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Phenylalanine meta ‐Hydroxylase: A Single Residue Mediates Mechanistic Control of Aromatic Amino Acid Hydroxylation
Author(s) -
Grüschow Sabine,
Sadler Joanna C.,
Sharratt Peter J.,
Goss Rebecca J. M.
Publication year - 2020
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201900320
Subject(s) - hydroxylation , chemistry , phenylalanine , stereochemistry , tyrosine , stereospecificity , deprotonation , enzyme , aromatic amino acids , amino acid , substrate (aquarium) , phenylalanine hydroxylase , electrophilic aromatic substitution , biocatalysis , biochemistry , organic chemistry , reaction mechanism , biology , catalysis , ion , ecology
The rare nonproteinogenic amino acid, meta ‐ l ‐tyrosine is biosynthetically intriguing. Whilst the biogenesis of tyrosine from phenylalanine is well characterised, the mechanistic basis for meta ‐hydroxylation is unknown. Herein, we report the analysis of 3‐hydroxylase (Phe3H) from Streptomyces coeruleorubidus . Insights from kinetic analyses of the wild‐type enzyme and key mutants as well as of the biocatalytic conversion of synthetic isotopically labelled substrates and fluorinated substrate analogues advance understanding of the process by which meta ‐hydroxylation is mediated, revealing T202 to play an important role. In the case of the WT enzyme, a deuterium label at the 3‐position is lost, whereas in in the T202A mutant 75 % retention is observed, with loss of stereospecificity. These data suggest that one of two possible mechanisms is at play; direct, enzyme‐catalysed deprotonation following electrophilic aromatic substitution or stereospecific loss of one proton after a 1,2‐hydride shift. Furthermore, our kinetic parameters for Phe3H show efficient regiospecific generation of meta ‐ l ‐tyrosine from phenylalanine and demonstrate the enzyme's ability to regiospecifically hydroxylate unnatural fluorinated substrates.