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Literature summary for 1.14.16.1 extracted from

  • Kino, K.; Hara, R.; Nozawa, A.
    Enhancement of L-tryptophan 5-hydroxylation activity by structure-based modification of L-phenylalanine 4-hydroxylase from Chromobacterium violaceum (2009), J. Biosci. Bioeng., 108, 184-189.
    View publication on PubMed
Search for more literature for 1.14.16.1

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli BL21(DE3) cells Chromobacterium violaceum

Protein Variants

Protein Variants Comment Organism
L101A the mutant shows 26% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
L101C the mutant shows 47% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
L101D the mutant shows 5% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
L101E the mutant shows 9% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
L101F the mutant shows 133% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
L101G the mutant shows 20% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
L101H the mutant shows 16% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
L101I the mutant shows 51% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
L101K the mutant shows 29% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
L101M the mutant shows 102% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
L101N the mutant shows 15% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
L101P the mutant shows 9% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
L101Q the mutant shows 30% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
L101R the mutant shows 29% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
L101S the mutant shows 28% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
L101T the mutant shows 26% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
L101V the mutant shows 26% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
L101W the mutant shows 55% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
L101Y the mutant shows 153% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
L101Y/W180F the double mutant displays higher L-tryptophan hydroxylation activity than the wild type enzyme with a 5.2fold increase in kcat Chromobacterium violaceum
W180A the mutant shows 66% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
W180C the mutant shows 119% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
W180D the mutant shows 3% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
W180E the mutant shows 6% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
W180F the mutant shows 204% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
W180G the mutant shows 8% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
W180H the mutant shows 73% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
W180I the mutant shows 113% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
W180K the mutant shows 4% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
W180L the mutant shows 174% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
W180M the mutant shows 166% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
W180N the mutant shows 49% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
W180P the mutant shows 15% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
W180Q the mutant shows 17% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
W180R the mutant shows 85% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
W180S the mutant shows 46% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
W180T the mutant shows 44% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
W180V the mutant shows 155% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum
W180Y the mutant shows 115% relative L-tryptophan hydroxylation activity compared to the wild type enzyme Chromobacterium violaceum

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.033
-
 L-phenylalanine mutant enzyme W180F, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
0.035
-
 5,6,7,8-tetrahydrobiopterin mutant enzyme W180F, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
0.086
-
 5,6,7,8-tetrahydrobiopterin mutant enzyme L101Y/W180F, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
0.099
-
 5,6,7,8-tetrahydrobiopterin mutant enzyme L101Y, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
0.111
-
 L-phenylalanine wild type enzyme, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
0.14
-
 5,6,7,8-tetrahydrobiopterin wild type enzyme, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
0.253
-
 L-phenylalanine mutant enzyme L101Y/W180F, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
0.478
-
 L-phenylalanine mutant enzyme L101Y, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
1
-
 L-tryptophan mutant enzyme W180F, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
3.44
-
 L-tryptophan wild type enzyme, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
3.54
-
 L-tryptophan mutant enzyme L101Y, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
4.06
-
 L-tryptophan mutant enzyme L101Y/W180F, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum

Metals/Ions

Metals/Ions Comment Organism Structure
Fe2+ required for activity Chromobacterium violaceum

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
34000
-
 SDS-PAGE Chromobacterium violaceum

Organism

Organism UniProt Comment Textmining
Chromobacterium violaceum P30967
-
-
Chromobacterium violaceum NBRC 12614 P30967
-
-

Purification (Commentary)

Purification (Comment) Organism
Ni-TED 2000 column chromatography, gel filtration Chromobacterium violaceum

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.307
-
 mutant enzyme W180F, at 30°C, using L-phenylalanine as substrate Chromobacterium violaceum
0.342
-
 mutant enzyme W180F, at 30°C, using 5,6,7,8-tetrahydrobiopterin as substrate Chromobacterium violaceum
0.347
-
 wild type enzyme, at 30°C, using 5,6,7,8-tetrahydrobiopterin as substrate Chromobacterium violaceum
0.69
-
 wild type enzyme, at 30°C, using L-tryptophan as substrate Chromobacterium violaceum
0.893
-
 mutant enzyme W180F, at 30°C, using L-tryptophan as substrate Chromobacterium violaceum
1
-
 mutant enzyme L101Y, at 30°C, using 5,6,7,8-tetrahydrobiopterin as substrate Chromobacterium violaceum
1
-
 mutant enzyme L101Y/W180F, at 30°C, using L-phenylalanine as substrate Chromobacterium violaceum
1.05
-
 mutant enzyme L101Y/W180F, at 30°C, using 5,6,7,8-tetrahydrobiopterin as substrate Chromobacterium violaceum
1.77
-
 mutant enzyme L101Y, at 30°C, using L-tryptophan as substrate Chromobacterium violaceum
3.37
-
 wild type enzyme, at 30°C, using L-phenylalanine as substrate Chromobacterium violaceum
3.62
-
 mutant enzyme L101Y/W180F, at 30°C, using L-tryptophan as substrate Chromobacterium violaceum
10.16
-
 mutant enzyme L101Y, at 30°C, using L-phenylalanine as substrate Chromobacterium violaceum

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
L-phenylalanine + 5,6,7,8-tetrahydrobiopterin + O2
-
 Chromobacterium violaceum L-tyrosine + 4a-hydroxytetrahydrobiopterin
-
 ?
L-phenylalanine + 5,6,7,8-tetrahydrobiopterin + O2
-
 Chromobacterium violaceum NBRC 12614 L-tyrosine + 4a-hydroxytetrahydrobiopterin
-
 ?
L-tryptophan + 5,6,7,8-tetrahydrobiopterin + O2 the activity for L-tryptophan is extremely low compared to L-phenylalanine activity levels Chromobacterium violaceum 5-hydroxy-L-tryptophan + 4a-hydroxytetrahydrobiopterin
-
 ?
L-tryptophan + 5,6,7,8-tetrahydrobiopterin + O2 the activity for L-tryptophan is extremely low compared to L-phenylalanine activity levels Chromobacterium violaceum NBRC 12614 5-hydroxy-L-tryptophan + 4a-hydroxytetrahydrobiopterin
-
 ?

Synonyms

Synonyms Comment Organism
L-phenylalanine 4-hydroxylase
-
 Chromobacterium violaceum
PAH
-
 Chromobacterium violaceum

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.18
-
 L-phenylalanine mutant enzyme W180F, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
0.2
-
 5,6,7,8-tetrahydrobiopterin mutant enzyme W180F, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
0.2
-
 5,6,7,8-tetrahydrobiopterin wild type enzyme, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
0.4
-
 L-tryptophan wild type enzyme, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
0.51
-
 L-tryptophan mutant enzyme W180F, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
0.57
-
 5,6,7,8-tetrahydrobiopterin mutant enzyme L101Y, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
0.57
-
 L-phenylalanine mutant enzyme L101Y/W180F, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
0.6
-
 5,6,7,8-tetrahydrobiopterin mutant enzyme L101Y/W180F, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
1.02
-
 L-tryptophan mutant enzyme L101Y, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
1.93
-
 L-phenylalanine wild type enzyme, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
2.08
-
 L-tryptophan mutant enzyme L101Y/W180F, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum
5.83
-
 L-phenylalanine mutant enzyme L101Y, at 30°C, in 50 mM HEPES-NaOH buffer (pH 7.5) Chromobacterium violaceum

Cofactor

Cofactor Comment Organism Structure
5,6,7,8-tetrahydro-L-biopterin
-
 Chromobacterium violaceum