Cloned (Comment) | Organism |
---|---|
sequence comparisons, recombinant expression of His-tagged wild-type and mutant enzymes | Oryza sativa |
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 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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Oryza sativa | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes by nickel affinity chromatography | Oryza sativa |
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 | 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 | 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 Maximum [°C] | Comment | Organism |
---|---|---|---|
29 | - |
assay at | Oryza sativa |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Oryza sativa |
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 | 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 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 |