Structural Investigations into the Stereochemistry and Activity of a Phenylalanine-2,3-aminomutase from Taxus chinensis

Abstract

Phenylalanine-2,3-aminomutase (PAM) from Taxus chinensis, a 4-methylidene-imidazole-5-one (MIO)-dependent enzyme, catalyzes the reversible conversion of (S)-α-phenylalanine into (R)-β-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 β-amino acids, which occur as frequent constituents of bioactive compounds. Different hypotheses have been formulated to explain the stereochemistry of the PAM-catalyzed reaction, but structural evidence for these hypotheses is lacking. Furthermore, it remains unclear how the PAM MIO group is formed from the three-amino acid (A-S-G) sequence motif. For these reasons, we elucidated PAM three-dimensional (3D) structures with a bound (R)-β-phenylalanine analogue and with bound trans-cinnamic acid. In addition, 3D structures of the (inactive) Y322A and N231A mutants of PAM were elucidated, which were found to be MIO-less. We conclude that 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 results also suggest that the N231 side chain promotes MIO group formation by increasing the nucleophilicity of the G177 N atom through acidification of the amide proton.

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

“…These findings agree with the experimental results found in X-ray structure of a TchPAM mutant (PDB ID: 4CQ5). 22 In the structure of this enzyme homologous to PcPAL, a continuous electron density between R354 and N384 and the carboxylate of the ligand was observed in the active site complexed with (E)-cinnamic acid and a water which was consistent with multiple ligand poses in a similar arrangement to that found in our models for 2a in PcPAL.…”

“…Numerous lines of evidence suggest that interactions between the amino group of the substrate L-Phe and the electrophilic MIO of PAL facilitate the ammonia-lyase reaction. 20 The crystal structures of phenylalanine 2,3-aminomutases from Pantoea agglomerans (PaPAM) complexed with L-phenylalanine 21 and from Taxus chinensis (TchPAM) complexed with L-α,α-difluoro-β-phenylalanine, 22 and the tyrosine 2,3-aminomutase from Streptomyces globisporus (SgTAM) cocrystallized with L-α,α-difluoro-β-tyrosine, 23 all with a covalent bond between the amino group of the substrate/inhibitor and the exocyclic methylene carbon of the MIO, supported a reaction mechanism proceeding through the so called N-MIO intermediate. This is also in agreement with QM/MM calculations on TAL 24 and PAL 19 supporting ammonia elimination via an N-MIO intermediate and suggesting the formation of similar N-MIO complexes as a common feature of the mechanisms for all MIO-enzymes.…”

Phenylalanine Ammonia‐Lyase‐Catalyzed Deamination of an Acyclic Amino Acid: Enzyme Mechanistic Studies Aided by a Novel Microreactor Filled with Magnetic Nanoparticles

“…X-Ray structural studies on various MIO enzymesr evealed that several substrates and substrate analoguesb ind covalently at their N-atoms to the MIO group in the 2-aminoindan-2-phosphonate in the RsTALm utant H89F, [12] (3R)-3-amino-2,2-difluoro-3-(4-hydroxyphenyl)propanoic acid and (3R)-3-amino-2,2-difluoro-3-(4-methoxyphenyl)propanoic acid in Streptomyces globisporus TAM, [43] l-Tyr in the Y63F (Tyr A ) mutant of SgTAM, [44] and (S)-3-amino-2,2-difluoro-3-phenylpropanoica cid in Taxus canadensis PAM. [45] Our detailed QM/MM calculations also indicate quite similar N-MIO intermediate structures for the reaction of PALw ith l-propargylglycine and with l-phenylalanine (Figures 2a nd S16).…”

Zymophore identification enables the discovery of novel phenylalanine ammonia lyase enzymes

“…These distal residues could coordinate breathing motions across the entire enzyme structure which may direct the malleability of the active site and modulate substrate accommodation. This has already been speculated within this class of enzymes, with the discovery that active site residues alone cannot discriminate closely related ammonia lyase and aminomutase enzymes in plants 53 . Although protein dynamics are known to influence enzyme activity 48 , 49 , there is little evidence of their consideration in biocatalyst or biotherapeutic research and development.…”