Literature summary for 5.4.3.10 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 .

Cloned(Commentary)

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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

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
N231A	site-directed mutagenesis, a cofactor MIO-less, catalytically inactive mutant	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
Y322A	site-directed mutagenesis, a cofactor MIO-less, catalytically inactive mutant	Taxus chinensis
Y80A	site-directed mutagenesis	Taxus chinensis

Natural Substrates/ Products (Substrates)

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

Organism

Organism	UniProt	Comment	Textmining
Taxus chinensis	Q68G83	i.e. Taxus chinensis	-

Purification (Commentary)

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 and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
D-alpha-phenylalanine	-	Taxus chinensis	L-beta-phenylalanine	-	r
D-alpha-phenylalanine	(S)-alpha-phenylalanine, reaction mechanism	Taxus chinensis	L-beta-phenylalanine	-	r
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	?	-	?
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

Subunits	Comment	Organism
homotetramer	4 * 75300, SDS-PAGE	Taxus chinensis
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

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

Cofactor

Cofactor	Comment	Organism	Structure
3,5-dihydro-5-methylidene-4H-imidazol-4-one	i.e. MIO, essential cofactor	Taxus chinensis
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

General Information	Comment	Organism
evolution	PAM enzyme structures comparisons, overview	Taxus chinensis
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