Information on EC 2.1.1.281 - phenylpyruvate C3-methyltransferase

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The expected taxonomic range for this enzyme is: Streptomyces hygroscopicus

EC NUMBER
COMMENTARY hide
2.1.1.281
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RECOMMENDED NAME
GeneOntology No.
phenylpyruvate C3-methyltransferase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
S-adenosyl-L-methionine + 3-phenylpyruvate = S-adenosyl-L-homocysteine + (3S)-2-oxo-3-phenylbutanoate
show the reaction diagram
SYSTEMATIC NAME
IUBMB Comments
S-adenosyl-L-methionine:2-oxo-3-phenylpropanoate C3-methyltransferase
The enzyme from the bacterium Streptomyces hygroscopicus NRRL3085 is involved in synthesis of the nonproteinogenic amino acid (2S,3S)-beta-methyl-phenylalanine, a building block of the antibiotic mannopeptimycin.
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
S-adenosyl-L-methionine + 2-oxo-3-phenylpropanoate
S-adenosyl-L-homocysteine + (3S)-2-oxo-3-methyl-3-phenylpropanoate
show the reaction diagram
S-adenosyl-L-methionine + 3-phenylpyruvate
S-adenosyl-L-homocysteine + (3R)-2-oxo-3-phenylbutanoate
show the reaction diagram
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
S-adenosyl-L-methionine + 2-oxo-3-phenylpropanoate
S-adenosyl-L-homocysteine + (3S)-2-oxo-3-methyl-3-phenylpropanoate
show the reaction diagram
S-adenosyl-L-methionine + 3-phenylpyruvate
S-adenosyl-L-homocysteine + (3R)-2-oxo-3-phenylbutanoate
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
S-adenosyl-L-methionine
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe2+
required, binding structure, overview
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.5
2-oxo-3-phenylpropanoate
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pH 8.0, 30C
0.014
S-adenosyl-L-methionine
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pH 8.0, 30C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.8
2-oxo-3-phenylpropanoate
Streptomyces hygroscopicus
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pH 8.0, 30C
8.15
S-adenosyl-L-methionine
Streptomyces hygroscopicus
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pH 8.0, 30C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.32
2-oxo-3-phenylpropanoate
Streptomyces hygroscopicus
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pH 8.0, 30C
4473
582
S-adenosyl-L-methionine
Streptomyces hygroscopicus
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pH 8.0, 30C
24
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
38000
2 * 38000, recombinant His6-tagged enzyme, SDS-PAGE
70200
recombinant His-tagged enzyme, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant His6-tagged enzyme, in apoform or in complex with S-adenosyl-L-methionine and phenylpyruvate, hanging drop vapour diffusion technique, mixing of 0.001 ml of 24 mg/ml protein in 50 mM HEPES, pH 7.5, 100 mM CaCl2, with 0.001 ml of reservoir solution containing 16% PEG 3350, 200 mM NaI, 20C, 10 days, X-ray diffraction structure determination and analysis at 2.0-2.5 A resolution, molecular replacement. From the collected crystal diffraction data four additional structures of MppJ in complex with phenlpyruvate, 4-hydroxyphenylpyruvate, S-adenosyl-L-methionine and phenylpyruvate or S-adenosyl-L-homocysteine and methylphenylpyruvate, although crystals of the apo form remain unobtainable
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant N-terminally His6-tagged enzyme by nickel affinity chromatography, ion-exchange chromatography, and ultrafiltration
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
gene mppJ from the mannopeptimycin-biosynthetic gene cluster, DNA and amino acid sequence determination and analysis, recombinant expression of N-terminally His6-tagged enzyme
His6-tagged enzyme is expressed in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C319A
site-directed mutagenesis, the mutant shows 39% reduced activity compared to the wild-type enzyme
D244E
site-directed mutagenesis, the mutant enzyme shows altered conformation and substrate binding structure compared to the wild-type enzyme
D244L
site-directed mutagenesis, the mutant enzyme shows altered conformation and substrate binding structure compared to the wild-type enzyme
R127L/D244E
site-directed mutagenesis, the mutant enzyme shows altered conformation and substrate binding structure compared to the wild-type enzyme
W99F
the mutant shows altered stereochemistry in the reaction compared to the wild-type enzyme
C319A
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site-directed mutagenesis, the mutant shows 39% reduced activity compared to the wild-type enzyme
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D244E
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site-directed mutagenesis, the mutant enzyme shows altered conformation and substrate binding structure compared to the wild-type enzyme
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D244L
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site-directed mutagenesis, the mutant enzyme shows altered conformation and substrate binding structure compared to the wild-type enzyme
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R127L/D244E
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site-directed mutagenesis, the mutant enzyme shows altered conformation and substrate binding structure compared to the wild-type enzyme
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W99F
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the mutant shows altered stereochemistry in the reaction compared to the wild-type enzyme
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