Structural characterization of the N-terminal autoregulatory sequence of phenylalanine hydroxylase

James Horne, Ian G. Jennings, Trazel Teh, Paul R. Gooley, Bostjan Kobe

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13 Citations (Scopus)

Abstract

Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine, and through phosphorylation by cAMP-dependent protein kinase at Serl 16 in the N-terminal autoregulatory sequence of the enzyme. The crystal structures of phosphorylated and unphosphorylated forms of the enzyme showed that, in the absence of phenylalanine, in both cases the N-terminal 18 residues including the phosphorylation site contained no interpretable electron density. We used nuclear magnetic resonance (NMR) spectroscopy to characterize this N-terminal region of the molecule in different stages of the regulatory pathway. A number of sharp resonances are observed in PAH with an intact N-terminal region, but no sharp resonances are present in a truncation mutant lacking the N-terminal 29 residues. The N-terminal sequence therefore represents a mobile flexible region of the molecule. The resonances become weaker after the addition of phenylalanine, indicating a loss of mobility. The peptides corresponding to residues 2-20 of PAH have different structural characteristics in the phosphorylated and unphosphorylated forms, with the former showing increased secondary structure. Our results support the model whereby upon phenylalanine binding, the mobile N-terminal 18 residues of PAH associate with the folded core of the molecule; phosphorylation may facilitate this interaction.

Original languageEnglish
Pages (from-to)2041-2047
Number of pages7
JournalProtein Science
Volume11
Issue number8
DOIs
Publication statusPublished - 30 Jul 2002
Externally publishedYes

Keywords

  • Autoregulatory sequence
  • Mutagenesis
  • Nuclear magnetic resonance (NMR)
  • Phenylalanine hydroxylase
  • Phosphorylation

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