European Biophysics Journal

, Volume 42, Issue 9, pp 691–708 | Cite as

An additional substrate binding site in a bacterial phenylalanine hydroxylase

  • Judith A. Ronau
  • Lake N. Paul
  • Julian E. Fuchs
  • Isaac R. Corn
  • Kyle T. Wagner
  • Klaus R. Liedl
  • Mahdi M. Abu-Omar
  • Chittaranjan DasEmail author
Original Paper


Phenylalanine hydroxylase (PAH) is a non-heme iron enzyme that catalyzes oxidation of phenylalanine to tyrosine, a reaction that must be kept under tight regulatory control. Mammalian PAH has a regulatory domain in which binding of the substrate leads to allosteric activation of the enzyme. However, the existence of PAH regulation in evolutionarily distant organisms, for example some bacteria in which it occurs, has so far been underappreciated. In an attempt to crystallographically characterize substrate binding by PAH from Chromobacterium violaceum, a single-domain monomeric enzyme, electron density for phenylalanine was observed at a distal site 15.7 Å from the active site. Isothermal titration calorimetry (ITC) experiments revealed a dissociation constant of 24 ± 1.1 μM for phenylalanine. Under the same conditions, ITC revealed no detectable binding for alanine, tyrosine, or isoleucine, indicating the distal site may be selective for phenylalanine. Point mutations of amino acid residues in the distal site that contact phenylalanine (F258A, Y155A, T254A) led to impaired binding, consistent with the presence of distal site binding in solution. Although kinetic analysis revealed that the distal site mutants suffer discernible loss of their catalytic activity, X-ray crystallographic analysis of Y155A and F258A, the two mutants with the most noticeable decrease in activity, revealed no discernible change in the structure of their active sites, suggesting that the effect of distal binding may result from protein dynamics in solution.


Phenylalanine hydroxylase Substrate affinity Distal substrate binding site Regulation of enzyme activity 



Phenylalanine hydroxylase


(6R)-5,6,7,8-tetrahydrobiopterin dihydrochloride




Phenylalanine hydroxylase from Chromobacterium violaceum


Human phenylalanine hydroxylase


Root-mean-square deviation













We are grateful to our hosts Michael Becker, Craig Ogata, Nukri Sanishvili, and Nagarajan Venugopalan at the GM/CA CAT beamline 23-ID-D at the Advanced Photon Source, Argonne National Laboratory. Use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the US DOE under contract no. DE-AC02-06CH11357. Julian E. Fuchs is a recipient of a DOC fellowship of the Austrian Academy of Sciences at the University of Innsbruck. The authors acknowledge the High Performance Computing at the University of Innsbruck for providing computer hardware resources for this project. This research was funded by the National Science Foundation (CHE-0749572 to M.M.A.O.) and the National Institutes of Health (1R01RR026273 to C.D.).

Supplementary material

249_2013_919_MOESM1_ESM.docx (11.5 mb)
Supplementary material 1 (DOCX 11777 kb)


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Copyright information

© European Biophysical Societies' Association 2013

Authors and Affiliations

  • Judith A. Ronau
    • 1
  • Lake N. Paul
    • 2
  • Julian E. Fuchs
    • 3
  • Isaac R. Corn
    • 1
  • Kyle T. Wagner
    • 1
  • Klaus R. Liedl
    • 3
  • Mahdi M. Abu-Omar
    • 1
  • Chittaranjan Das
    • 1
    Email author
  1. 1.Brown Laboratory of Chemistry, Department of ChemistryPurdue UniversityWest LafayetteUSA
  2. 2.Bindley Biosciences CenterPurdue UniversityWest LafayetteUSA
  3. 3.Institute of General, Inorganic and Theoretical Chemistry, and Center for Molecular Biosciences Innsbruck (CMBI)University of InnsbruckInnsbruckAustria

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