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 (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 | 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 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 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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Chloroflexus aurantiacus | A9W9U6 | two isozymes | - |
Chloroflexus aurantiacus J-10-fl | A9W9U6 | two isozymes | - |
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 | 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 | 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 | Comment | Organism |
---|---|---|
4-OT | - |
Chloroflexus aurantiacus |
4-oxalocrotonate tautomerase | - |
Chloroflexus aurantiacus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
24 | - |
assay at | Chloroflexus aurantiacus |
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 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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.3 | - |
assay at | Chloroflexus aurantiacus |
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 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 |