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Mapping the hydrophobic substrate binding site of phenylalanine ammonia lyase from petroselinum crispum

DOI: 10.1021/acscatal.9b02108 DOI Help

Authors: Emma Zsófia Aletta Nagy (University of Cluj-Napoca) , Souad Diana Tork (University of Cluj-Napoca) , Pauline A. Lang (University of Oxford) , Alina Filip (University of Cluj-Napoca) , Florin Dan Irimie (University of Cluj-Napoca) , László Poppe (Budapest University of Technology and Economics) , Monica Ioana Toşa (University of Cluj-Napoca) , Christopher J. Schofield (University of Oxford) , Jurgen Brem (University of Oxford) , Csaba Paizs (University of Cluj-Napoca) , Laszlo Csaba Bencze (University of Cluj-Napoca)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Catalysis

State: Published (Approved)
Published: August 2019
Diamond Proposal Number(s): 19458

Abstract: Modification of the hydrophobic binding pocket of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) enables increased activity and selectivity towards phenylalanines and cinnamic acids mono-substituted with both electron donating (-CH3, -OCH3) and electron withdrawing (-CF3, -Br) groups at all positions (o-, m-, p-) of their aromatic ring. The results reveal specific residues involved in accommodating substituents at o-, m-, p-positions of the substrate’s phenyl ring. The predicted interactions were validated by crystallographic analysis of the binding mode of para-methoxy cinnamic acid complexed at the active site of PcPAL. The biocatalytic utility of the tailored PcPAL mutants was demonstrated by the efficient preparative scale synthesis of (S)-m-bromo-phenylalanine (ee: > 99%, yield: 60%) and (R)-p-methyl-phenylalanine (ee: 97%, yield: 49%), using the corresponding ammonia addition and ammonia elimination reactions catalyzed by the L134A and I460V PcPAL variants, respectively. Overall, the results reveal the potential for structure based protein engineering of PALs to provide enzymes with enhanced catalytic properties and which are specifically tailored for differently substituted phenylalanine analogues of high synthetic value.

Journal Keywords: biocatalysis; protein engineering; phenylalanine ammonia-lyases; active site modification; D- and L-phenylalanine analogues

Subject Areas: Chemistry


Instruments: I03-Macromolecular Crystallography