Information on EC 1.14.19.58 - tryptophan 5-halogenase

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The expected taxonomic range for this enzyme is: Actinobacteria

EC NUMBER
COMMENTARY hide
1.14.19.58
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RECOMMENDED NAME
GeneOntology No.
tryptophan 5-halogenase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
L-tryptophan + FADH2 + chloride + O2 + H+ = 5-chloro-L-tryptophan + FAD + 2 H2O
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
halogenation
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SYSTEMATIC NAME
IUBMB Comments
L-tryptophan:FADH2 oxidoreductase (5-halogenating)
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
3-indolepropionate + FADH2 + chloride + O2 + H+
6-chloro-3-indolepropionate + 5-chloro-3-indolepropionate + FAD + 2 H2O
show the reaction diagram
E9P162
57% conversion by the wild-type enzyme, 75% 5-chlorination is by mutant L460F/P461E/P462T in comparison to 90% 6-chlorination by the wild-type SttH
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-
?
anthranilamide + FADH2 + chloride + O2 + H+
5-chloro-anthranilamide + FAD + 2 H2O
show the reaction diagram
E9P162
43% conversion by the wild-type enzyme
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-
?
anthranilate + FADH2 + chloride + O2 + H+
5-chloro-anthranilate + FAD + 2 H2O
show the reaction diagram
E9P162
1.1% conversion by the wild-type enzyme
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-
?
indole-3-acetic acid + FADH2 + chloride + O2 + H+
5-chloroindole-3-acetic acid + FAD + 2 H2O
show the reaction diagram
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-
-
-
?
kynurenine + FADH2 + chloride + O2 + H+
5-chloro-kynurenine + FAD + 2 H2O
show the reaction diagram
E9P162
79% conversion by the wild-type enzyme
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-
?
L-tryptophan + FADH2 + Br- + O2 + H+
5-bromo-L-tryptophan + FAD + 2 H2O
show the reaction diagram
L-tryptophan + FADH2 + chloride + O2 + H+
5-chloro-L-tryptophan + FAD + 2 H2O
show the reaction diagram
L-tryptophan + FADH2 + Cl- + O2 + H+
5-chloro-L-tryptophan + FAD + 2 H2O
show the reaction diagram
lantibiotic NAI-107 + FADH2-[tryptophan] + Cl- + O2 + H+
lantibiotic NAI-107-[5-chlorotryptophan] + FAD + 2 H2O
show the reaction diagram
N-phenylanthranilate + FADH2 + chloride + O2 + H+
5-chloro-N-phenylanthranilate + FAD + 2 H2O
show the reaction diagram
E9P162
20% conversion by the wild-type enzyme
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
L-tryptophan + FADH2 + Br- + O2 + H+
5-bromo-L-tryptophan + FAD + 2 H2O
show the reaction diagram
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-
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?
L-tryptophan + FADH2 + Cl- + O2 + H+
5-chloro-L-tryptophan + FAD + 2 H2O
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
L-tryptophan
additional information
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no substrate inhibition by tryptophan up to 2 mM
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.075
anthranilamide
E9P162
wild-type enzyme, pH and temperature not specified in the publication
0.241
kynurenine
E9P162
wild-type enzyme, pH and temperature not specified in the publication
0.071 - 0.15
L-tryptophan
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.02
anthranilamide
E9P162
wild-type enzyme, pH and temperature not specified in the publication
0.0085
kynurenine
E9P162
wild-type enzyme, pH and temperature not specified in the publication
0.00813
L-tryptophan
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pH 7.2, 30°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.019
anthranilamide
E9P162
wild-type enzyme, pH and temperature not specified in the publication
0.035
kynurenine
E9P162
wild-type enzyme, pH and temperature not specified in the publication
0.01 - 0.54
L-tryptophan
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant His6-tagged enzyme with bound FAD, hanging drop vapour diffusion method, mixing of 22 mg/ml protein in 20 mM HEPES, pH 8.0, and 150 mM KCl, with reservoir solution containing 12.5% MPD, 12.5% PEG 3350, 0.1 M MES/imidazole, pH 6.5, and 0.09 M NPS (1:1:1 NaNO3, Na2HPO4, (NH4)2SO4), X-ray diffraction structure determination and analysis at 1.85 A resolution
sitting-drop vapor-diffusion method at 20°C, crystal structure of a tryptophan 5-halogenase (PyrH) bound to tryptophan and FAD
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0
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half-life 1848 min
22
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half-life 517 min
30
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half-life 43 min
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant His6-tagged enzyme from Escherichia coli by nickel affinity chromatography and gel filtration
recombinant protein
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using a nickel-chelating HisTrap HP column
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Arabidopsis thaliana
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expression in Escherichia coli
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for expression in Pseudomonas fluorescens
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gene mibH, DNA and amino acid sequence determination and analysis, phylogenetic analysis, recombinant expression of His6-tagged enzyme in Escherichia coli, coexpression of mibH with the chaperones groES/EL and gene mibS
His-tagged apoPyrH was expressed in Pseudomonas fluorescens BL915
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overexpression in Pseudomonas fluorescens BL915 DELTAORF1
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E46D
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mutant enzyme folds properly
E46Q
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mutant enzyme folds properly
F49A/Y454F
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mutant enzyme is incorrectly folded
E46D
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mutant enzyme folds properly
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E46Q
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mutant enzyme folds properly
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F49A/Y454F
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mutant enzyme is incorrectly folded
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L460F/P461E/P462T
E9P162
site-directed, structure-guided mutagenesis, the mutant exhibit similar activity to the wild-type SttH, with tryptophan as a substrate, but shows a complete switch in regioselectivity compared to the wild-type enzyme without impacting on catalytic efficiency: 75% 5-chlorination is observed for the substrate 3-indolepropionate with the mutant in comparison to 90% 6-chlorination of 3-indolepropionate for the wild-type SttH. SttH is more like PyrH than PrnA, with insertions present in PyrH and SttH between residues SttH 155 and 167 and a deletion between SttH 457 and 464 compared with PrnA, the mutation, the only differences evident in the active-site region between the structures of PyrH and SttH are those of PyrH residues F451, E452 and T453 and SttH L460, P461 and P462. These residues are of particular interest because they are in close proximity to the active site in PyrH and SttH, and are positioned directly above the alpha-amino acid moiety of the substrate, tryptophan. Moreover, there isa loop insertion in PrnA in this region that is suggested to contribute to its regioselectivity. Each of these residues is mutated in SttH to the corresponding residue in PyrH, that is, L460F, P461E and P462T. Individually, each mutation reduces the relative activity of the enzyme with tryptophan, but does not have a significant effect on the observed regioselectivity, with 6-chlorotryptophan remaining the major product
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pharmacology
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5-Br- and 5-Cl-tryptophan could presumably be applied as a pharmacologically attractive precursor of serotonin
synthesis