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. 2011 Jun 21;50(24):5401-3.
doi: 10.1021/bi200733c. Epub 2011 May 31.

Identification of phenylalanine 3-hydroxylase for meta-tyrosine biosynthesis

Wenjun Zhang  1 , Brian D Ames, Christopher T Walsh
Affiliations

Identification of phenylalanine 3-hydroxylase for meta-tyrosine biosynthesis

Wenjun Zhang et al. Biochemistry. .

Abstract

Phenylalanine hydroxylase (PheH) is an iron(II)-dependent enzyme that catalyzes the hydroxylation of aromatic amino acid l-phenylalanine (L-Phe) to l-tyrosine (L-Tyr). The enzymatic modification has been demonstrated to be highly regiospecific, forming proteinogenic para-Tyr (p-Tyr) exclusively. Here we biochemically characterized the first example of a phenylalanine 3-hydroxylase (Phe3H) that catalyzes the synthesis of meta-Tyr (m-Tyr) from Phe. Subsequent mutagenesis studies revealed that two residues in the active site of Phe3H (Cys187 and Thr202) contribute to C-3 rather than C-4 hydroxylation of the phenyl ring. This work sets the stage for the mechanistic and structural study of regiospecific control of the substrate hydroxylation by PheH.

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Figures

Figure 1
Figure 1
Examples of m-Tyr (highlighted in red) containing bacterial secondary metabolites.
Figure 2
Figure 2
Characterization of PacX as a phenylalanine-3-hydroxylase. a) Schematic of PheH catalyzed reactions. b) Fluorescence chromatograms (λex 270 nm and λem 310 nm) showing PacX catalyzed production of m-Tyr with various control reactions omitting one of the components indicated. c) Determination of PacX kinetic parameters by HPLC assays monitoring the formation of m-Tyr. Parameters were determined for l-Phe (top panel) by fixing the 6-MePH4 concentration at 0.5 mM, while parameters were determined for 6-MePH4 (bottom panel) by fixing the l-Phe concentration at 5 mM.
Figure 3
Figure 3
Mutagenesis studies on the regiospecific hydroxylation control of PacX. a) Fluorescence chromatograms showing production of m-Tyr and p-Tyr catalyzed by wild-type PacX and mutants. b) Three-dimensional structural model of PacX active site (Phe3H). The structure of PacX was modeled based on Phe4H (PDB 1KW0) (23) using the online program SWISS-MODEL. The five residues that form the hydrophobic cage around the aromatic side chain of l-Phe are underlined.
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