Characterization of metal ligand mutants of phenylalanine hydroxylase: Insights into the plasticity of a 2-histidine-1-carboxylate triad

Abstract

The iron atom in the nonheme iron monooxygenase phenylalanine hydroxylase is bound on one face by His285, His290, and Glu330. This arrangement of metal ligands is conserved in the other aromatic amino acid hydroxylases, tyrosine hydroxylase and tryptophan hydroxylase. A similar 2-His-1-carboxylate facial triad of two histidines and an acidic residue are the ligands to the iron in other nonheme iron enzymes, including the α-ketoglutarate dependent hydroxylases and the extradiol dioxygenases. Previous studies of the effects of conservative mutations of the iron ligands in tyrosine hydroxylase established that there is some plasticity in the nature of the ligands and that the three ligands differ in their sensitivity to mutagenesis. To determine the generality of this finding for enzymes containing a 2-His-1-carboxylate facial triad, the His285, His290, and Glu330 in rat phenylalanine hydroxylase were mutated to glutamine, glutamate, and histidine. All of the mutant proteins had low but measurable activities for tyrosine formation. In general, mutation of Glu330 had the greatest effect on activity and mutation of His290 the least. All of the mutations resulted in an excess of tetrahydropterin oxidized relative to tyrosine formation, with mutation of His285 having the greatest effect on the coupling of the two partial reactions. The H285Q enzyme had the highest activity as tetrahydropterin oxidase at 20% the wild-type value. All of the mutations greatly decreased the affinity for iron, with mutation of Glu330 the most deleterious. The results complement previous results with tyrosine hydroxylase in establishing the plasticity of the individual iron ligands in this enzyme family.Phenylalanine hydroxylase (PheH) 1 is a tetrahydropterin-dependent nonheme enzyme that catalyzes the hydroxylation of phenylalanine to tyrosine, an important pathway for phenylalanine catabolism (Scheme 1) [1]. As a liver enzyme, PheH is critical for catabolizing excess phenylalanine in the diet. Consequently, a deficiency in PheH results in the debilitating disease phenylketonuria [2]. PheH belongs to the small family of aromatic amino acid hydroxylases, all of which use tetrahydrobiopterin as the source of electrons to hydroxylate the aromatic ring of an aromatic amino acid. Tyrosine hydroxylase (TyrH) and tryptophan †

Regulation of phenylalanine hydroxylase: Conformational changes upon phosphorylation detected by H/D exchange and mass spectrometry

“…Wild-type rat PheH was expressed in E. coli and purified as previously described [24, 25]. The apo-enzyme was reconstituted with ferrous iron under argon immediately before use [16].…”

Regulation of Phenylalanine Hydroxylase: Conformational Changes Upon Phenylalanine Binding Detected by Hydrogen/Deuterium Exchange and Mass Spectrometry

“…Protein Purification and Preparation. The purification of wild-type rat PheH and the catalytic domain (Δ117PheH) were described previously (19,24). To remove the ferric iron present in the enzyme when purified, 45 mL concentrated wild-type PheH (about 2 mg/mL) in 50 mM Hepes, pH 7.0, 15% glycerol, 1 μM pepstatin A, and 1 μM leupeptin was dialyzed overnight against 1 L of the same buffer plus 50 mM EDTA and 50 mM nitrilotriacetic acid (NTA), pH 7.0, with one buffer change.…”

Scallop phenylalanine hydroxylase implicates in immune response and can be induced by human TNF-α

“…The ACT domain was an N-terminal regulatory domain to promote allosteric effect, and the Biopterin_H domain was a C-terminal catalytic domain responsible for the conversion from phenylalanine to tyrosine [5,7]. In the Biopterin_H domain of CfPAH, there was a conserved Biopterin-dependent aromatic amino acid hydroxylases signature sequence (from Pro287 to Pro298) with two histidines to bind an iron atom through the form of 2-histidine-1-carboxylate triad [9]. It suggested that CfPAH could possess the similar structure and function to previously reported PAHs.…”

“…The homologous catalytic Biopterin_H domains contain an iron-bound motif and a tetramerization motif, and all the residues are required for the determination of substrate specificity and catalytic function [9e11]. For example, the iron is bound to the 2-His-1-carboxylate facial triad constituted by two histidines and an acidic residue in catalytic domain of PAH [9]. PAHs catalyze the reaction from phenylalanine to tyrosine mainly in the liver [12].…”