PDBsum entry 4jpy

Oxidoreductase

PDB id: 4jpy

Contents

Protein chain

268 a.a.

Ligands

PHE

Metals

_FE

Waters ×25

PDB id:

4jpy

Name:

Oxidoreductase

Title:

Iron and phenylalanine bound crystal structure of phenylalanine hydroxylase from chromobacterium violaceum

Structure:

Phenylalanine-4-hydroxylase. Chain: a. Synonym: pah, phe-4-monooxygenase. Engineered: yes

Source:

Chromobacterium violaceum. Organism_taxid: 243365. Strain: atcc 12472 / dsm 30191 / jcm 1249 / nbrc 12614 / ncimb 9131 / nctc 9757. Gene: phha, cv_3180. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Resolution:

2.13Å R-factor: 0.206 R-free: 0.251

Authors:

J.A.Ronau,M.M.Abu-Omar,C.Das

Key ref:

J.A.Ronau et al. (2013). An additional substrate binding site in a bacterial phenylalanine hydroxylase. Eur Biophys J, 42, 691-708. PubMed id: 23860686 DOI: 10.1007/s00249-013-0919-8

Date:

19-Mar-13 Release date: 07-Aug-13

Protein chain

P30967  (PH4H_CHRVO) -  Phenylalanine-4-hydroxylase from Chromobacterium violaceum (strain ATCC 12472 / DSM 30191 / JCM 1249 / NBRC 12614 / NCIMB 9131 / NCTC 9757)

Seq: 297 a.a.

Struc: 268 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.1.14.16.1 - phenylalanine 4-monooxygenase.
• Pathway: Phenylalanine and Tyrosine Biosynthesis
• Reaction: (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin + L-phenylalanine + O2 = (4aS,6R)-4a-hydroxy-L-erythro-5,6,7,8-tetrahydrobiopterin + L-tyrosine

(6R)-L-erythro-5,6,7,8-tetrahydrobiopterin + L-phenylalanine (Bound ligand (Het Group name = PHE) corresponds exactly) + O2 = (4aS,6R)-4a-hydroxy-L-erythro-5,6,7,8-tetrahydrobiopterin + L-tyrosine

• Cofactor: Fe cation

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1007/s00249-013-0919-8

Eur Biophys J 42:691-708 (2013)

PubMed id: 23860686

An additional substrate binding site in a bacterial phenylalanine hydroxylase.

J.A.Ronau, L.N.Paul, J.E.Fuchs, I.R.Corn, K.T.Wagner, K.R.Liedl, M.M.Abu-Omar, C.Das.

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.