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

László Csaba Bencze, Alina Filip, Gergely Bánóczi, Monica Ioana Toşa, Florin Dan Irimie, Ákos Gellért, László Poppe, Csaba Paizs

Research output: Contribution to journalArticle

10 Citations (Scopus)


This study focuses on the expansion of the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) towards the l-enantiomers of racemic styrylalanines rac-1a-d-which are less studied and synthetically challenging unnatural amino acids-by reshaping the aromatic binding pocket of the active site of PcPAL by point mutations. Ammonia elimination from l-styrylalanine (l-1a) catalyzed by non-mutated PcPAL (wt-PcPAL) took place with a 777-fold lower kcat/KM value than the deamination of the natural substrate, l-Phe. Computer modeling of the reactions catalyzed by wt-PcPAL indicated an unproductive and two major catalytically active conformations and detrimental interactions between the aromatic moiety of l-styrylalanine, l-1a, and the phenyl ring of the residue F137 in the aromatic binding region of the active site. Replacing the residue F137 by smaller hydrophobic residues resulted in a small mutant library (F137X-PcPAL, X being V, A, and G), from which F137V-PcPAL could transform l-styrylalanine with comparable activity to that of the wt-PcPAL with l-Phe. Furthermore, F137V-PcPAL showed superior catalytic efficiency in the ammonia elimination reaction of several racemic styrylalanine derivatives (rac-1a-d) providing access to d-1a-d by kinetic resolution, even though the d-enantiomers proved to be reversible inhibitors. The enhanced catalytic efficiency of F137V-PcPAL towards racemic styrylalanines rac-1a-d could be rationalized by molecular modeling, indicating the more relaxed enzyme-substrate complexes and the promotion of conformations with higher catalytic activities as the main reasons. Unfortunately, ammonia addition onto the corresponding styrylacrylates 2a-d failed with both wt-PcPAL and F137V-PcPAL. The low equilibrium constant of the ammonia addition, the poor ligand binding affinities of 2a-d, and the non-productive binding states of the unsaturated ligands 2a-d within the active sites of either wt-PcPAL or F137V-PcPAL-as indicated by molecular modeling-might be responsible for the inactivity of the PcPAL variants in the reverse reaction. Modeling predicted that the F137V mutation is beneficial for the KRs of 4-fluoro-, 4-cyano- and 4-bromostyrylalanines, but non-effective for the KR process of 4-trifluoromethylstyrylalanine.

Original languageEnglish
Pages (from-to)3717-3727
Number of pages11
JournalOrganic and Biomolecular Chemistry
Issue number17
Publication statusPublished - Jan 1 2017

ASJC Scopus subject areas

  • Biochemistry
  • Physical and Theoretical Chemistry
  • Organic Chemistry

Fingerprint Dive into the research topics of 'Expanding the substrate scope of phenylalanine ammonia-lyase from: Petroselinum crispum towards styrylalanines'. Together they form a unique fingerprint.

  • Cite this

    Bencze, L. C., Filip, A., Bánóczi, G., Toşa, M. I., Irimie, F. D., Gellért, Á., Poppe, L., & Paizs, C. (2017). Expanding the substrate scope of phenylalanine ammonia-lyase from: Petroselinum crispum towards styrylalanines. Organic and Biomolecular Chemistry, 15(17), 3717-3727. https://doi.org/10.1039/c7ob00562h