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Literature summary extracted from

  • Zhu, L.; Ge, F.; Li, W.; Song, P.; Tang, H.; Tao, Y.; Liu, Y.; Du, G.
    One step synthesis of unnatural beta-arylalanines using mutant phenylalanine aminomutase from Taxus chinensis with high beta-regioselectivity (2018), Enzyme Microb. Technol., 114, 22-28 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
5.4.3.10 recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain JM109 Taxus chinensis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
5.4.3.10 purified recombinant wild-type PAM in complex with (R)-beta-phenylalanine or with L-beta-Phe analogue (S)-3-amino-2,2-difluoro-phenylpropanoic acid, enzyme mutant Y80A complexed with (S)-3-amino-2,2-difluoro-phenylpropanoic acid, and MIO-less enzyme mutants N231A and Y322A, crystallization from 0.2-0.5 mM TCEP, pH 7.0, with or without 2.0-20 mM ligand, X-ray diffraction structure determination and analysis at 1.85-2.20 A resolution Taxus chinensis

Protein Variants

EC Number Protein Variants Comment Organism
5.4.3.10 L108E site-directed mutagenesis, the mutant shows reduced activity with trans-cinnamate compared to wild-type enzyme Taxus chinensis
5.4.3.10 L108E/N458F site-directed mutagenesis, the mutant shows reduced activity with trans-cinnamate compared to wild-type enzyme Taxus chinensis
5.4.3.10 N231A site-directed mutagenesis, a cofactor MIO-less, catalytically inactive mutant Taxus chinensis
5.4.3.10 N458F site-directed mutagenesis, the mutant shows reduced activity with trans-cinnamate compared to wild-type enzyme Taxus chinensis
5.4.3.10 N458L site-directed mutagenesis, the mutant shows reduced activity with trans-cinnamate compared to wild-type enzyme Taxus chinensis
5.4.3.10 Y322A site-directed mutagenesis, a cofactor MIO-less, catalytically inactive mutant Taxus chinensis
5.4.3.10 Y80A site-directed mutagenesis Taxus chinensis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
5.4.3.10 D-alpha-phenylalanine Taxus chinensis
-
 L-beta-phenylalanine
-
 r

Organism

EC Number Organism UniProt Comment Textmining
5.4.3.10 Taxus chinensis Q68G83 i.e. Taxus chinensis
-

Purification (Commentary)

EC Number Purification (Comment) Organism
5.4.3.10 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and dialysis Taxus chinensis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
5.4.3.10 D-alpha-phenylalanine
-
 Taxus chinensis L-beta-phenylalanine
-
 r
5.4.3.10 D-alpha-phenylalanine (S)-alpha-phenylalanine, reaction mechanism Taxus chinensis L-beta-phenylalanine
-
 r
5.4.3.10 additional information phenylalanine-2,3-aminomutase (PAM) from Taxus chinensis, a 4-methylidene-imidazole-5-one (MIO)-dependent enzyme, catalyzes the reversible conversion of (S)-alpha-phenylalanine into (R)-beta-phenylalanine via trans-cinnamic acid. The enzyme also catalyzes the direct addition of ammonia to trans-cinnamic acid, a reaction that can be used for the preparation of beta-amino acids, cf. EC 4.3.1.24, phenylalanine ammonia-lyase Taxus chinensis ?
-
 ?
5.4.3.10 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 beta-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 also catalyzes the regioselective hydroamination of trans-cinnamic acid (t-CA) to yield L-beta-Phe, TcPAL, EC 4.3.1.24. The final product mixture consists of both alpha- and beta-Phe owing to low regioselectivity of the enzyme Taxus chinensis ?
-
 ?

Subunits

EC Number Subunits Comment Organism
5.4.3.10 homotetramer 4 * 75300, SDS-PAGE Taxus chinensis
5.4.3.10 More analysis of enzyme PAM three-dimensional structures with a bound (R)-beta-phenylalanine analogue and with bound trans-cinnamic acid, and of cofactor MIO-less enzyme mutant structures, overview Taxus chinensis

Synonyms

EC Number Synonyms Comment Organism
5.4.3.10 More cf. EC 4.3.1.24, phenylalanine ammonium-lyase Taxus chinensis
5.4.3.10 PAM
-
 Taxus chinensis
5.4.3.10 phenylalanine aminomutase
-
 Taxus chinensis

Cofactor

EC Number Cofactor Comment Organism Structure
5.4.3.10 3,5-dihydro-5-methylidene-4H-imidazol-4-one i.e. MIO, essential cofactor Taxus chinensis
5.4.3.10 3,5-dihydro-5-methylidene-4H-imidazol-4-one i.e. MIO, essential cofactor, residues Y322 and N231 are essential for MIO group formation Taxus chinensis

General Information

EC Number General Information Comment Organism
5.4.3.10 evolution PAM enzyme structures comparisons, overview Taxus chinensis
5.4.3.10 additional information the stereochemistry of the PAM-catalyzed reaction originates from the enzyme's ability to bind trans-cinnamic acid in two different orientations, with either the si,si face or the re,re face directed toward the MIO group, as evidenced by two distinct carboxylate binding modes. The N231 side chain promotes prosthetic MIO group formation by increasing the nucleophilicity of the G177 N atom through acidification of the amide proton. PAM enzyme structures comparisons, overview Taxus chinensis