The enzyme contains the cofactor 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO). This unique cofactor is formed autocatalytically by cyclization and dehydration of the three amino-acid residues alanine, serine and glycine. cf. EC 5.4.3.11, phenylalanine aminomutase (D-beta-phenylalanine forming).
The enzyme contains the cofactor 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO). This unique cofactor is formed autocatalytically by cyclization and dehydration of the three amino-acid residues alanine, serine and glycine. cf. EC 5.4.3.11, phenylalanine aminomutase (D-beta-phenylalanine forming).
(E)-cinnamate is both a substrate and an intermediate of the reaction. To account for the distinct (3alpha)-beta-amino acid stereochemistry catalyzed by the enzyme, the cinnamate skeleton must rotate the C1-Calpha and Cipso-Cbeta bonds 180° in the active site prior to exchange and rebinding of theNH2/H pair to the cinnamate
(E)-cinnamate is both a substrate and an intermediate of the reaction. To account for the distinct (3alpha)-beta-amino acid stereochemistry catalyzed by the enzyme, the cinnamate skeleton must rotate the C1-Calpha and Cipso-Cbeta bonds 180° in the active site prior to exchange and rebinding of theNH2/H pair to the cinnamate
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
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
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
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
structural determinant that dictates the activity differences between a phenylalanine ammonia lyase (PAL, EC 4.3.1.24) and aminomutase (PAM), overview. An inner loop region closes the active sites of both PAM and PAL. The inner loop is a structural determinant of the lyase and mutase activities of PAM. Three-dimensional structure comparisons of Taxus canadensis PAM with PAM from Taxus chinensis and phenylalanine ammonia lyase from Petroselinum crispum (PcPAL)
structural determinant that dictates the activity differences between a phenylalanine ammonia lyase (PAL, EC 4.3.1.24) and aminomutase (PAM), overview. An inner loop region closes the active sites of both PAM and PAL. The inner loop is a structural determinant of the lyase and mutase activities of PAM. Three-dimensional structure comparisons of Taxus chinensis PAM with PAM from Taxus canadensis and phenylalanine ammonia lyase from Petroselinum crispum (PcPAL)
the enzyme belongs to the MIO-dependent aminomutases. Aminomutases (defined as isomerases mediating intramolecular transfer of amino groups) catalyze the synthetically challenging shift of an amine group along a saturated carbon chain, typically of an amino acid. PAMs and tyrosine aminomutases (TAMs) share the same structure, mechanistic pathway, and characteristics of phenylalanine ammonia-lyases (PALs), histidine ammonia-lyases (HALs), and tyrosine ammonia-lyases (TALs), being all members of the same MIO-dependent enzyme family, also called class I lyase-like enzymes
the enzyme belongs to the MIO-dependent aminomutases. Aminomutases (defined as isomerases mediating intramolecular transfer of amino groups) catalyze the synthetically challenging shift of an amine group along a saturated carbon chain, typically of an amino acid. PAMs and tyrosine aminomutases (TAMs) share the same structure, mechanistic pathway, and characteristics of phenylalanine ammonia-lyases (PALs), histidine ammonia-lyases (HALs), and tyrosine ammonia-lyases (TALs), being all members of the same MIO-dependent enzyme family, also called class I lyase-like enzymes
the enzyme belongs to the MIO-dependent aminomutases. Aminomutases (defined as isomerases mediating intramolecular transfer of amino groups) catalyze the synthetically challenging shift of an amine group along a saturated carbon chain, typically of an amino acid. PAMs and tyrosine aminomutases (TAMs) share the same structure, mechanistic pathway, and characteristics of phenylalanine ammonia-lyases (PALs), histidine ammonia-lyases (HALs), and tyrosine ammonia-lyases (TALs), being all members of the same MIO-dependent enzyme family, also called class I lyase-like enzymes
the enzyme belongs to the MIO-dependent aminomutases. Aminomutases (defined as isomerases mediating intramolecular transfer of amino groups) catalyze the synthetically challenging shift of an amine group along a saturated carbon chain, typically of an amino acid. PAMs and tyrosine aminomutases (TAMs) share the same structure, mechanistic pathway, and characteristics of phenylalanine ammonia-lyases (PALs), histidine ammonia-lyases (HALs), and tyrosine ammonia-lyases (TALs), being all members of the same MIO-dependent enzyme family, also called class I lyase-like enzymes
phenylalanine aminomutase (PAM) catalyzes the 2,3-shift of the alpha-amino group of L-phenylalanine and L-tyrosine to afford beta-phenylalanine. Biocatalytic strategies for the production of (R)- or (S)-beta-arylalanines employing enzymes with enantiocomplementary aminomutase activity
phenylalanine aminomutase (PAM) catalyzes the 2,3-shift of the alpha-amino group of L-phenylalanine and L-tyrosine to afford beta-phenylalanine. Biocatalytic strategies for the production of (R)- or (S)-beta-arylalanines employing enzymes with enantiocomplementary aminomutase activity. (R)-beta-phenylalanine is a precursor in biosynthesis of taxol in Taxus species
phenylalanine aminomutase (PAM) catalyzes the 2,3-shift of the alpha-amino group of L-phenylalanine and L-tyrosine to afford beta-phenylalanine. Biocatalytic strategies for the production of (R)- or (S)-beta-arylalanines employing enzymes with enantiocomplementary aminomutase activity. Metabolites containing (R)-beta-phenylalanine are chemically similar astins from the plant Aster tataricus
phenylalanine aminomutase (PAM) catalyzes the 2,3-shift of the alpha-amino group of L-phenylalanine and L-tyrosine to afford beta-phenylalanine. Biocatalytic strategies for the production of (R)- or (S)-beta-arylalanines employing enzymes with enantiocomplementary aminomutase activity. Metabolites containing (R)-beta-phenylalanine are chemically similar cyclochlorotines from the plant Talaromyces islandicum
the inner loop is a structural determinant of the lyase and mutase activities of PAM. Three-dimensional structure comparisons of Taxus chinensis PAM with PAM from Taxus canadensis and phenylalanine ammonia lyase from Petroselinum crispum (PcPAL). The latter contains an open inner loop conformation. The active-site inner loop, which contains the catalytic base Tyr, appears more rigid in PAM and more open or flexible in PAL. The rigidity of this loop in PAM is considered crucial for sequestering the trans-cinnamic acid and MIO-amine adduct in the active site to promote readdition of the amino-group to either the alpha- or beta-carbon positions of trans-cinnamic acid. Molecular dynamic simulations
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
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
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
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
to 2.38 A resolution, space group C2. A (E)-cinnamate molecule is bound in the active site, lying above the 4-methylidene-1H-imidazol-5(4H)-one cofactor and under a loop region that includes residues 80-97, which define the top of the active site. The (E)-cinnamate molecule lies about 3.4 A above the methylidene carbon of the 4-methylidene-1H-imidazol-5(4H)-one moiety. The carboxylate of the cinnamate makes a salt bridge interaction with a strongly conserved residue R325, which serves to position the product in the active site. The plane of the aromatic ring of the cinnamate is displaced about 20° from the perpendicular relative to the pi-bond plane of the propenoate carboncarbon double bond. The aromatic ring is bound relatively loosely in the active site, making only one direct hydrophobic interaction with residue L104
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
site-directed mutagenesis, the mutant shows an altered structure compared to wild-type enzyme, the mutant has a significant increase in 310-helix and beta-ladder, compared to wild-type, with the highest coil percent among the PAM variants and improved activity. The mutant shows reduced temperature stability and optimum for PAM activity, in contrast to PAL activity
site-directed mutagenesis, the mutant shows an altered structure compared to wild-type enzyme, the mutant has a significant increase in 310-helix and beta-ladder compared to wild-type. The mutant shows reduced temperature stability and optimum for PAM activity, in contrast to PAL activity
mutation of the inner loop region, that closes the active site of PAM, within PAM (PAM residues 77-97) in a stepwise approach. Almost all of the single loop mutations trigger a lyase phenotype in PAM. Experimental and computational evidence suggest that the induced lyase features result from inner loop mobility enhancements, which are possibly caused by a 310-helix cluster, flanking alpha-helices, and hydrophobic interactions. The application of wild-type PAM for the synthesis of beta-amino acids is hindered by low reaction rates and the mixture of alpha-Phe and beta-Phe generated from the asymmetric synthetic route. Molecular dynamic simulations