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show all sequences of 5.3.2.6

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

Burks, E.A.; Fleming, C.D.; Mesecar, A.D.; Whitman, C.P.; Pegan, S.D.; Biochemistry 49, 5016-5027 (2010)

Data extracted from this reference:

Cloned(Commentary)
Commentary
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
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
Engineering
Amino acid exchange
Commentary
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
Commentary
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]
Commentary
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
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
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
Primary Accession No. (UniProt)
Commentary
Textmining
Chloroflexus aurantiacus
A9W9U6
two isozymes
-
Chloroflexus aurantiacus J-10-fl
A9W9U6
two isozymes
-
Purification (Commentary)
Commentary
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
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2-hydroxy-2,4-hexadienedioate
i.e. 2-hydroxymuconate
718874
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
718874
Chloroflexus aurantiacus
2-oxo-3-hexenedioate
-
-
-
r
2-hydroxy-2,4-hexadienedioate
i.e. 2-hydroxymuconate
718874
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
718874
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
718874
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
718874
Chloroflexus aurantiacus J-10-fl
?
-
-
-
-
phenylenolpyruvate
-
718874
Chloroflexus aurantiacus
phenylpyruvate
-
-
-
r
phenylenolpyruvate
-
718874
Chloroflexus aurantiacus J-10-fl
phenylpyruvate
-
-
-
r
Subunits
Subunits
Commentary
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
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
24
-
assay at
Chloroflexus aurantiacus
Temperature Stability [°C]
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
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
Commentary
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
Commentary
Organism
7.3
-
assay at
Chloroflexus aurantiacus
Cloned(Commentary) (protein specific)
Commentary
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) (protein specific)
Crystallization
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
Engineering (protein specific)
Amino acid exchange
Commentary
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] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
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] (protein specific)
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
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) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
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
Purification (Commentary) (protein specific)
Commentary
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) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2-hydroxy-2,4-hexadienedioate
i.e. 2-hydroxymuconate
718874
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
718874
Chloroflexus aurantiacus
2-oxo-3-hexenedioate
-
-
-
r
2-hydroxy-2,4-hexadienedioate
i.e. 2-hydroxymuconate
718874
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
718874
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
718874
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
718874
Chloroflexus aurantiacus J-10-fl
?
-
-
-
-
phenylenolpyruvate
-
718874
Chloroflexus aurantiacus
phenylpyruvate
-
-
-
r
phenylenolpyruvate
-
718874
Chloroflexus aurantiacus J-10-fl
phenylpyruvate
-
-
-
r
Subunits (protein specific)
Subunits
Commentary
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
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
24
-
assay at
Chloroflexus aurantiacus
Temperature Stability [°C] (protein specific)
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
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] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
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 (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.3
-
assay at
Chloroflexus aurantiacus
General Information
General Information
Commentary
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
General Information (protein specific)
General Information
Commentary
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
Commentary
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
KCat/KM [mM/s] (protein specific)
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
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
Other publictions for EC 5.3.2.6
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
747133
Stack
Inactivation of 4-oxalocroton ...
Leptothrix cholodnii, Leptothrix cholodnii SP-6, Pseudomonas putida
Biochemistry
57
1012-1021
2018
-
-
-
-
-
-
18
-
-
-
-
3
-
4
-
-
2
-
-
-
-
-
3
1
-
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-
-
-
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-
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-
-
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-
18
-
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3
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2
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-
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-
3
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
749336
Diemen
The S. aureus 4-oxalocrotonat ...
Staphylococcus aureus, Staphylococcus aureus MRSA252
Sci. Rep.
7
1745
2017
-
-
1
-
3
-
-
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-
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2
-
5
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2
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1
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3
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2
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-
2
-
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-
-
-
-
-
-
-
-
-
-
-
-
-
748503
Lazic
-
Importance of N-terminal prol ...
Pseudomonas putida
J. Serb. Chem. Soc.
81
871-881
2016
-
-
1
-
-
-
-
-
-
-
-
1
-
1
-
-
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-
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4
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1
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1
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-
-
-
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
748742
van der Meer
Using mutability landscapes o ...
Pseudomonas putida
Nat. Commun.
7
10911
2016
-
-
1
1
6
-
-
-
-
-
-
2
-
1
-
-
1
-
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-
-
-
10
-
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-
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-
1
-
1
6
-
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-
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-
-
-
2
-
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-
1
-
-
-
-
10
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
747290
Djokic
Immobilization of Escherichia ...
Pseudomonas putida
Bioprocess Biosyst. Eng.
38
2389-2395
2015
-
-
1
-
-
-
-
-
-
-
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1
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3
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-
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2
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1
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1
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-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
747537
Poddar
Evidence for the formation of ...
Pseudomonas putida
ChemBioChem
16
738-741
2015
-
-
-
-
-
-
1
-
-
-
-
1
-
1
-
-
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-
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2
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1
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1
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-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
746910
Huddleston
Identification and characteri ...
Pseudomonas putida
Arch. Biochem. Biophys.
564
189-196
2014
-
-
-
-
-
-
1
-
-
-
-
1
-
1
-
-
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2
-
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-
-
1
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1
-
-
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-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
747782
Baas
Demethionylation of Pro-1 var ...
Pseudomonas putida
FEBS Open Bio
4
651-658
2014
-
-
1
-
-
-
-
1
-
-
-
1
-
2
-
-
-
-
-
-
-
-
1
1
-
-
-
1
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
1
-
-
-
1
-
-
-
-
-
-
-
-
1
1
-
-
-
1
-
-
-
-
-
-
-
-
1
1
749284
Radivojevic
-
Synthesis of gamma-nitroaldeh ...
Pseudomonas putida
RSC Adv.
4
60502-60510
2014
-
-
1
-
-
-
-
-
-
-
-
1
-
1
-
-
-
-
-
-
-
-
7
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
7
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
726816
Terrell
Structural and kinetic charact ...
Methylibium petroleiphilum
Arch. Biochem. Biophys.
537
113-124
2013
-
-
1
1
-
-
-
4
-
-
-
1
-
2
-
-
1
-
-
-
-
-
3
-
-
-
-
4
-
-
-
-
-
-
-
-
-
1
-
1
-
-
-
-
-
4
-
-
-
1
-
-
-
1
-
-
-
-
3
-
-
-
-
4
-
-
-
-
-
-
-
-
4
4
727187
Narancic
Highly efficient Michael-type ...
Pseudomonas putida
Biores. Technol.
142
462-468
2013
-
-
1
-
-
-
-
-
-
-
1
1
-
2
-
-
-
-
-
1
-
-
5
1
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
-
-
1
-
-
5
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
727314
Miao
Promiscuous catalysis of asymm ...
Bacillus subtilis
ChemBioChem
14
191-194
2013
-
-
-
-
-
-
-
-
-
-
-
1
-
1
-
-
-
-
-
-
-
-
8
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
8
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
727327
Geertsema
Biocatalytic Michael-type addi ...
Escherichia coli
Chemistry
19
14407-14410
2013
-
-
-
-
-
-
-
-
-
-
-
1
-
2
-
-
-
-
-
-
-
-
8
-
-
-
-
-
-
-
-
-
-
-
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-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
8
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
720309
Wu
Catalytic mechanism of 4-oxalo ...
Pseudomonas putida
J. Phys. Chem. B
116
6889-6897
2012
-
-
-
-
-
-
-
-
-
-
-
1
-
1
-
-
-
1
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
3
3
-
-
-
726964
Burks
Kinetic, crystallographic, and ...
Pseudomonas putida
Biochemistry
50
7600-7611
2011
-
-
1
1
4
-
1
6
-
-
-
1
-
1
-
-
1
-
-
-
-
-
8
-
-
-
-
6
-
-
-
-
-
-
-
-
-
1
-
1
4
-
-
1
-
6
-
-
-
1
-
-
-
1
-
-
-
-
8
-
-
-
-
6
-
-
-
-
-
-
-
-
6
6
718874
Burks
Kinetic and structural charact ...
Chloroflexus aurantiacus, Chloroflexus aurantiacus J-10-fl
Biochemistry
49
5016-5027
2010
-
-
1
1
5
-
-
12
-
-
3
2
-
5
-
-
1
-
-
-
-
-
8
2
1
-
1
12
1
-
-
-
-
-
-
-
-
1
-
1
5
-
-
-
-
12
-
-
3
2
-
-
-
1
-
-
-
-
8
2
1
-
1
12
1
-
-
-
-
3
3
-
12
12
721792
Almrud
Kinetic and structural charact ...
Archaeoglobus fulgidus, Helicobacter pylori
Bioorg. Chem.
38
252-259
2010
-
-
2
2
-
-
-
8
-
-
-
-
-
4
-
-
2
-
-
-
-
-
8
2
2
-
-
7
2
-
-
-
-
-
-
-
-
2
-
2
-
-
-
-
-
8
-
-
-
-
-
-
-
2
-
-
-
-
8
2
2
-
-
7
2
-
-
-
-
-
-
-
8
8
704310
Kasai
Uncovering the protocatechuate ...
Paenibacillus sp., Paenibacillus sp. JJ-1b
J. Bacteriol.
191
6758-6768
2009
-
-
1
-
-
-
-
-
-
-
-
2
-
2
-
-
-
-
-
1
-
-
2
-
1
-
-
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
1
-
-
2
-
1
-
-
-
1
-
-
-
-
1
1
-
-
-
718847
Wang
Kinetic and stereochemical ana ...
Bacillus subtilis, Pseudomonas putida, Pseudomonas putida mt-2 / ATCC 33015 / DSM 3931 / NCIB 12182 / NCIMB 12182
Biochemistry
46
11919-11929
2007
-
-
1
-
2
-
-
-
-
-
-
4
-
21
-
-
1
-
-
-
-
-
10
2
2
-
-
-
2
-
-
-
-
-
-
-
-
1
-
-
2
-
-
-
-
-
-
-
-
4
-
-
-
1
-
-
-
-
10
2
2
-
-
-
2
-
-
-
-
1
1
-
-
-
718844
Poelarends
Evolution of enzymatic activit ...
Pseudomonas putida, Pseudomonas putida mt-2 / ATCC 33015 / DSM 3931 / NCIB 12182 / NCIMB 12182
Biochemistry
45
7700-7708
2006
-
-
1
-
3
-
-
5
-
-
-
4
-
21
-
-
1
-
-
-
-
-
4
1
1
-
-
4
1
-
-
-
-
-
-
-
-
1
-
-
3
-
-
-
-
5
-
-
-
4
-
-
-
1
-
-
-
-
4
1
1
-
-
4
1
-
-
-
-
1
1
-
4
4
718840
Azurmendi
Half-of-the-sites binding of r ...
Pseudomonas putida
Biochemistry
44
7725-7737
2005
-
-
-
-
1
-
4
1
-
-
-
-
-
1
-
-
-
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
1
-
-
4
1
1
-
-
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
1
1
-
-
-
718830
Harris
Kinetic, stereochemical, and s ...
Pseudomonas putida, Pseudomonas putida mt-2 / ATCC 33015 / DSM 3931 / NCIB 12182 / NCIMB 12182
Biochemistry
38
12343-12357
1999
-
-
1
-
5
-
1
6
-
-
-
2
-
19
-
-
1
1
-
-
-
-
6
1
1
-
-
7
1
-
-
-
-
-
-
-
-
1
-
-
5
-
-
1
-
6
-
-
-
2
-
-
-
1
-
-
-
-
6
1
1
-
-
7
1
-
-
-
-
2
2
-
7
7
718825
Subramanya
Enzymatic ketonization of 2-hy ...
Pseudomonas sp., Pseudomonas sp. CF 600
Biochemistry
35
792-802
1996
-
-
-
1
-
-
-
-
-
-
-
-
-
2
-
-
-
1
-
-
-
-
2
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
718826
Stivers
Catalytic role of the amino-te ...
Pseudomonas sp., Pseudomonas sp. CF 600
Biochemistry
35
803-813
1996
-
-
-
-
-
-
1
-
-
-
-
-
-
9
-
-
-
-
-
-
-
-
2
1
1
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
1
1
-
-
-
1
-
-
-
-
1
1
-
-
-
719636
Fitzgerald
-
Total chemical synthesis and c ...
Pseudomonas putida, Pseudomonas putida mt-2 / ATCC 33015 / DSM 3931 / NCIB 12182 / NCIMB 12182
J. Am. Chem. Soc.
117
11075-11080
1995
-
-
-
-
-
-
-
2
-
-
-
2
-
19
-
-
-
-
-
-
-
-
4
1
1
-
-
2
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
2
-
-
-
-
-
-
-
-
4
1
1
-
-
2
1
-
-
-
-
1
1
-
2
2
719634
Whitman
-
Stereospecific ketonization of ...
Escherichia coli, Escherichia coli C, Pseudomonas putida, Pseudomonas putida mt-2 / ATCC 33015 / DSM 3931 / NCIB 12182 / NCIMB 12182
J. Am. Chem. Soc.
114
10104-10110
1992
-
-
-
-
-
-
-
-
-
-
1
-
-
27
-
-
1
-
-
-
-
-
4
2
2
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
1
-
-
-
-
4
2
2
-
-
-
2
-
-
-
-
4
4
-
-
-
719633
Whitman
-
Chemical and enzymatic ketoniz ...
Pseudomonas putida, Pseudomonas putida mt-2 / ATCC 33015 / DSM 3931 / NCIB 12182 / NCIMB 12182
J. Am. Chem. Soc.
113
3154-3162
1991
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1
-
-
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3
-
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2
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19
-
-
1
-
-
-
-
-
10
-
1
-
-
2
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
3
-
-
-
2
-
-
-
1
-
-
-
-
10
-
1
-
-
2
1
-
-
-
-
2
2
-
2
2