(S)-Styryl-α-alanine Used To Probe the Intermolecular Mechanism of an Intramolecular MIO-Aminomutase

Abstract

A Taxus canadensis phenylalanine aminomutase (TcPAM) catalyzes the isomerization of (S)-α- to (R)-β-phenylalanine, making (E)-cinnamate (~10%) as a byproduct at steady state. A currently accepted mechanism for TcPAM suggests that the amino group is transferred from the substrate to a prosthetic group comprised of an amino acid triad in the active site and then principally rebinds to the carbon skeleton of the cinnamate intermediate to complete the α-β isomerization. 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 (k(cat) 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 (K(I)) for selected acrylates ranging from 0.6 to 106 μM were obtained, and each had a lower K(I) 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 K(I) 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.

Expanding the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum towards styrylalanines

“…It was already shown that phenylalanine 2,3-aminomutase from Taxus canadensis (TcaPAM) could transform (S)α-styrylalanine (L-1a) into (2E,4E)-5-phenylpenta-2,4-dienoic acid (2a) as the major product. 28 Considering the high structural and sequence similarities between TcaPAM and PcPAL, it seemed commendable to explore the potential of PcPAL in the kinetic resolution of α-styrylalanines and stereoselective amination of (2E,4E)-styrylacrylates in the reverse reaction (Scheme 1).…”

A new member of the 4-methylideneimidazole-5-one–containing aminomutase family from the enediyne kedarcidin biosynthetic pathway

“…Mechanistic and structural characterizations of the MIO-containing aminomutases as a family have unveiled much unique chemistry, enzymology, and structural biology (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), and exploitation of these enzymes as biocatalysts has provided access to α-and β-amino acids, which are difficult to prepare by other means (4,37). However, ʟ-phenylalanine and ʟ-tyrosine remain the only two known natural substrates (3)(4)(5).…”

“…The MIO-containing aminomutases have been exploited as biocatalysts to generate novel α-and β-amino acids (8,37). SgcC4 exhibits substrate promiscuity, including L-dopa and L-3-chlorotyrosine, albeit with significantly reduced activity (8).…”

“…SgcC4 exhibits substrate promiscuity, including L-dopa and L-3-chlorotyrosine, albeit with significantly reduced activity (8). The PAMs have been demonstrated to accept a broad range of substrate analogs (37). Although KedY4 has shown no activity toward L-tyrosine and 2-aza-L-phenylalanine, the limited availability of other pyridine-containing L-α-amino acids has precluded testing of KedY4 substrate promiscuity; the similar kinetic parameters of KedY4 and SgcC4 would support such an exploration.…”

Kinetic Resolution of Aromatic β‐Amino Acids Using a Combination of Phenylalanine Ammonia Lyase and Aminomutase Biocatalysts

“…[2][3][4] A PAM-like enzyme is also proposed to form part of the biosynthesis of the fungal toxin cyclochlorotine in Talaromyces islandisum based on identification of a candidate predicted open reading frame within the biosynthetic gene cluster. [5] The lack of characterisation with this class of enzyme is surprising given the detailed structural and biochemical knowledge of known enzymes [6][7][8][9][10] and the wealth of organisms shown to produce βphenylalanine-containing compounds. [2,[11][12][13][14] Progress in this area is likely hindered by the poor quality of enzyme sequence annotations in biological databases a fact which itself precludes correct identification biosynthetic pathways for known and novel bioactive molecules applicable to medical research.…”