title: Expanding the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum towards styrylalanines.
abstract: 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/KMvalue 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.
authors.affiliation: Biocatalysis and Biotransformation Research Centre, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University of Cluj-Napoca, Arany János Str. 11, RO-400028 Cluj-Napoca, Romania. paizs@chem.ubbcluj.ro.
authors.forename: László Csaba
authors.initials: LC
authors.lastname: Bencze
authors.affiliation: Biocatalysis and Biotransformation Research Centre, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University of Cluj-Napoca, Arany János Str. 11, RO-400028 Cluj-Napoca, Romania. paizs@chem.ubbcluj.ro.
authors.forename: Alina
authors.initials: A
authors.lastname: Filip
authors.affiliation: Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Műegyetem rkp. 3, H-1111 Budapest, Hungary. poppe@mail.bme.hu.
authors.forename: Gergely
authors.initials: G
authors.lastname: Bánóczi
authors.affiliation: Biocatalysis and Biotransformation Research Centre, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University of Cluj-Napoca, Arany János Str. 11, RO-400028 Cluj-Napoca, Romania. paizs@chem.ubbcluj.ro.
authors.forename: Monica Ioana
authors.initials: MI
authors.lastname: Toşa
authors.affiliation: Biocatalysis and Biotransformation Research Centre, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University of Cluj-Napoca, Arany János Str. 11, RO-400028 Cluj-Napoca, Romania. paizs@chem.ubbcluj.ro.
authors.forename: Florin Dan
authors.initials: FD
authors.lastname: Irimie
authors.affiliation: Agricultural Institute, Centre of Agricultural Research, Hungarian Academy of Sciences, Brunszvik u. 2, H-2462 Martonvásár, Hungary.
authors.forename: Ákos
authors.initials: Á
authors.lastname: Gellért
authors.affiliation: Biocatalysis and Biotransformation Research Centre, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University of Cluj-Napoca, Arany János Str. 11, RO-400028 Cluj-Napoca, Romania. paizs@chem.ubbcluj.ro and Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Műegyetem rkp. 3, H-1111 Budapest, Hungary. poppe@mail.bme.hu and SynBiocat Ltd, Lövőház u. 19/1, H-1024 Budapest, Hungary.
authors.forename: László
authors.identifier: http://orcid.org/0000-0002-9957-1203
authors.initials: L
authors.lastname: Poppe
authors.affiliation: Biocatalysis and Biotransformation Research Centre, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University of Cluj-Napoca, Arany János Str. 11, RO-400028 Cluj-Napoca, Romania. paizs@chem.ubbcluj.ro.
authors.forename: Csaba
authors.identifier: http://orcid.org/0000-0002-7403-7098
authors.initials: C
authors.lastname: Paizs
mesh_terms: D000409:Alanine / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D028528:Petroselinum / Q000201:enzymology*; D010650:Phenylalanine Ammonia-Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D010650:Phenylalanine Ammonia-Lyase; D000409:Alanine