479064-87-4Relevant articles and documents
The bacterial ammonia lyase EncP: A tunable biocatalyst for the synthesis of unnatural amino acids
Weise, Nicholas J.,Parmeggiani, Fabio,Ahmed, Syed T.,Turner, Nicholas J.
supporting information, p. 12977 - 12983 (2015/10/28)
Enzymes of the class I lyase-like family catalyze the asymmetric addition of ammonia to arylacrylates, yielding high value amino acids as products. Recent examples include the use of phenylalanine ammonia lyases (PALs), either alone or as a gateway to deracemization cascades (giving (S)- or (R)-α-phenylalanine derivatives, respectively), and also eukaryotic phenylalanine aminomutases (PAMs) for the synthesis of the (R)-β-products. Herein, we present the investigation of another family member, EncP from Streptomyces maritimus, thereby expanding the biocatalytic toolbox and enabling the production of the missing (S)-β-isomer. EncP was found to convert a range of arylacrylates to a mixture of (S)-α- and (S)-β-arylalanines, with regioselectivity correlating to the strength of electron-withdrawing/-donating groups on the ring of each substrate. The low regioselectivity of the wild-type enzyme was addressed via structure-based rational design to generate three variants with altered preference for either α- or β-products. By examining various biocatalyst/substrate combinations, it was demonstrated that the amination pattern of the reaction could be tuned to achieve selectivities between 99:1 and 1:99 for β:α-product ratios as desired.
Mechanistic, mutational, and structural evaluation of a taxus phenylalanine aminomutase
Feng, Lei,Wanninayake, Udayanga,Strom, Susan,Geiger, James,Walker, Kevin D.
experimental part, p. 2919 - 2930 (2012/07/14)
The structure of a phenylalanine aminomutase (TcPAM) from Taxus canadensis has been determined at 2.4 A resolution. The active site of the TcPAM contains the signature 4-methylidene-1H-imidazol-5(4H)-one prosthesis, observed in all catalysts of the class I lyase-like family. This catalyst isomerizes (S)-α-phenylalanine to the (R)-β-isomer by exchange of the NH 2/H pair. The stereochemistry of the TcPAM reaction product is opposite of the (S)-β-tyrosine made by the mechanistically related tyrosine aminomutase (SgTAM) from Streptomyces globisporus. Since TcPAM and SgTAM share similar tertiary- and quaternary-structures and have several highly conserved aliphatic residues positioned analogously in their active sites for substrate recognition, the divergent product stereochemistries of these catalysts likely cannot be explained by differences in active site architecture. The active site of the TcPAM structure also is in complex with (E)-cinnamate; the latter functions as both a substrate and an intermediate. To account for the distinct (3R)-β-amino acid stereochemistry catalyzed by TcPAM, the cinnamate skeleton must rotate the C1-Cα and C ipso-β bonds 180° in the active site prior to exchange and rebinding of the NH2/H pair to the cinnamate, an event that is not required for the corresponding acrylate intermediate in the SgTAM reaction. Moreover, the aromatic ring of the intermediate makes only one direct hydrophobic interaction with Leu-104. A L104A mutant of TcPAM demonstrated an ～1.5-fold increase in kcat and a decrease in KM values for sterically demanding 3′-methyl-α-phenylalanine and styryl-α-alanine substrates, compared to the kinetic parameters for TcPAM. These parameters did not change significantly for the mutant with 4′-methyl-α-phenylalanine compared to those for TcPAM.(Figure Presented)
Enhanced conversion of racemic α-arylalanines to (R)-β- arylalanines by coupled racemase/aminomutase catalysis
Cox, Brad M.,Bilsborrow, Joshua B.,Walker, Kevin D.
experimental part, p. 6953 - 6959 (2009/12/25)
(Graph Presented) The Taxus phenylalanine aminomutase (PAM) enzyme converts several (S)-α-arylalanines to their corresponding (R)-β- arylalanines. After incubating various racemic substrateswith 100 μg of PAM for 20 h at 31°C, each (S)-α-arylalanine was enantioselectively isomerized to its corresponding (R)-β-product. With racemic starting materials, the ratio of (R)-β-arylalanine product to the (S)-α-substrate ranged between 0.4 and 1.8, and the remaining nonproductive (R)-α-arylalanine became enriched. To utilize the (R)-α-isomer, the catalysis of a promiscuous alanine racemase from Pseudomonas putida (KT2440) was coupled with that of PAM to increase the production of enantiopure (R)-β-arylalanines from racemic α-arylalanine substrates. The inclusion of a biocatalytic racemization along with the PAM-catalyzed reactionmoderately increased the overall reaction yield of enantiopure β-arylalanines between 4% and 19% (depending on the arylalanine), which corresponded to as much as a 63% increase compared to the turnover with the aminomutase reaction alone. The use of these biocatalysts, in tandem, could potentially find application in the production of chiral β-arylalanine building blocks, particularly, as refinements to the process are made that increase reaction flux, such as by selectively removing the desired (R)-β-arylalanine product from the reaction mixture. 2009 American Chemical Society.
