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Literature summary for 5.3.2.6 extracted from

  • Poelarends, G.J.; Almrud, J.J.; Serrano, H.; Darty, J.E.; Johnson, W.H.; Hackert, M.L.; Whitman, C.P.
    Evolution of enzymatic activity in the tautomerase superfamily: mechanistic and structural consequences of the L8R mutation in 4-oxalocrotonate tautomerase (2006), Biochemistry, 45, 7700-7708.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 5.3.2.6

Cloned(Commentary)

Cloned (Comment) Organism
overexpression of wild-type and mutant enzymes in Escherichia coli strain BL21-Gold(DE3) Pseudomonas putida

Protein Variants

Protein Variants Comment Organism
alphaI52E site-directed mutagenesis, active site mutant,the mutant shows improved trans-3-chloroacrylic acid dehalogenase activity with a 36fold increase in kcat/Km, largely due to a 110fold decrease in Km, and diminished 4-oxalocrotonate tautomerase activity. The negatively charged group may hinder the formation of the enolate intermediate and may contribute to a decrease in kcat Pseudomonas putida
alphaL8R site-directed mutagenesis, active site mutant, the mutant shows improved trans-3-chloroacrylic acid dehalogenase activity with a 50fold increase in kcat/Km, primarily from an 8.8fold increase in kcat, and diminished 4-oxalocrotonate tautomerase activity with a 5fold decrease in kcat/Km. The increased CaaD activity of L8R-4-OT does not substantially diminish the original 4-OT activity Pseudomonas putida
alphaL8R/I52E site-directed mutagenesis, active site mutant, the mutant shows improved trans-3-chloroacrylic acid dehalogenase activity with a 32fold increase in kcat/Km, largely due to a 23fold decrease in Km, and diminished 4-oxalocrotonate tautomerase activity with a 1700fold decrease in kcat/Km Pseudomonas putida

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information steady-state kinetics of wild-type and mutant 4-OTs for trans-3-chloroacrylic acid dehalogenase activity, pH 8.2, 23°C, overview Pseudomonas putida
0.017
-
 2-hydroxymuconate mutant 4-OT L8R, pH 7.3, 23°C Pseudomonas putida
0.027
-
 2-hydroxymuconate mutant 4-OT L8R/I52E, pH 7.3, 23°C Pseudomonas putida
0.062
-
 2-hydroxymuconate mutant 4-OT I52E, pH 7.3, 23°C Pseudomonas putida
0.18
-
 2-hydroxymuconate wild-type 4-OT, pH 7.3, 23°C Pseudomonas putida

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2-hydroxymuconate Pseudomonas putida ketonization 2-oxo-3-hexenedioate
-
 ?
2-hydroxymuconate Pseudomonas putida mt-2 / ATCC 33015 / DSM 3931 / NCIB 12182 / NCIMB 12182 ketonization 2-oxo-3-hexenedioate
-
 ?
2-oxo-4-hexenedioate Pseudomonas putida via dienol intermediate 2-hydroxymuconate 2-oxo-3-hexenedioate
-
 ?
2-oxo-4-hexenedioate Pseudomonas putida mt-2 / ATCC 33015 / DSM 3931 / NCIB 12182 / NCIMB 12182 via dienol intermediate 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 mutant enzymes from Escherichia coli strain BL21-Gold(DE3) to over 95% purity Pseudomonas putida

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-hydroxymuconate ketonization Pseudomonas putida 2-oxo-3-hexenedioate
-
 ?
2-hydroxymuconate ketonization Pseudomonas putida mt-2 / ATCC 33015 / DSM 3931 / NCIB 12182 / NCIMB 12182 2-oxo-3-hexenedioate
-
 ?
2-oxo-4-hexenedioate via dienol intermediate 2-hydroxymuconate Pseudomonas putida 2-oxo-3-hexenedioate
-
 ?
2-oxo-4-hexenedioate via dienol intermediate 2-hydroxymuconate Pseudomonas putida mt-2 / ATCC 33015 / DSM 3931 / NCIB 12182 / NCIMB 12182 2-oxo-3-hexenedioate
-
 ?

Subunits

Subunits Comment Organism
More structure determinatin and analysis by NMR of wild-type and mutant L8R 4-OTs, overview Pseudomonas putida

Synonyms

Synonyms Comment Organism
4-OT
-
 Pseudomonas putida
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
0.3
-
 2-hydroxymuconate mutant 4-OT L8R/I52E, pH 7.3, 23°C Pseudomonas putida
32
-
 2-hydroxymuconate mutant 4-OT I52E, pH 7.3, 23°C Pseudomonas putida
61
-
 2-hydroxymuconate mutant 4-OT L8R, pH 7.3, 23°C Pseudomonas putida
3500
-
 2-hydroxymuconate wild-type 4-OT, pH 7.3, 23°C Pseudomonas putida

pH Optimum

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

General Information

General Information Comment Organism
malfunction introduction of polar residues into the active site produces significant decreases in kcat and Km Pseudomonas putida

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
11
-
 2-hydroxymuconate mutant 4-OT L8R/I52E, pH 7.3, 23°C Pseudomonas putida
520
-
 2-hydroxymuconate mutant 4-OT I52E, pH 7.3, 23°C Pseudomonas putida
3600
-
 2-hydroxymuconate mutant 4-OT L8R, pH 7.3, 23°C Pseudomonas putida
19000
-
 2-hydroxymuconate wild-type 4-OT, pH 7.3, 23°C Pseudomonas putida