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Literature summary extracted from

  • Baas, B.J.; Zandvoort, E.; Wasiel, A.A.; Quax, W.J.; Poelarends, G.J.
    Characterization of a newly identified mycobacterial tautomerase with promiscuous dehalogenase and hydratase activities reveals a functional link to a recently diverged cis-3-chloroacrylic acid dehalogenase (2011), Biochemistry, 50, 2889-2899.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
5.3.2.1 expressed in Escherichia coli BL21(DE3) cells Mycolicibacterium smegmatis

Protein Variants

EC Number Protein Variants Comment Organism
5.3.2.1 P1A for phenylenolpyruvate, the P1A mutant shows a 41fold decrease in kcat and a 14fold decrease in Km, resulting in an about 3fold decrease in kcat/Km. For (4-hydroxyphenyl)enolpyruvate, the P1A mutant shows a 16fold decrease in kcat, whereas the Km is not significantly affected, resulting in a 16fold decrease in kcat/Km. The P1A mutant has no detectable dehalogenase activity toward cis-3-chloroacrylate and trans-3-chloroacrylate, and no detectable activity toward 2-oxo-3-pentynoate (at pH 7.3) Mycolicibacterium smegmatis

Inhibitors

EC Number Inhibitors Comment Organism Structure
5.3.2.1 2-Oxo-3-pentynoate at pH 8.5, CCH2 is inactivated by 2-oxo-3-pentynoate due to the covalent modification of Pro-1 Mycolicibacterium smegmatis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
5.3.2.1 0.11
-
 phenylenolpyruvate wild type enzyme cis-CAAD, in 10 mM Na2HPO4 buffer, pH 7.3, at 22°C Mycolicibacterium smegmatis
5.3.2.1 0.152
-
 cis-3-chloroacrylate wild type enzyme cis-CAAD, in 10 mM Na2HPO4 buffer, pH 7.3, at 22°C Mycolicibacterium smegmatis
5.3.2.1 0.25
-
 phenylenolpyruvate mutant enzyme CCH2-P1A, in 10 mM Na2HPO4 buffer, pH 7.3, at 22°C Mycolicibacterium smegmatis
5.3.2.1 0.79
-
 (4-hydroxyphenyl)enolpyruvate wild type enzyme CCH2-P1A, in 10 mM Na2HPO4 buffer, pH 7.3, at 22°C Mycolicibacterium smegmatis
5.3.2.1 0.89
-
 (4-hydroxyphenyl)enolpyruvate wild type enzyme CCH2, in 10 mM Na2HPO4 buffer, pH 7.3, at 22°C Mycolicibacterium smegmatis
5.3.2.1 3.5
-
 phenylenolpyruvate wild type enzyme CCH2, in 10 mM Na2HPO4 buffer, pH 7.3, at 22°C Mycolicibacterium smegmatis
5.3.2.1 96
-
 trans-3-chloroacrylate wild type enzyme CCH2, in in 50 mM Tris-SO4 buffer (pH 8.0), at 22°C Mycolicibacterium smegmatis

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
5.3.2.1 16093
-
 3 * 16093, electrospray ionization mass spectrometry Mycolicibacterium smegmatis
5.3.2.1 16223
-
 3 * 16223, calculated from amino acid sequence Mycolicibacterium smegmatis
5.3.2.1 48000
-
 gel filtration Mycolicibacterium smegmatis

Organism

EC Number Organism UniProt Comment Textmining
5.3.2.1 Mycolicibacterium smegmatis
-
-
-
5.3.2.1 Mycolicibacterium smegmatis mc(2)155
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
5.3.2.1 Ni-Sepharose column chromatography Mycolicibacterium smegmatis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
5.3.2.1 (4-hydroxyphenyl)enolpyruvate
-
 Mycolicibacterium smegmatis (4-hydroxyphenyl)pyruvate
-
 ?
5.3.2.1 (4-hydroxyphenyl)enolpyruvate
-
 Mycolicibacterium smegmatis mc(2)155 (4-hydroxyphenyl)pyruvate
-
 ?
5.3.2.1 2-oxo-3-pentynoate at pH 6.5, CCH2 exhibits hydratase activity and converts 2-oxo-3-pentynoate to acetopyruvate Mycolicibacterium smegmatis acetopyruvate + ?
-
 ?
5.3.2.1 2-oxo-3-pentynoate at pH 6.5, CCH2 exhibits hydratase activity and converts 2-oxo-3-pentynoate to acetopyruvate Mycolicibacterium smegmatis mc(2)155 acetopyruvate + ?
-
 ?
5.3.2.1 cis-3-chloroacrylate the reaction is performed by cis-CAAD Mycolicibacterium smegmatis malonate semialdehyde + HCl
-
 ?
5.3.2.1 cis-3-chloroacrylate the reaction is performed by cis-CAAD Mycolicibacterium smegmatis mc(2)155 malonate semialdehyde + HCl
-
 ?
5.3.2.1 additional information CCH2 functions as an efficient PPT and exhibits low-level promiscuous dehalogenase activity, processing both cis- and trans-3-chloroacrylic acid Mycolicibacterium smegmatis ?
-
 ?
5.3.2.1 additional information CCH2 functions as an efficient PPT and exhibits low-level promiscuous dehalogenase activity, processing both cis- and trans-3-chloroacrylic acid Mycolicibacterium smegmatis mc(2)155 ?
-
 ?
5.3.2.1 phenylenolpyruvate
-
 Mycolicibacterium smegmatis phenylpyruvate
-
 ?
5.3.2.1 phenylenolpyruvate
-
 Mycolicibacterium smegmatis mc(2)155 phenylpyruvate
-
 ?
5.3.2.1 trans-3-chloroacrylate the reaction is performed by CAAD Mycolicibacterium smegmatis malonate semialdehyde + HCl
-
 ?

Subunits

EC Number Subunits Comment Organism
5.3.2.1 homotrimer 3 * 16093, electrospray ionization mass spectrometry Mycolicibacterium smegmatis
5.3.2.1 homotrimer 3 * 16223, calculated from amino acid sequence Mycolicibacterium smegmatis

Synonyms

EC Number Synonyms Comment Organism
5.3.2.1 CaaD
-
 Mycolicibacterium smegmatis
5.3.2.1 CCH2
-
 Mycolicibacterium smegmatis
5.3.2.1 cis-3-chloroacrylic acid dehalogenase
-
 Mycolicibacterium smegmatis
5.3.2.1 cis-CaaD
-
 Mycolicibacterium smegmatis
5.3.2.1 phenyl(enol)pyruvate tautomerase
-
 Mycolicibacterium smegmatis
5.3.2.1 PPT
-
 Mycolicibacterium smegmatis
5.3.2.1 trans-3-chloroacrylic acid dehalogenase
-
 Mycolicibacterium smegmatis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
5.3.2.1 0.0004
-
 trans-3-chloroacrylate wild type enzyme CCH2, in in 50 mM Tris-SO4 buffer (pH 8.0), at 22°C Mycolicibacterium smegmatis
5.3.2.1 0.2
-
 (4-hydroxyphenyl)enolpyruvate wild type enzyme CCH2-P1A, in 10 mM Na2HPO4 buffer, pH 7.3, at 22°C Mycolicibacterium smegmatis
5.3.2.1 0.2
-
 phenylenolpyruvate wild type enzyme cis-CAAD, in 10 mM Na2HPO4 buffer, pH 7.3, at 22°C Mycolicibacterium smegmatis
5.3.2.1 0.8
-
 phenylenolpyruvate mutant enzyme CCH2-P1A, in 10 mM Na2HPO4 buffer, pH 7.3, at 22°C Mycolicibacterium smegmatis
5.3.2.1 3.2
-
 (4-hydroxyphenyl)enolpyruvate wild type enzyme CCH2, in 10 mM Na2HPO4 buffer, pH 7.3, at 22°C Mycolicibacterium smegmatis
5.3.2.1 4.6
-
 cis-3-chloroacrylate wild type enzyme cis-CAAD, in 10 mM Na2HPO4 buffer, pH 7.3, at 22°C Mycolicibacterium smegmatis
5.3.2.1 34
-
 phenylenolpyruvate wild type enzyme CCH2, in 10 mM Na2HPO4 buffer, pH 7.3, at 22°C Mycolicibacterium smegmatis

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
5.3.2.1 1.6
-
 cis-3-chloroacrylate wild type enzyme CCH2, in in 50 mM Tris-SO4 buffer (pH 8.0), at 22°C Mycolicibacterium smegmatis
5.3.2.1 1.8
-
 phenylenolpyruvate wild type enzyme cis-CAAD, in 10 mM Na2HPO4 buffer, pH 7.3, at 22°C Mycolicibacterium smegmatis
5.3.2.1 2.5
-
 (4-hydroxyphenyl)enolpyruvate wild type enzyme CCH2-P1A, in 10 mM Na2HPO4 buffer, pH 7.3, at 22°C Mycolicibacterium smegmatis
5.3.2.1 3.3
-
 phenylenolpyruvate mutant enzyme CCH2-P1A, in 10 mM Na2HPO4 buffer, pH 7.3, at 22°C Mycolicibacterium smegmatis
5.3.2.1 3.5
-
 (4-hydroxyphenyl)enolpyruvate wild type enzyme CCH2, in 10 mM Na2HPO4 buffer, pH 7.3, at 22°C Mycolicibacterium smegmatis
5.3.2.1 4
-
 trans-3-chloroacrylate wild type enzyme CCH2, in in 50 mM Tris-SO4 buffer (pH 8.0), at 22°C Mycolicibacterium smegmatis
5.3.2.1 9.8
-
 phenylenolpyruvate wild type enzyme CCH2, in 10 mM Na2HPO4 buffer, pH 7.3, at 22°C Mycolicibacterium smegmatis
5.3.2.1 30
-
 cis-3-chloroacrylate wild type enzyme cis-CAAD, in 10 mM Na2HPO4 buffer, pH 7.3, at 22°C Mycolicibacterium smegmatis