Phenylalanine meta‐hydroxylase: A single residue mediates mechanistic control of aromatic amino acid hydroxylation

Sabine Grüschow, Joanna C. Sadler, Peter J. Sharratt, Rebecca J. M. Goss

Research output: Contribution to journalArticlepeer-review


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.
Original languageEnglish
Early online date18 Jul 2019
Publication statusE-pub ahead of print - 18 Jul 2019


  • biocatalysis
  • enzyme mechanisms
  • hydroxylases
  • meta-tyrosine biosynthesis
  • 1,2-hydride (NIH) shift


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