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Literature summary extracted from

  • Shah, D.D.; Conrad, J.A.; Heinz, B.; Brownlee, J.M.; Moran, G.R.
    Evidence for the mechanism of hydroxylation by 4-hydroxyphenylpyruvate dioxygenase and hydroxymandelate synthase from intermediate partitioning in active site variants (2011), Biochemistry, 50, 7694-7704.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.13.11.27 expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Streptomyces avermitilis
1.13.11.46 expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Amycolatopsis orientalis

Protein Variants

EC Number Protein Variants Comment Organism
1.13.11.27 N245I site-directed mutagenesis, the mutant enzyme produces 13% homogentisate and 87% 4-hydroxyphenylacetate, which differs from the wild-type activity Streptomyces avermitilis
1.13.11.27 N245Q site-directed mutagenesis, the mutant enzyme produces 44.5% homogentisate and 52% 4-hydroxyphenylacetate, and 4% quinolacetate, which differs from the wild-type activity Streptomyces avermitilis
1.13.11.27 P243T site-directed mutagenesis, the mutant enzyme produces 13% homogentisate, 8.7% 4-hydroxyphenylacetate, and 78% quinolacetate, which differs from the wild-type activity Streptomyces avermitilis
1.13.11.27 S230A site-directed mutagenesis, the mutant enzyme produces 11.6% homogentisate, 30.6% 4-hydroxyphenylacetate, and 57.8% quinolacetate, which differs from the wild-type activity Streptomyces avermitilis
1.13.11.46 I216N site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme, the mutant produces 90% 4-hydroxmandelate and 10% 4-hydroxyphenylacetate from 4-hydroxyphenylpyruvate, which differs from the wild-type, that does not produce any 4-hydroxyphenylacetate Amycolatopsis orientalis
1.13.11.46 S201A site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme, the mutant produces 88% 4-hydroxmandelate and 12% 4-hydroxyphenylacetate from 4-hydroxyphenylpyruvate, which differs from the wild-type, that does not produce any 4-hydroxyphenylacetate Amycolatopsis orientalis
1.13.11.46 T214P site-directed mutagenesis, mutant shows decreased catalytic efficiency compared to the wild-type enzyme Amycolatopsis orientalis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.13.11.27 0.0087
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, mutant P243T Streptomyces avermitilis
1.13.11.27 0.015
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, mutant N245Q Streptomyces avermitilis
1.13.11.27 0.027
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, wild-type enzyme and mutant N245I Streptomyces avermitilis
1.13.11.27 0.047
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, mutant S230A Streptomyces avermitilis
1.13.11.46 0.0143
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, mutant I216N Amycolatopsis orientalis
1.13.11.46 0.0205
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, mutant T214P Amycolatopsis orientalis
1.13.11.46 0.028
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, mutant S201A Amycolatopsis orientalis
1.13.11.46 0.0476
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, wild-type enzyme Amycolatopsis orientalis

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.13.11.46 Fe2+ a Fe(II)-dependent dioxygenase Amycolatopsis orientalis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.13.11.27 4-hydroxyphenylpyruvate + O2 Streptomyces avermitilis
-
homogentisate + CO2 + 4-hydroxyphenylacetate the wild-type enzyme produces 85% homogentisate and 15% 4-hydroxyphenylacetate ?
1.13.11.46 4-hydroxyphenylpyruvate + O2 Amycolatopsis orientalis
-
4-hydroxymandelate + CO2
-
?

Organism

EC Number Organism UniProt Comment Textmining
1.13.11.27 Streptomyces avermitilis
-
-
-
1.13.11.46 Amycolatopsis orientalis
-
gene AOHMS
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.13.11.27 recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by ammonium sulfate fractionation, anion exchange chromatography, and gel filtration Streptomyces avermitilis
1.13.11.46 recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by ammonium sulfate fractionation, anion exchange chromatography, and gel filtration Amycolatopsis orientalis

Reaction

EC Number Reaction Comment Organism Reaction ID
1.13.11.27 4-hydroxyphenylpyruvate + O2 = homogentisate + CO2 reaction mechanism, overview Streptomyces avermitilis
1.13.11.46 4-hydroxyphenylpyruvate + O2 = (S)-4-hydroxymandelate + CO2 reaction mechanism, overview Amycolatopsis orientalis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.13.11.27 4-hydroxyphenylpyruvate + O2
-
Streptomyces avermitilis homogentisate + CO2 + 4-hydroxyphenylacetate the wild-type enzyme produces 85% homogentisate and 15% 4-hydroxyphenylacetate ?
1.13.11.46 4-hydroxyphenylpyruvate + O2
-
Amycolatopsis orientalis 4-hydroxymandelate + CO2
-
?

Synonyms

EC Number Synonyms Comment Organism
1.13.11.27 HPPD
-
Streptomyces avermitilis
1.13.11.46 hydroxymandelate synthase
-
Amycolatopsis orientalis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.13.11.27 25
-
assay at Streptomyces avermitilis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.13.11.27 0.03
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, mutant N245Q Streptomyces avermitilis
1.13.11.27 0.64
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, mutant N245I Streptomyces avermitilis
1.13.11.27 2.1
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, mutant P243T Streptomyces avermitilis
1.13.11.27 2.8
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, mutant S230A Streptomyces avermitilis
1.13.11.27 6.8
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, wild-type enzyme Streptomyces avermitilis
1.13.11.46 1.2
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, mutant T214P Amycolatopsis orientalis
1.13.11.46 1.4
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, mutant I216N Amycolatopsis orientalis
1.13.11.46 3
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, mutant S201A Amycolatopsis orientalis
1.13.11.46 3.7
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, wild-type enzyme Amycolatopsis orientalis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.13.11.27 7
-
assay at Streptomyces avermitilis

General Information

EC Number General Information Comment Organism
1.13.11.46 evolution 4-hydroxyphenylpyruvate dioxygenase, HPPD, EC 1.13.11.27, and hydroxymandelate synthase catalyze similar reactions using the same substrates, 4-hydroxyphenylpyruvate and dioxygen. Initially, both enzymes reduce and activate dioxygen in order to decarboxylate 4-hydroxyphenylpyruvate, yielding 4-hydroxyphenylacetate, CO2, and an activated oxo intermediate. Both enzymes then hydroxylate 4-hydroxyphenylacetate but do so in different positions Amycolatopsis orientalis

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.13.11.27 2.4
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, mutant N245Q Streptomyces avermitilis
1.13.11.27 24
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, mutant N245I Streptomyces avermitilis
1.13.11.27 60
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, mutant S230A Streptomyces avermitilis
1.13.11.27 241
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, mutant P243T Streptomyces avermitilis
1.13.11.27 254
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, wild-type enzyme Streptomyces avermitilis
1.13.11.46 57.5
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, mutant T214P Amycolatopsis orientalis
1.13.11.46 77
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, wild-type enzyme Amycolatopsis orientalis
1.13.11.46 98.5
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, mutant I216N Amycolatopsis orientalis
1.13.11.46 108
-
4-hydroxyphenylpyruvate pH 7.0, 25°C, mutant S201A Amycolatopsis orientalis