in catalytic efficiency Methanothermobacter thermautotrophicus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0018
-
orotidine 5'-phosphate wild-type, pH 7.1, 25°C Methanothermobacter thermautotrophicus
0.0055
-
orotidine 5'-phosphate mutant Y206F, pH 7.1, 25°C Methanothermobacter thermautotrophicus
0.024
-
orotidine 5'-phosphate mutant V182A, pH 7.1, 25°C Methanothermobacter thermautotrophicus
0.03
-
orotidine 5'-phosphate mutant R160A, pH 7.1, 25°C Methanothermobacter thermautotrophicus
0.033
-
orotidine 5'-phosphate mutant T159V, pH 7.1, 25°C Methanothermobacter thermautotrophicus
0.1
-
orotidine 5'-phosphate mutant V182A/Y206F, pH 7.1, 25°C Methanothermobacter thermautotrophicus
0.1 1 orotidine 5'-phosphate mutant Q185A, pH 7.1, 25°C Methanothermobacter thermautotrophicus
0.13
-
orotidine 5'-phosphate mutant R160A/Y206F, pH 7.1, 25°C Methanothermobacter thermautotrophicus
0.3
-
orotidine 5'-phosphate mutant T159V/Y206F, pH 7.1, 25°C Methanothermobacter thermautotrophicus
0.6
-
orotidine 5'-phosphate mutant R160A/V182A, pH 7.1, 25°C Methanothermobacter thermautotrophicus
0.7
-
orotidine 5'-phosphate mutant T159V/V182A, pH 7.1, 25°C Methanothermobacter thermautotrophicus
0.98
-
orotidine 5'-phosphate mutant R203A, pH 7.1, 25°C Methanothermobacter thermautotrophicus
1.9
-
orotidine 5'-phosphate mutant T159V/V182A/Y206F, pH 7.1, 25°C Methanothermobacter thermautotrophicus

Organism

Organism UniProt Comment Textmining
Methanothermobacter thermautotrophicus O26232
-
-
Methanothermobacter thermautotrophicus DSM 1053 O26232
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
Orotidine 5'-phosphate the remote 5'-phosphate group of the substrate activates the enzyme 240 millionfold, the activation corresponds to an intrinsic binding energy of 11.4 kcal/mol. This intrinsic binding energy is used to allow interactions both near the N-terminus of the active site loop and across the domain interface that stabilize both the Ec-S and Ec-S* complexes relative to the Eo-S complex Methanothermobacter thermautotrophicus UMP + CO2
-
?
Orotidine 5'-phosphate the remote 5'-phosphate group of the substrate activates the enzyme 240 millionfold, the activation corresponds to an intrinsic binding energy of 11.4 kcal/mol. This intrinsic binding energy is used to allow interactions both near the N-terminus of the active site loop and across the domain interface that stabilize both the Ec-S and Ec-S* complexes relative to the Eo-S complex Methanothermobacter thermautotrophicus DSM 1053 UMP + CO2
-
?

Synonyms

Synonyms Comment Organism
OMPDC
-
Methanothermobacter thermautotrophicus
Orotidine 5'-monophosphate decarboxylase
-
Methanothermobacter thermautotrophicus
PyrF
-
Methanothermobacter thermautotrophicus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Methanothermobacter thermautotrophicus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.24
-
orotidine 5'-phosphate mutant T159V/V182A/Y206F, pH 7.1, 25°C Methanothermobacter thermautotrophicus
0.56
-
orotidine 5'-phosphate mutant T159V/V182A, pH 7.1, 25°C Methanothermobacter thermautotrophicus
1
-
orotidine 5'-phosphate mutant R160A/V182A, pH 7.1, 25°C Methanothermobacter thermautotrophicus
1.1
-
orotidine 5'-phosphate mutant R160A/Y206F, pH 7.1, 25°C Methanothermobacter thermautotrophicus
1.1
-
orotidine 5'-phosphate mutant T159V/Y206F, pH 7.1, 25°C Methanothermobacter thermautotrophicus
1.4
-
orotidine 5'-phosphate mutant Q185A, pH 7.1, 25°C Methanothermobacter thermautotrophicus
1.5
-
orotidine 5'-phosphate mutant R203A, pH 7.1, 25°C Methanothermobacter thermautotrophicus
1.5
-
orotidine 5'-phosphate mutant V182A/Y206F, pH 7.1, 25°C Methanothermobacter thermautotrophicus
2
-
orotidine 5'-phosphate mutant R160A, pH 7.1, 25°C Methanothermobacter thermautotrophicus
2.3
-
orotidine 5'-phosphate mutant T159V, pH 7.1, 25°C Methanothermobacter thermautotrophicus
3
-
orotidine 5'-phosphate mutant V182A, pH 7.1, 25°C Methanothermobacter thermautotrophicus
4.1
-
orotidine 5'-phosphate mutant Y206F, pH 7.1, 25°C Methanothermobacter thermautotrophicus
5.3
-
orotidine 5'-phosphate wild-type, pH 7.1, 25°C Methanothermobacter thermautotrophicus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.1
-
assay at Methanothermobacter thermautotrophicus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.13
-
orotidine 5'-phosphate mutant T159V/V182A/Y206F, pH 7.1, 25°C Methanothermobacter thermautotrophicus
0.8
-
orotidine 5'-phosphate mutant T159V/V182A, pH 7.1, 25°C Methanothermobacter thermautotrophicus
1.5
-
orotidine 5'-phosphate mutant R203A, pH 7.1, 25°C Methanothermobacter thermautotrophicus
1.7
-
orotidine 5'-phosphate mutant R160A/V182A, pH 7.1, 25°C Methanothermobacter thermautotrophicus
3.7
-
orotidine 5'-phosphate mutant T159V/Y206F, pH 7.1, 25°C Methanothermobacter thermautotrophicus
8.5
-
orotidine 5'-phosphate mutant R160A/Y206F, pH 7.1, 25°C Methanothermobacter thermautotrophicus
13
-
orotidine 5'-phosphate mutant Q185A, pH 7.1, 25°C Methanothermobacter thermautotrophicus
15
-
orotidine 5'-phosphate mutant V182A/Y206F, pH 7.1, 25°C Methanothermobacter thermautotrophicus
67
-
orotidine 5'-phosphate mutant R160A, pH 7.1, 25°C Methanothermobacter thermautotrophicus
70
-
orotidine 5'-phosphate mutant T159V, pH 7.1, 25°C Methanothermobacter thermautotrophicus
130
-
orotidine 5'-phosphate mutant V182A, pH 7.1, 25°C Methanothermobacter thermautotrophicus
750
-
orotidine 5'-phosphate mutant Y206F, pH 7.1, 25°C Methanothermobacter thermautotrophicus
2900
-
orotidine 5'-phosphate wild-type, pH 7.1, 25°C Methanothermobacter thermautotrophicus