Literature summary for 5.4.3.10 extracted from

Feng, L.; Wanninayake, U.; Strom, S.; Geiger, J.; Walker, K.
Mechanistic, mutational, and structural evaluation of a taxus phenylalanine aminomutase (2011), Biochemistry, 50, 2919-2930.

Activating Compound

Activating Compound	Comment	Organism	Structure
4-methylidene-1H-imidazol-5(4H)-one	-	Taxus canadensis

Cloned(Commentary)

Cloned (Comment)	Organism
expression in Escherichia coli	Taxus canadensis

Crystallization (Commentary)

Crystallization (Comment)	Organism
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	Taxus canadensis

Crystallization (Comment)

Organism

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

Taxus canadensis

Protein Variants

Protein Variants	Comment	Organism
L104A	1.5-fold increase in kcat and a decrease in KM values for 3'-methyl-alpha-phenylalanine and styryl-alpha-alanine substrates	Taxus canadensis

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.057	-	L-phenylalanine	wild-type, pH 8.5, 31°C	Taxus canadensis
0.073	-	4'-methyl-alpha-phenylalanine	mutant L104A, pH 8.5, 31°C	Taxus canadensis
0.083	-	3'-methyl-alpha-phenylalanine	mutant L104A, pH 8.5, 31°C	Taxus canadensis
0.091	-	4'-methyl-alpha-phenylalanine	wild-type, pH 8.5, 31°C	Taxus canadensis
0.12	-	styryl-alpha-alanine	mutant L104A, pH 8.5, 31°C	Taxus canadensis
0.136	-	L-phenylalanine	mutant L104A, pH 8.5, 31°C	Taxus canadensis
0.25	-	styryl-alpha-alanine	wild-type, pH 8.5, 31°C	Taxus canadensis
0.397	-	3'-methyl-alpha-phenylalanine	wild-type, pH 8.5, 31°C	Taxus canadensis

Organism

Organism	UniProt	Comment	Textmining
Taxus canadensis	Q6GZ04	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
3'-methyl-alpha-phenylalanine	-	Taxus canadensis	3'-methyl-beta-phenylalanine	plus 3'-methylcinnamate, product of ammonia lyase reaction. Distribution of 3'-methyl-beta-phenylalanine and 3'-methylcinnamate is at about 1:1	?
4'-methyl-alpha-phenylalanine	-	Taxus canadensis	4'-methyl-beta-phenylalanine	plus 4'-methylcinnamate, product of ammonia lyase reaction	?
L-phenylalanine	-	Taxus canadensis	L-beta-phenylalanine	-	?
additional information	(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	Taxus canadensis	?	-	?
styryl-alpha-alanine	-	Taxus canadensis	5-phenyl-(2E,4E)-pentadienoate	-	?

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.0002	-	styryl-alpha-alanine	wild-type, pH 8.5, 31°C	Taxus canadensis
0.003	-	L-phenylalanine	mutant L104A, pH 8.5, 31°C	Taxus canadensis
0.003	-	styryl-alpha-alanine	mutant L104A, pH 8.5, 31°C	Taxus canadensis
0.02	-	4'-methyl-alpha-phenylalanine	mutant L104A, pH 8.5, 31°C	Taxus canadensis
0.022	-	3'-methyl-alpha-phenylalanine	wild-type, pH 8.5, 31°C	Taxus canadensis
0.028	-	3'-methyl-alpha-phenylalanine	mutant L104A, pH 8.5, 31°C	Taxus canadensis
0.03	-	4'-methyl-alpha-phenylalanine	wild-type, pH 8.5, 31°C	Taxus canadensis
0.053	-	L-phenylalanine	wild-type, pH 8.5, 31°C	Taxus canadensis

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.022	-	L-phenylalanine	mutant L104A, pH 8.5, 31°C	Taxus canadensis
0.025	-	styryl-alpha-alanine	mutant L104A, pH 8.5, 31°C	Taxus canadensis
0.055	-	3'-methyl-alpha-phenylalanine	wild-type, pH 8.5, 31°C	Taxus canadensis
0.27	-	4'-methyl-alpha-phenylalanine	mutant L104A, pH 8.5, 31°C	Taxus canadensis
0.33	-	4'-methyl-alpha-phenylalanine	wild-type, pH 8.5, 31°C	Taxus canadensis
0.34	-	3'-methyl-alpha-phenylalanine	mutant L104A, pH 8.5, 31°C	Taxus canadensis
0.93	-	L-phenylalanine	wild-type, pH 8.5, 31°C	Taxus canadensis