Cloned (Comment) | Organism |
---|---|
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain JM109 | Taxus chinensis |
Protein Variants | Comment | Organism |
---|---|---|
L108E | site-directed mutagenesis, the mutant shows reduced activity with trans-cinnamate compared to wild-type enzyme | Taxus chinensis |
L108E/N458F | site-directed mutagenesis, the mutant shows reduced activity with trans-cinnamate compared to wild-type enzyme | Taxus chinensis |
N458F | site-directed mutagenesis, the mutant shows reduced activity with trans-cinnamate compared to wild-type enzyme | Taxus chinensis |
N458L | site-directed mutagenesis, the mutant shows reduced activity with trans-cinnamate compared to wild-type enzyme | Taxus chinensis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | amination reaction kinetic parameters of wild-type PAM and mutants, all with an enantiomeric excess of over 99% (ee). Michaelis-Menten kinetics of formation of alpha- and beta-phenylalanine from trans-cinnamate | Taxus chinensis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
trans-cinnamate + NH3 | Taxus chinensis | - |
L-phenylalanine | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Taxus chinensis | Q68G83 | i.e. Taxus chinensis | - |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and dialysis | Taxus chinensis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | TcPAM catalyzes the isomerization of alpha-phenylalanine to beta-phenylalanine through exchanging the position of the amine group (Calpha -> Cbeta) and pro-3S hydrogen proton (Cbeta -> Calpha) with retention of the configuration at the reaction termini, which requires reorientation after deamination of L-phenylalanine to trans-cinnamic acid in which the reface of the Cbeta and the si-face of the Cbeta carton atoms are positioned for amine readdition and reprotonation. The enzyme TcPAM (EC 5.4.3.10) also catalyzes the regioselective hydroamination of transcinnamic acid (t-CA) to yield L-beta-Phe, TcPAL. The final product mixture consists of both alpha- and beta-Phe owing to low regioselectivity of the enzyme. Substrate docking | Taxus chinensis | ? | - |
? | |
trans-cinnamate + NH3 | - |
Taxus chinensis | L-phenylalanine | - |
r | |
trans-cinnamate + NH3 | - |
Taxus chinensis | L-alpha-phenylalanine + L-beta-phenylalanine | - |
r |
Synonyms | Comment | Organism |
---|---|---|
More | cf. EC 5.4.3.10, phenylalanine aminomutase | Taxus chinensis |
PAL | - |
Taxus chinensis |
phenylalanine ammonia lyase | - |
Taxus chinensis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
40 | - |
assay at | Taxus chinensis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Taxus chinensis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
3,5-dihydro-5-methylidene-4H-imidazol-4-one | i.e. MIO, essential cofactor | Taxus chinensis |