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Literature summary for 5.4.3.6 extracted from

  • Walter, T.; Wijewardena, D.; Walker, K.
    Mutation of aryl binding pocket residues results in an unexpected activity switch in an Oryza sativa tyrosine aminomutase (2016), Biochemistry, 55, 3497-3503 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
sequence comparisons, recombinant expression of His-tagged wild-type and mutant enzymes Oryza sativa

Protein Variants

Protein Variants Comment Organism
N446K site-directed mutagenesis, the mutant shows increased activity with phenylalanine and halogenated phenylalanine substrates compared to the wild-type enzyme, the mutant activity is reduced at converting alpha-tyrosine to its beta-isomer and 4-coumarate (ratio 22:78) compared to the wild-type, the mutant binds alpha-phenylalanine about 9fold better than wild-type OsTAM, total turnover rate of the mutant for converting alpha-phenylalanine to both beta-phenylalanine and cinnamate is about 4fold greater than the wild-type OsTAM rate for making beta-phenylalanine and cinnamate. Mutation of Asn446 of OsTAM to a Lys residue creates an active site bearing the characteristic triad sequence ([aromatic amino acid]-Leu-Lys) conserved in all PALs and thus explains in part why N446K-OsTAM has increased PAL activity compared to wild-type OsTAM Oryza sativa
Y125C site-directed mutagenesis, the mutant shows altered activity with phenylalanine and halogenated phenylalanine substrates compared to the wild-type enzyme, mutant activity is reduced at converting alpha-tyrosine to its beta-isomer and 4-coumarate (ratio 2:98) compared to the wild-type, the mutant binds alpha-phenylalanine about 9fold better than wild-type OsTAM, total turnover rate of the mutant for converting alpha-phenylalanine to both beta-phenylalanine and cinnamate is about 4fold greater than the wild-type OsTAM rate for making beta-phenylalanine and cinnamate Oryza sativa
Y125C/N446K site-directed mutagenesis, inactive mutant with L-tyrosine, the mutant binds alpha-phenylalanine about 9fold better than wild-type OsTAM, the mutant converts alpha-phenylalanine to both beta-phenylalanine and cinnamate Oryza sativa

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.49
-
L-phenylalanine alpha-phenylalanine, pH 8.0, 29°C, recombinant mutant Y125C/N446K Oryza sativa
0.51
-
L-tyrosine alpha-tyrosine, pH 8.0, 29°C, recombinant mutant Y125C Oryza sativa
0.54
-
L-tyrosine alpha-tyrosine, pH 8.0, 29°C, recombinant wild-type enzyme Oryza sativa
0.64
-
L-tyrosine alpha-tyrosine, pH 8.0, 29°C, recombinant mutant N446K Oryza sativa
0.72
-
L-phenylalanine alpha-phenylalanine, pH 8.0, 29°C, recombinant mutant N446K Oryza sativa
1
-
L-phenylalanine alpha-phenylalanine, pH 8.0, 29°C, recombinant mutant Y125C Oryza sativa
9
-
L-phenylalanine alpha-phenylalanine, pH 8.0, 29°C, recombinant wild-type enzyme Oryza sativa

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-tyrosine Oryza sativa Oryza sativa enzyme OsTAM makes 75% beta-tyrosine and 25% 4-coumarate from alpha-tyrosine 3-amino-3-(4-hydroxyphenyl)propanoate
-
?

Organism

Organism UniProt Comment Textmining
Oryza sativa
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes by nickel affinity chromatography Oryza sativa

Reaction

Reaction Comment Organism Reaction ID
L-tyrosine = 3-amino-3-(4-hydroxyphenyl)propanoate MIO (3,5-dihydro-5-methylidene-4H-imidazol-4-one)-dependent aminomutases begin their reactions by adding the amino group of the substrate to the methylidene carbon of the MIO prosthesis. The enzyme removes the NH2/H pair from the substrate, yielding an NH2-MIO adduct, a tightly bound acrylate intermediate, and protonated catalytic Tyr Oryza sativa

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
L-phenylalanine 3% relative to the activity with L-tyrosine of wild-type enzyme Oryza sativa L-beta-phenylalanine
-
?
L-tyrosine Oryza sativa enzyme OsTAM makes 75% beta-tyrosine and 25% 4-coumarate from alpha-tyrosine Oryza sativa 3-amino-3-(4-hydroxyphenyl)propanoate
-
?
L-tyrosine wild-type OsTAM from Oryza sativa preferentially isomerizes (2S)-alpha-tyrosine to (3R)-beta-tyrosine with an enantioselectivity (94% ee) Oryza sativa 3-amino-3-(4-hydroxyphenyl)propanoate
-
?
additional information substrate specificity of recombinant wild-type and mutant enzymes, overview Oryza sativa ?
-
?

Synonyms

Synonyms Comment Organism
3,5-dihydro-5-methylidine-4H-imidazol-4-one-dependent tyrosine aminomutase
-
Oryza sativa
MIO-dependent tyrosine aminomutase
-
Oryza sativa
OsTAM
-
Oryza sativa
TAM
-
Oryza sativa
tyrosine aminomutase
-
Oryza sativa

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
29
-
assay at Oryza sativa

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.0000021
-
L-tyrosine alpha-tyrosine, pH 8.0, 29°C, recombinant mutant N446K Oryza sativa
0.000035
-
L-phenylalanine alpha-phenylalanine, pH 8.0, 29°C, recombinant mutant Y125C Oryza sativa
0.000052
-
L-phenylalanine alpha-phenylalanine, pH 8.0, 29°C, recombinant mutant Y125C/N446K Oryza sativa
0.000053
-
L-tyrosine alpha-tyrosine, pH 8.0, 29°C, recombinant mutant Y125C Oryza sativa
0.00008
-
L-phenylalanine alpha-phenylalanine, pH 8.0, 29°C, recombinant mutant N446K Oryza sativa
0.00016
-
L-phenylalanine alpha-phenylalanine, pH 8.0, 29°C, recombinant wild-type enzyme Oryza sativa
0.005
-
L-tyrosine alpha-tyrosine, pH 8.0, 29°C, recombinant wild-type enzyme Oryza sativa

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Oryza sativa

Cofactor

Cofactor Comment Organism Structure
3,5-dihydro-5-methylidene-4H-imidazol-4-one i.e. MIO, dependent on. The MIO group is formed by condensation and cyclization of backbone residues of an (A, T, or S)-Ser-Gly triad in the active site. The MIO N-alkylates the NH2 of the alpha-amino acid substrates and promotes the removal of an intermediary NH2-MIO adduct. Concomitant removal of a beta-proton from the substrate (NH2-MIO adduct) by a catalytic tyrosine yields an acrylate intermediate Oryza sativa

General Information

General Information Comment Organism
additional information residues Y125 and N446 not only play a central role in substrate selectivity but, in part, also set the intrinsic reactivity of OsTAM Oryza sativa

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.000018
-
L-phenylalanine alpha-phenylalanine, pH 8.0, 29°C, recombinant wild-type enzyme Oryza sativa
0.00003
-
L-tyrosine alpha-tyrosine, pH 8.0, 29°C, recombinant mutant N446K Oryza sativa
0.000035
-
L-phenylalanine alpha-phenylalanine, pH 8.0, 29°C, recombinant mutant Y125C Oryza sativa
0.0001
-
L-tyrosine alpha-tyrosine, pH 8.0, 29°C, recombinant mutant Y125C Oryza sativa
0.00011
-
L-phenylalanine alpha-phenylalanine, pH 8.0, 29°C, recombinant mutant N446K Oryza sativa
0.00011
-
L-phenylalanine alpha-phenylalanine, pH 8.0, 29°C, recombinant mutant Y125C/N446K Oryza sativa
0.0093
-
L-tyrosine alpha-tyrosine, pH 8.0, 29°C, recombinant wild-type enzyme Oryza sativa