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Phenylalanine aminomutase from Taxus chinensis (TchPAM) is employed in the biosynthesis of the widely used antitumor drug paclitaxel. TchPAM has received substantial attention due to its strict enantioselectivity towards (R)-β-phenylalanine, in contrast to the bacterial enzymes classified as EC 5.4.3.11 which are (S)-selective for this substrate. However, the understanding of the isomerization mechanism of the reorientation and rearrangement reactions in TchPAM might support and promote further research on expanding the scope of the substrate and thus the establishment of large-scale production of potential synthesis for drug development. Upon conservation analysis, computational simulation, and mutagenesis experiments, we report a mutant from TchPAM, which can catalyze the amination reaction of trans-p-hydroxycinnamic acid to (R)- and (S)-β-tyrosine. We propose a mechanism for the function of the highly conserved residues L179, N458, and Q459 in the active site of TchPAM. This work highlights the importance of the hydrophobic residues in the active site, including the residues L104, L108, and I431, for maintaining the strict enantioselectivity of TchPAM, and the importance of these residues for substrate specificity and activation by altering the substrate binding position or varying the location of neighboring residues. Furthermore, an explanation of (R)-selectivity in TchPAM is proposed based on the mutagenesis study of these hydrophobic residues. In summary, these studies support the future exploitation of the rational engineering of corresponding enzymes with MIO moiety (3,5-dihydro-5-methylidene-4H-imidazole-4-one) such as ammonia lyases and aminomutases of aromatic amino acids. |
