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Literature summary for 5.3.2.6 extracted from

  • Harris, T.K.; Czerwinski, R.M.; Johnson, W.H.; Legler, P.M.; Abeygunawardana, C.; Massiah, M.A.; Stivers, J.T.; Whitman, C.P.; Mildvan, A.S.
    Kinetic, stereochemical, and structural effects of mutations of the active site arginine residues in 4-oxalocrotonate tautomerase (1999), Biochemistry, 38, 12343-12357.
    View publication on PubMed
Search for more literature for 5.3.2.6

Cloned(Commentary)

Cloned (Comment) Organism
overexpression of wild-type and mutants in Escherichia coli strain BL21(DE3)pLysS, subcloning in Escherichia coli strain JM109 Pseudomonas putida

Protein Variants

Protein Variants Comment Organism
R11A site-directed mutagenesis, no kinetic effects of the R11A mutation, the stereoselectivity of the R11A-catalyzed protonation at C-5 of the dicarboxylate substrate decreases, while the stereoselectivity of protonation at C-3 of the monocarboxylate substrate increases in comparison with wild-type 4-OT Pseudomonas putida
R11A/R39A site-directed mutagenesis, inactive mutant Pseudomonas putida
R39A site-directed mutagenesis, with 2-hydroxymuconate the R39A mutant shows decreased kcat by 125fold and increased Km by 1.5fold Pseudomonas putida
R39Q site-directed mutagenesis, with 2-hydroxymuconate the R39Q mutant shows decreased kcat by 389fold and increased Km by 2.6fold, only the tight binding sites function catalytically in the R39Q mutant, structural changes in the R39Q mutant were mainly in the beta-hairpin from residues 50 to 57 which covers the active site Pseudomonas putida
R61A site-directed mutagenesis, Arg61 mutation does not affect either substrate binding or catalysis Pseudomonas putida

Inhibitors

Inhibitors Comment Organism Structure
2-Oxo-3-pentynoate mechanism-based inhibitor Pseudomonas putida

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information steady-state kinetics, overview Pseudomonas putida
0.18
-
 2-hydroxy-2,4-hexadienedioate pH 7.3, 23°C, wild-type enzyme Pseudomonas putida
0.29
-
 2-hydroxy-2,4-hexadienedioate pH 7.3, 23°C, mutant R11A/R39A Pseudomonas putida
0.29
-
 2-hydroxy-2,4-hexadienedioate pH 7.3, 23°C, mutant R39A Pseudomonas putida
0.47
-
 2-hydroxy-2,4-hexadienedioate pH 7.3, 23°C, mutant R39Q Pseudomonas putida
1.11
-
 2-hydroxy-2,4-pentadienoate pH 7.3, 23°C, mutant R11A Pseudomonas putida
1.11
-
 2-hydroxy-2,4-pentadienoate pH 7.3, 23°C, wild-type enzyme Pseudomonas putida
1.6
-
 2-hydroxy-2,4-hexadienedioate pH 7.3, 23°C, mutant R11A Pseudomonas putida

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2-oxo-4-hexenedioate Pseudomonas putida isomerization of unconjugated 2-oxo acids such as 2-oxo-4-hexenedioate, to its conjugated isomer via dienol intermediate 2-hydroxy-2,4-hexadienedioate, i.e. 2-hydroxymuconate 2-oxo-3-hexenedioate
-
 ?
2-oxo-4-hexenedioate Pseudomonas putida mt-2 / ATCC 33015 / DSM 3931 / NCIB 12182 / NCIMB 12182 isomerization of unconjugated 2-oxo acids such as 2-oxo-4-hexenedioate, to its conjugated isomer via dienol intermediate 2-hydroxy-2,4-hexadienedioate, i.e. 2-hydroxymuconate 2-oxo-3-hexenedioate
-
 ?

Organism

Organism UniProt Comment Textmining
Pseudomonas putida
-
-
-
Pseudomonas putida mt-2 / ATCC 33015 / DSM 3931 / NCIB 12182 / NCIMB 12182
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutants from Escherichia coli strain BL21(DE3)pLysS to homogeneity Pseudomonas putida

Reaction

Reaction Comment Organism Reaction ID
(2Z,4E)-2-hydroxyhexa-2,4-dienedioate = (3E)-2-oxohex-3-enedioate catalytic reaction mechanism, detailed overview Pseudomonas putida
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-hydroxy-2,4-hexadienedioate
-
 Pseudomonas putida 2-oxo-3-hexenedioate
-
 ?
2-hydroxy-2,4-hexadienedioate
-
 Pseudomonas putida mt-2 / ATCC 33015 / DSM 3931 / NCIB 12182 / NCIMB 12182 2-oxo-3-hexenedioate
-
 ?
2-oxo-4-hexenedioate isomerization of unconjugated 2-oxo acids such as 2-oxo-4-hexenedioate, to its conjugated isomer via dienol intermediate 2-hydroxy-2,4-hexadienedioate, i.e. 2-hydroxymuconate Pseudomonas putida 2-oxo-3-hexenedioate
-
 ?
2-oxo-4-hexenedioate isomerization of unconjugated 2-oxo acids such as 2-oxo-4-hexenedioate, to its conjugated isomer via dienol intermediate 2-hydroxy-2,4-hexadienedioate, i.e. 2-hydroxymuconate Pseudomonas putida mt-2 / ATCC 33015 / DSM 3931 / NCIB 12182 / NCIMB 12182 2-oxo-3-hexenedioate
-
 ?

Subunits

Subunits Comment Organism
More structure-function relationship, NMR analysis, overview Pseudomonas putida

Synonyms

Synonyms Comment Organism
4-oxalocrotonate tautomerase
-
 Pseudomonas putida

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
23
-
 assay at Pseudomonas putida

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.4
-
 2-hydroxy-2,4-hexadienedioate below, pH 7.3, 23°C, mutant R11A/R39A Pseudomonas putida
0.4
-
 2-hydroxy-2,4-pentadienoate pH 7.3, 23°C, wild-type enzyme Pseudomonas putida
1
-
 2-hydroxy-2,4-pentadienoate pH 7.3, 23°C, mutant R11A Pseudomonas putida
2 8 2-hydroxy-2,4-hexadienedioate pH 7.3, 23°C, mutant R39A Pseudomonas putida
9
-
 2-hydroxy-2,4-hexadienedioate pH 7.3, 23°C, mutant R39Q Pseudomonas putida
40
-
 2-hydroxy-2,4-hexadienedioate pH 7.3, 23°C, mutant R11A Pseudomonas putida
3500
-
 2-hydroxy-2,4-hexadienedioate pH 7.3, 23°C, wild-type enzyme Pseudomonas putida

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.3
-
 assay at Pseudomonas putida

General Information

General Information Comment Organism
metabolism the enzyme is part of a degradative pathway that converts various aromatic hydrocarbons to intermediates in the Krebs cycle Pseudomonas putida
additional information structure-function relationship, spectroscopic NMR analysis, detailed overview. Three arginine residues, Arg11, Arg39, and Arg61, are localized in the active site of 4-oxalocrotonate tautomerase. Importance of Arg11 in properly orienting the dicarboxylate substrate by interacting with the charged 6-carboxylate group. Arg39 interacts with the 1-carboxylate and the 2-keto group of the substrate to promote carbonyl polarization and catalysis, while Pro-1 transfers protons from C-3 to C-5. Arg61 does not play a significant role in either substrate binding or catalysis Pseudomonas putida

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.36
-
 2-hydroxy-2,4-pentadienoate pH 7.3, 23°C, wild-type enzyme Pseudomonas putida
0.9
-
 2-hydroxy-2,4-pentadienoate pH 7.3, 23°C, mutant R11A Pseudomonas putida
19
-
 2-hydroxy-2,4-hexadienedioate pH 7.3, 23°C, mutant R39Q Pseudomonas putida
25
-
 2-hydroxy-2,4-hexadienedioate pH 7.3, 23°C, mutant R11A Pseudomonas putida
97
-
 2-hydroxy-2,4-hexadienedioate pH 7.3, 23°C, mutant R39A Pseudomonas putida
12000
-
 2-hydroxy-2,4-hexadienedioate pH 7.3, 23°C, mutant R11A/R39A Pseudomonas putida
19000
-
 2-hydroxy-2,4-hexadienedioate pH 7.3, 23°C, wild-type enzyme Pseudomonas putida