title: Posttranslational hydroxylation of human phenylalanine hydroxylase is a novel example of enzyme self-repair within the second coordination sphere of catalytic iron.
abstract: Phenylalanine hydroxylase, a mononuclear non-heme iron enzyme, catalyzes the hydroxylation of phenylalanine to tyrosine in the presence of oxygen and reduced pterin cofactor. X-ray structural studies have established the coordination around the iron metal center and point to significant interactions within the second coordination sphere. One such interaction involves Tyr325 in human phenylalanine hydroxylase (hPAH), which forms a hydrogen-bonding network with an aqua ligand on iron and the pterin cofactor. The full-length tetramer (1-452) and truncated dimer (117-424) Tyr325Phe hPAH mutant enzymes showed similar kinetics, thermal stabilities, and oligomerization profiles as their corresponding wild-type proteins. The possibility of in vivo posttranslational hydroxylation that would restore the activity of hPAH was examined by mass spectrometry on the trypsin digested full-length (1-452) hPAH Tyr325Phe point mutant. The amino acid tags obtained by ESI-MS/MS confirmed the presence of a Phe325 in the peptide corresponding to the doubly charged precursor ion at m/z 916.4 (L A T I F W F T V E F G L C K), and its hydroxylated counterpart in the peptide corresponding to the m/z 924.4 (L A T I F-OH W F T V E F G L C K) byproduct ion series comprising the fragments y(5)-y(12). Furthermore, the point mutation Tyr325Ala resulted in an enzyme that was totally inactive and did not display any evidence of hydroxylation. These results demonstrate the importance of Tyr325 for proper conformation of the active site, substrate binding, and catalysis. The rescue of the Tyr325Phe mutant in hPAH via self-hydroxylation presents a novel example of oxidative repair on the molecular level.
authors.affiliation: Department of Chemistry, University of California, Los Angeles, California 90095-1569, USA.
authors.forename: Sylvia Daoud
authors.initials: SD
authors.lastname: Kinzie
authors.forename: Mario
authors.initials: M
authors.lastname: Thevis
authors.forename: Khanh
authors.initials: K
authors.lastname: Ngo
authors.forename: Julian
authors.initials: J
authors.lastname: Whitelegge
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Loo
authors.forename: Mahdi M
authors.initials: MM
authors.lastname: Abu-Omar
mesh_terms: D000595:Amino Acid Sequence; D004795:Enzyme Stability; D006801:Humans; D006860:Hydrogen Bonding; D006900:Hydroxylation; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019159:Nonheme Iron Proteins / Q000737:chemistry / Q000378:metabolism*; D010651:Phenylalanine Hydroxylase / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational
chemical_list: D019159:Nonheme Iron Proteins; D010651:Phenylalanine Hydroxylase