β-Amino acids are biologically active compounds of interest in medicinal chemistry, which are used as precursors for the biosynthesis of several biologically active compounds such as taxol, andrimid, chondromides and C-1027. Also they are used as important precursors for the synthesis of β-lactams and β-peptides. A class I lyase-like family of aminomutases isomerizes (S)-α-arylalanines to the corresponding β-amino acids by exchange of the NH2/H pair. This family uses a 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) group within the active site to initiate the reaction. The structures of a phenylalanine aminomutases from Taxus canadensis (TcPAM) and from Pantoea agglomerans (PaPAM) have been determined at 2.4 Å and 1.7 Å resolution, respectively. TcPAM isomerizes (S)-α-phenylalanine to the (R)-β-isomer, and PaPAM isomerizes (S)-α-phenylalanine to the (S)-β-isomer by exchange of the NH2/H pair. Both enzymes share similar tertiary- and quaternary-structures and have several highly conserved aliphatic residues positioned analogously in their active sites for substrate recognition. The active site of the TcPAM structure was solved in complex with (E)-cinnamate, which functions as both a substrate and an intermediate. The structure of PaPAM was solved as α- and β-phenylpropanoid adducts, presumably with (S)-α- and (S)-β-phenylalanine. These covalent intermediates provide strong evidence that PaPAM reacts by an alkylamine elimination pathway (Hoffmann-type or E2 elimination process), which involves covalent attachment between MIO cofactor and the amino group of the substrate or the product. The results indicate that the conformation of the carbon skeleton of the (S)-phenylalanine substrate remains as one rotamer after elimination of the NH2/H pair. It is presumed that the the exocyclic C-N bond of the amine-MIO adduct rotates into position below the α- and β-carbon atoms of the cinnamate intermediate to complete the isomerization reaction. Thus, the X-ray crystal structure of PaPAM also confirms the inversion of configuration at each migration terminus during the isomerization of the α-amino acid substrate into its β-isomer.To account for the distinct (3R)-β-amino acid stereochemistry catalyzed by TcPAM, the cinnamate skeleton must rotate the C1−Cα and Cβ−Cipso 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 PaPAM reaction. Moreover, the aromatic ring of the intermediate makes only one direct hydrophobic interaction with Leu-104. A L104A mutant of TcPAM demonstrated a ~ 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 wild type TcPAM. These parameters did not change significantly for the mutant with 4′-methyl-α-phenylalanine when compared to those for TcPAM.TcPAM catalyzes the isomerization of (S)-α- to (R)-β-phenylalanine, making (E)-cinnamate (~10%) as a by-product at steady state. In contrast, when (S)-styryl-α-alanine is used as a substrate, TcPAM produces (2E,4E)-styrylacrylate as the major product (>99%) and (R)-styryl-β-alanine (<1%). Comparison of the rates of conversion of the natural substrate (S)-α-phenylalanine and (S)-styryl-α-alanine to their corresponding products (kcat values of 0.053 ± 0.001 and 0.082 ± 0.002 s-1, respectively) catalyzed by TcPAM suggests that the amino group resides in the active site longer than styrylacrylate. To demonstrate this principle, inhibition constants (KI) for selected acrylates ranging from 0.6 to 106 μM were obtained, and each had a lower KI compared to that of (2E,4E)-styrylacrylate (337 ± 12 μM). Evaluation of the inhibition constants and the rates at which both the α/β-amino acids (between 7 and 80% yield) and styrylacrylate were made from a corresponding arylacrylate and styryl-α-alanine, respectively, by TcPAM catalysis revealed that the reaction progress was largely dependent on the KI of the acrylate. Bicyclic amino donor substrates also transferred their amino groups to an arylacrylate, demonstrating for the first time that ring-fused amino acids are productive substrates in the TcPAM-catalyzed reaction.Burst-phase kinetic analysis was used to evaluate the deamination rate of the aminated-methylidene imidazolone (NH2-MIO) adduct of TcPAM. The kinetic parameters were interrogated by a non-natural substrate (S)-styryl-α-alanine that yielded a chromophoric styrylacrylate product upon deamination by the aminomutase. Transient inactivation of the enzyme by the NH2-MIO adduct intermediate resulted in an initial burst of product, with reactivation by deamination of the adduct. This study validated the rate constants of a kinetic model, demonstrating that the NH2-MIO adduct and cinnamate intermediate are sufficiently retained in the active site to catalyze the natural α- to β-phenylalanine isomerization.The cryptic stereochemistry of the TcPAM and PaPAM has been evaluated. TcPAM retains the stereochemistry at Cα and Cβ during the isomerization, while PaPAM inverts the configurationat these migration termini. However, the stereochemical course is not completely understood for the tyrosine aminomutase (TAM) reactions. To further understand the overall mechanism of the MIO-based aminomutases, the cryptic stereochemistry of the CcTAM mechanism was studied. (2S,3S)-[2,3-2H2]- and (2S,3R)-[3-2H]-α-tyrosine were stereoselectively synthesized from unlabeled (or [2H]-labeled) (4'-hydroxyphenyl)acrylic acids by reduction with D2 (or H2) gas and a chiral Rh-Prophos catalyst. GC-MS analysis of the [2H]-β-tyrosine biosynthesized by CcTAM informed that the α-amino group was transferred to Cβ of the phenylpropanoid skeleton with retention of configuration. These labeled substrates also identified that the pro-(3S) proton exchanges with protons from the bulk media during its migration to Cα during catalysis. 1H- and 2H-NMR analyses of the [2H]-β-tyrosine derived from (2S)-[3,3-2H2]-α-tyrosine by CcTAM catalysis showed that the migratory proton attached to Cα of the product also with retention of configuration. CcTAM is stereoselective for (R)-β-tyrosine (85%), yet also forms the (S)-β-tyrosine enantiomer (15%) through inversion of configuration at both Cα and Cβ, as described herein. The proportion of the (S)-β-isomer made by CcTAM during steady state interestingly increased with solvent pH, and this effect on the proposed reaction mechanism is also discussed
Read
