4JPY: Iron And Phenylalanine Bound Crystal Structure Of Phenylalanine Hydroxylase From Chromobacterium Violaceum

Citation:
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 A from the active site. Isothermal titration calorimetry (ITC) experiments revealed a dissociation constant of 24 +/- 1.1 muM 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.
PDB ID: 4JPYDownload
MMDB ID: 112466
PDB Deposition Date: 2013/3/19
Updated in MMDB: 2013/08
Experimental Method:
x-ray diffraction
Resolution: 2.13  Å
Source Organism:
Similar Structures:
Biological Unit for 4JPY: monomeric; determined by author
Molecular Components in 4JPY
Label Count Molecule
Protein (1 molecule)
1
Phenylalanine-4-hydroxylase(Gene symbol: CV_RS15610)
Molecule annotation
Chemicals (2 molecules)
1
1
2
1
* Click molecule labels to explore molecular sequence information.

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