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An additional substrate binding site in a bacterial phenylalanine hydroxylase

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Abstract

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.

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Abbreviations

PAH:

Phenylalanine hydroxylase

BH4 :

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

PKU:

Phenylketonuria

cPAH:

Phenylalanine hydroxylase from Chromobacterium violaceum

hPAH:

Human phenylalanine hydroxylase

RMSD:

Root-mean-square deviation

THA:

3-(2-thienyl)-l-alanine

NLE:

l-Norleucine

Fe:

Iron

Co:

Cobalt

DMPH4 :

6,7-Dimethyltetrahydropterin

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Acknowledgments

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.).

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Authors and Affiliations

  1. Brown Laboratory of Chemistry, Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN, 47907, USA

    Judith A. Ronau, Isaac R. Corn, Kyle T. Wagner, Mahdi M. Abu-Omar & Chittaranjan Das

  2. Bindley Biosciences Center, Purdue University, West Lafayette, IN, 47907, USA

    Lake N. Paul

  3. Institute of General, Inorganic and Theoretical Chemistry, and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80/82, 6020, Innsbruck, Austria

    Julian E. Fuchs & Klaus R. Liedl

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  1. Judith A. Ronau
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Correspondence to Chittaranjan Das.

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Ronau, J.A., Paul, L.N., Fuchs, J.E. et al. An additional substrate binding site in a bacterial phenylalanine hydroxylase. Eur Biophys J 42, 691–708 (2013). https://doi.org/10.1007/s00249-013-0919-8

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