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Literature summary for 5.3.2.6 extracted from

  • Burks, E.A.; Fleming, C.D.; Mesecar, A.D.; Whitman, C.P.; Pegan, S.D.
    Kinetic and structural characterization of a heterohexamer 4-oxalocrotonate tautomerase from Chloroflexus aurantiacus J-10-fl: implications for functional and structural diversity in the tautomerase superfamily (2010), Biochemistry, 49, 5016-5027.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
cloning of alpha- and beta-subunits of hh4-OT from genomic DNA, recombinant overexpression of wild-type hh4-OT, and of mutants in Escherichia coli Chloroflexus aurantiacus

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant hh4-OT, by sitting drop vapour diffusion method, 0.001 ml of 20 mg/mL protein in 10 mM Na/KPO4 buffer, pH 7.3, is mixed in a 1:1 ratio with precipitant against a 0.1 ml reservoir solution, and by hanging drop vapor diffusion method with a 0.5 ml reservoir and 0.004 ml hanging drops that contain protein and precipitant, 0.25 M (NH4)2SO4 and 4% PEG 4000, in a 1:1 ratio, 3-5 days, X-ray diffraction structure determination and analysis at 2.41 A resolution Chloroflexus aurantiacus

Protein Variants

Protein Variants Comment Organism
alphaR12A site-directed mutagenesis, the mutant shows highly reduced catalytic efficiency compared to the wild-type hh4-OT Chloroflexus aurantiacus
alphaR40A site-directed mutagenesis, the mutant shows reduced catalytic efficiency compared to the wild-type hh4-OT Chloroflexus aurantiacus
betaP1A site-directed mutagenesis, the mutant shows highly reduced catalytic efficiency compared to the wild-type hh4-OT Chloroflexus aurantiacus
betaR11A site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type hh4-OT Chloroflexus aurantiacus
betaR39A site-directed mutagenesis, the mutant shows reduced catalytic efficiency compared to the wild-type hh4-OT Chloroflexus aurantiacus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.017
-
2-hydroxy-2,4-hexadienedioate pH 7.3, 24°C, recombinant hh4-OT mutant betaP1A Chloroflexus aurantiacus
0.063
-
phenylenolpyruvate pH 7.3, 24°C, recombinant hh4-OT mutant betaP1A Chloroflexus aurantiacus
0.069
-
2-hydroxy-2,4-hexadienedioate pH 7.3, 24°C, recombinant hh4-OT mutant betaR11A Chloroflexus aurantiacus
0.07
-
2-hydroxy-2,4-hexadienedioate pH 7.3, 24°C, recombinant wild-type hh4-OT Chloroflexus aurantiacus
0.121
-
phenylenolpyruvate pH 7.3, 24°C, recombinant wild-type hh4-OT Chloroflexus aurantiacus
0.135
-
2-hydroxy-2,4-hexadienedioate pH 7.3, 24°C, recombinant hh4-OT mutant betaR39A Chloroflexus aurantiacus
0.143
-
phenylenolpyruvate pH 7.3, 24°C, recombinant hh4-OT mutant alphaR12A Chloroflexus aurantiacus
0.152
-
phenylenolpyruvate pH 7.3, 24°C, recombinant hh4-OT mutant alphaR40A Chloroflexus aurantiacus
0.159
-
phenylenolpyruvate pH 7.3, 24°C, recombinant hh4-OT mutant betaR39A Chloroflexus aurantiacus
0.198
-
phenylenolpyruvate pH 7.3, 24°C, recombinant hh4-OT mutant betaR11A Chloroflexus aurantiacus
0.345
-
2-hydroxy-2,4-hexadienedioate pH 7.3, 24°C, recombinant hh4-OT mutant alphaR40A Chloroflexus aurantiacus
1.033
-
2-hydroxy-2,4-hexadienedioate pH 7.3, 24°C, recombinant hh4-OT mutant alphaR12A Chloroflexus aurantiacus

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
7732
-
3* 7730, alpha-subunit, + 3 * 7963, beta-subunit, mass spectrometry, 3 * 7732, alpha-subunit + 3 * 8096, beta-subunit, sequence calculation, functional heterohexamer OT, hh4-OT, composed of three alphabeta dimers, the fully functional enzyme requires both subunits Chloroflexus aurantiacus
7963
-
3* 7730, alpha-subunit, + 3 * 7963, beta-subunit, mass spectrometry, 3 * 7732, alpha-subunit + 3 * 8096, beta-subunit, sequence calculation, functional heterohexamer OT, hh4-OT, composed of three alphabeta dimers, the fully functional enzyme requires both subunits Chloroflexus aurantiacus
8096
-
3* 7730, alpha-subunit, + 3 * 7963, beta-subunit, mass spectrometry, 3 * 7732, alpha-subunit + 3 * 8096, beta-subunit, sequence calculation, functional heterohexamer OT, hh4-OT, composed of three alphabeta dimers, the fully functional enzyme requires both subunits Chloroflexus aurantiacus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2-hydroxy-2,4-hexadienedioate Chloroflexus aurantiacus i.e. 2-hydroxymuconate 2-oxo-3-hexenedioate
-
r
2-hydroxy-2,4-hexadienedioate Chloroflexus aurantiacus J-10-fl i.e. 2-hydroxymuconate 2-oxo-3-hexenedioate
-
r

Organism

Organism UniProt Comment Textmining
Chloroflexus aurantiacus A9W9U6 two isozymes
-
Chloroflexus aurantiacus J-10-fl A9W9U6 two isozymes
-

Purification (Commentary)

Purification (Comment) Organism
recombinant hh4-OT wild-type and mutants from Escherichia coli by heat treatment, anion exchange chromatography, and gel-filtration Chloroflexus aurantiacus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-hydroxy-2,4-hexadienedioate i.e. 2-hydroxymuconate Chloroflexus aurantiacus 2-oxo-3-hexenedioate
-
r
2-hydroxy-2,4-hexadienedioate i.e. 2-hydroxymuconate, the enzyme catalyzes the conversion of 2-hydroxy-2,4-hexadienedioate (or 2-hydroxymuconate) to 2-oxo-3-hexenedioate, where Pro-1 functions as a general base and shuttles a proton from the 2-hydroxyl group of the substrate to the C-5 position of the product. hh4-OT requires the amino-terminal proline and two arginines for the conversion of 2-hydroxymuconate to the product Chloroflexus aurantiacus 2-oxo-3-hexenedioate
-
r
2-hydroxy-2,4-hexadienedioate i.e. 2-hydroxymuconate Chloroflexus aurantiacus J-10-fl 2-oxo-3-hexenedioate
-
r
2-hydroxy-2,4-hexadienedioate i.e. 2-hydroxymuconate, the enzyme catalyzes the conversion of 2-hydroxy-2,4-hexadienedioate (or 2-hydroxymuconate) to 2-oxo-3-hexenedioate, where Pro-1 functions as a general base and shuttles a proton from the 2-hydroxyl group of the substrate to the C-5 position of the product. hh4-OT requires the amino-terminal proline and two arginines for the conversion of 2-hydroxymuconate to the product Chloroflexus aurantiacus J-10-fl 2-oxo-3-hexenedioate
-
r
additional information hh4-OT does not exhibit the low-level activity of another tautomerase superfamily member, the heterohexamer trans-3-chloroacrylic acid dehalogenase, no activity with trans-3-chloroacrylic acid Chloroflexus aurantiacus ?
-
?
additional information hh4-OT does not exhibit the low-level activity of another tautomerase superfamily member, the heterohexamer trans-3-chloroacrylic acid dehalogenase, no activity with trans-3-chloroacrylic acid Chloroflexus aurantiacus J-10-fl ?
-
?
phenylenolpyruvate
-
Chloroflexus aurantiacus phenylpyruvate
-
r
phenylenolpyruvate
-
Chloroflexus aurantiacus J-10-fl phenylpyruvate
-
r

Subunits

Subunits Comment Organism
heterohexamer 3* 7730, alpha-subunit, + 3 * 7963, beta-subunit, mass spectrometry, 3 * 7732, alpha-subunit + 3 * 8096, beta-subunit, sequence calculation, functional heterohexamer OT, hh4-OT, composed of three alphabeta dimers, the fully functional enzyme requires both subunits Chloroflexus aurantiacus
More beta-subunit hh4-OT homology modeling derived from MODELER using the Pseudomonas sp. CF600 4OT isozyme, PDB entry 1OTF Chloroflexus aurantiacus

Synonyms

Synonyms Comment Organism
4-OT
-
Chloroflexus aurantiacus
4-oxalocrotonate tautomerase
-
Chloroflexus aurantiacus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
24
-
assay at Chloroflexus aurantiacus

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
additional information
-
higher thermostability of the hh4-OT for stability in the thermophilic environment of the organism, overview Chloroflexus aurantiacus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.2
-
phenylenolpyruvate pH 7.3, 24°C, recombinant hh4-OT mutant betaP1A Chloroflexus aurantiacus
0.6
-
phenylenolpyruvate pH 7.3, 24°C, recombinant hh4-OT mutant alphaR12A Chloroflexus aurantiacus
3 6 2-hydroxy-2,4-hexadienedioate pH 7.3, 24°C, recombinant hh4-OT mutant betaP1A Chloroflexus aurantiacus
11
-
phenylenolpyruvate pH 7.3, 24°C, recombinant hh4-OT mutant betaR39A Chloroflexus aurantiacus
13
-
phenylenolpyruvate pH 7.3, 24°C, recombinant wild-type hh4-OT Chloroflexus aurantiacus
17
-
phenylenolpyruvate pH 7.3, 24°C, recombinant hh4-OT mutant betaR11A Chloroflexus aurantiacus
43
-
2-hydroxy-2,4-hexadienedioate pH 7.3, 24°C, recombinant hh4-OT mutant alphaR12A Chloroflexus aurantiacus
44
-
phenylenolpyruvate pH 7.3, 24°C, recombinant hh4-OT mutant alphaR40A Chloroflexus aurantiacus
65
-
2-hydroxy-2,4-hexadienedioate pH 7.3, 24°C, recombinant hh4-OT mutant alphaR40A Chloroflexus aurantiacus
733
-
2-hydroxy-2,4-hexadienedioate pH 7.3, 24°C, recombinant hh4-OT mutant betaR39A Chloroflexus aurantiacus
3000
-
2-hydroxy-2,4-hexadienedioate pH 7.3, 24°C, recombinant wild-type hh4-OT Chloroflexus aurantiacus
3500
-
2-hydroxy-2,4-hexadienedioate pH 7.3, 24°C, recombinant hh4-OT mutant betaR11A Chloroflexus aurantiacus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.3
-
assay at Chloroflexus aurantiacus

General Information

General Information Comment Organism
evolution the enzyme is a member of the tautomerase superfamily Chloroflexus aurantiacus
additional information the fully functional enzyme requires both subunits, active site structure and function of hh4-OT, overview. Three type II sites are formed at the other end of the heterodimeric unit interface around betaPro-1. As with the type I sites, two of the type II active site sides are composed of loops, but these loops are contributed from different subunits, that is, the alphaA'beta2' loop in the beta-subunit and the beta1alphaA loop in the alpha-subunit. The third side of the type II active site is composed of the intramonomeric alpha-subunit beta2-beta3 loop instead of a 310 helix Chloroflexus aurantiacus
physiological function 4-oxalocrotonate tautomerase isozymes play prominent roles in the bacterial utilization of aromatic hydrocarbons as sole carbon sources Chloroflexus aurantiacus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.2
-
phenylenolpyruvate pH 7.3, 24°C, recombinant hh4-OT mutant betaP1A Chloroflexus aurantiacus
4.2
-
phenylenolpyruvate pH 7.3, 24°C, recombinant hh4-OT mutant alphaR12A Chloroflexus aurantiacus
42
-
2-hydroxy-2,4-hexadienedioate pH 7.3, 24°C, recombinant hh4-OT mutant alphaR12A Chloroflexus aurantiacus
70
-
phenylenolpyruvate pH 7.3, 24°C, recombinant hh4-OT mutant betaR39A Chloroflexus aurantiacus
86
-
phenylenolpyruvate pH 7.3, 24°C, recombinant hh4-OT mutant betaR11A Chloroflexus aurantiacus
110
-
phenylenolpyruvate pH 7.3, 24°C, recombinant wild-type hh4-OT Chloroflexus aurantiacus
190
-
2-hydroxy-2,4-hexadienedioate pH 7.3, 24°C, recombinant hh4-OT mutant alphaR40A Chloroflexus aurantiacus
290
-
phenylenolpyruvate pH 7.3, 24°C, recombinant hh4-OT mutant alphaR40A Chloroflexus aurantiacus
2100
-
2-hydroxy-2,4-hexadienedioate pH 7.3, 24°C, recombinant hh4-OT mutant betaP1A Chloroflexus aurantiacus
5400
-
2-hydroxy-2,4-hexadienedioate pH 7.3, 24°C, recombinant hh4-OT mutant betaR39A Chloroflexus aurantiacus
43000
-
2-hydroxy-2,4-hexadienedioate pH 7.3, 24°C, recombinant wild-type hh4-OT Chloroflexus aurantiacus
51000
-
2-hydroxy-2,4-hexadienedioate pH 7.3, 24°C, recombinant hh4-OT mutant betaR11A Chloroflexus aurantiacus