Phenylalanine Ammonia Lyase Catalyzed Synthesis of Amino Acids by an MIO-Cofactor Independent Pathway

Authors

  • Sarah L. Lovelock,

    1. School of Chemistry, University of Manchester, Manchester Institute of Biotechnology, 131 Princess Street, Manchester, M1 7DN (UK)
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  • Dr. Richard C. Lloyd,

    1. Dr. Reddy's Laboratories, Chirotech Technology Centre, 410 Cambridge Science Park, Milton Road, Cambridge, CB4 0PE (UK)
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  • Prof. Nicholas J. Turner

    Corresponding author
    1. School of Chemistry, University of Manchester, Manchester Institute of Biotechnology, 131 Princess Street, Manchester, M1 7DN (UK)
    • School of Chemistry, University of Manchester, Manchester Institute of Biotechnology, 131 Princess Street, Manchester, M1 7DN (UK)===

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  • Financial support from the Centre of Excellence in Biocatalysis, Biotransformation and Biocatalytic Manufacture (CoEBio3: www.coebio3.org) and a Royal Society Wolfson Research Merit Award are gratefully acknowledged.

Abstract

Phenylalanine ammonia lyases (PALs) belong to a family of 4-methylideneimidazole-5-one (MIO) cofactor dependent enzymes which are responsible for the conversion of L-phenylalanine into trans-cinnamic acid in eukaryotic and prokaryotic organisms. Under conditions of high ammonia concentration, this deamination reaction is reversible and hence there is considerable interest in the development of PALs as biocatalysts for the enantioselective synthesis of non-natural amino acids. Herein the discovery of a previously unobserved competing MIO-independent reaction pathway, which proceeds in a non-stereoselective manner and results in the generation of both L- and D-phenylalanine derivatives, is described. The mechanism of the MIO-independent pathway is explored through isotopic-labeling studies and mutagenesis of key active-site residues. The results obtained are consistent with amino acid deamination occurring by a stepwise E1cB elimination mechanism.

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