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.

The active site residue tyrosine 325 influences iron binding and coupling efficiency in human phenylalanine hydroxylase

“…The specific activities for the enzyme forms prior to and after preincubation with L-Phe are summarized in Table 2. As expected [9], Y325F showed indistinguishable properties to wt-PAH. On the other hand, the mutant Y325L appears to be appropriate to investigate the early suspected role of Tyr325 on the structure and function of the enzyme [4].…”

“…Despite the early suspected role of Tyr325 in the function of PAH [32], the conservative mutation to Phe is not appropriate to investigate the functional and structural importance of this residue due to the reversal of the mutant back to wt-PAH by posttranslational hydroxylation [9]. Accordingly, the mutant Y325F prepared in this work in full-length PAH showed indistinguishable properties to wt-PAH.…”

“…Notably, the conservative mutation Tyr to Phe resulted in enzymes of similar activity and affinity for the natural substrates. This intriguing result was explained by Kinzie et al showing that the Y325F mutant in human PAH was rescued by self-hydroxylation [9]. Posttranslational self-hydroxylation reactions occur in several non-heme iron enzymes (for review see [10]).…”

Aberrant activity of the DNA repair enzyme AlkB

“…In addition to the hydroxylation of a Trp side chain noted above for AlkB and previously described for TfdA [10], a tyrosine residue is hydroxylated to form a catecholate derivative in TauD [11,12] and phenylalanine hydroxylation has been noted in both tyrosine hydroxylase [20,21] and the Y325F variant of phenylalanine hydroxylase [22]. Furthermore, both tyrosine and phenylalanine hydroxylation have been observed in various mutants of the R2 subunit of ribonucleotide reductase, a dinuclear iron protein [23][24][25][26].…”

Altering Conserved Lipid Binding Sites in Cytochrome c Oxidase of Rhodobacter sphaeroides Perturbs the Interaction between Subunits I and III and Promotes Suicide Inactivation of the Enzyme

“…Slow proton uptake by the D pathway increases the probability of suicide inactivation at the O 2 reduction site, apparently because the slow arrival of protons extends the lifetime of the reactive oxoferryl intermediate (Fe 4+ dO 2-) and increases the chances that it may hydroxylate nearby residues (14). Such side reactions by oxoferryl intermediates have been documented in other proteins (45)(46)(47). The removal of subunit III directly impacts the D pathway, slowing the rate of proton uptake 20-fold at pH 7.4 (34).…”