Phenylalanine Hydroxylase from Chromobacterium violaceum

Abstract

؊1 under these conditions. Glutathione, mercaptoethanol, and dihydrolipoate support the hydroxylation of phenylalanine essentially as well as DTT. Incubation of copper-depleted hydroxylase with FeSO 4 , phenylalanine, and DTT followed by gel permeation chromatography leads to an iron-hydroxylase containing approximately 1 molecule of iron per molecule of enzyme. The iron-hydroxylase displays an optical absorption band extending from 300 to 600 nm, and it catalyzes the hydroxylation of phenylalanine at the same maximum rate as the iron-activated hydroxylase but does not require added Fe 2؉ . We conclude that iron participates in the hydroxylation of phenylalanine. Iron is not required for the oxidation of DMPH 4 , although it may exert a modest acceleration effect. A hypothetical mechanism is presented wherein the reaction of iron with the putative 4a-hydroperoxy-DMPH 4 leads to 4a-hydroxy-DMPH 4 and a high valent iron-oxy species. The iron-oxy species is postulated to react with phenylalanine in the hydroxylation process.Phenylalanine hydroxylases are tetrahydropterin-dependent monooxygenases that catalyze the hydroxylation of phenylalanine by dioxygen to produce tyrosine, with the concomitant two-electron oxidation of a tetrahydropterin cofactor. The overall transformations of the cofactor are illustrated in Scheme 1. A quinonoid form of the dihydropterin can be observed spectrophotometrically as an intermediate but spontaneously undergoes isomerization to the corresponding 7,8-dihydropterin. Recycling of the cofactor between its dihydro-and tetrahydroforms can be brought about by reducing agents such as DTT (1).1 Phenylalanine hydroxylases exist in many species from bacteria to humans and play important roles in aromatic amino acid metabolism. A genetic deficiency in phenylalanine hydroxylase activity in humans results in phenylketonuria, which is associated with mental retardation during development, as well as behavioral disorders (1, 2). Phenylalanine hydroxylases purified from mammalian sources are homotetrameric proteins that require iron for activity (3, 4). The precise mechanism by which iron-containing phenylalanine hydroxylases use dioxygen to hydroxylate an aromatic substrate is not completely understood, despite investigations extending over more than two decades (5).A phenylalanine hydroxylase purified from Chromobacterium violaceum is a monomeric protein that contains approximately one Cu(II)/molecule (6, 7). However, copper does not support activity (8). Amino acid sequence alignments reveal an overall identity of approximately 22% to the catalytic domains of mammalian phenylalanine hydroxylases (9), which suggests similarities in three-dimensional structure. Among the mechanistic questions surrounding this enzyme are whether a redox-active metal center is required for its activity, how the molecular oxygen is activated to form a hydroxylating intermediate, and whether it shares a common mechanism with the mammalian enzymes.We here describe the cloning of a gene encoding a phenylalanine hydroxyla...

Mechanism of Aromatic Amino Acid Hydroxylation

“…Consistent with such an expectation, all three hydroxylases have been shown to catalyze benzylic hydroxylation of methylated aromatic amino acids (54,85,88). In addition, PheH has been reported to catalyze aliphatic hydroxylation (21,46) and epoxidation (23). Only in the case of benzylic hydroxylation by TyrH has the mechanism been investigated in detail.…”

“…The C. violaceum enzyme has been isolated with copper instead of iron (20), and this observation led to the proposal that the bacterial enzyme does not require any metal for activity (21,22). More recently, C. violaceum PheH was shown to have an absolute requirement for iron for tyrosine formation (23), in agreement with the metal requirements of the eukaryotic enzymes. There have been no other reports to date of a pterin dependent hydroxylase catalyzing amino acid hydroxylation using a metal other than iron.…”

“…However, in the presence of Co(II), this mutant protein will catalyze tyrosine dependent tetrahydropterin oxidation at 14% the rate seen with Fe(II), although it does not catalyze DOPA formation (72). The iron-free C. violaceum PheH was not metal-free but contained substoichiometric amounts of nickel and copper (23).…”

“…In the absence of direct detection of the intermediates proposed for the two different mechanisms, one criterion for clarifying the role of the iron in the initial reaction between tetrahydropterin is to determine whether these enzymes can catalyze a reaction between tetrahydropterin and oxygen in the absence of iron. The metal-free PheH from C. violaceum has been reported to catalyze the oxidation of dimethyltetrahydropterin to quinonoid dihydropterin and hydrogen peroxide at about 5% the turnover of the iron-containing enzyme (23), suggesting that the metal is not absolutely required. In contrast, several mutant forms of TyrH in which the metal affinity has been decreased by mutation of the one of the metal ligands still show an absolute requirement for metal to catalyze any tetrahydropterin consumption (35).…”

“…Hydrogen peroxide is produced by this unproductive reaction, consistent with the breakdown of a peroxypterin intermediate (68). In addition, C. violaceum PheH is reported to form hydrogen peroxide in the presence of phenylalanine if the metal-free enzyme is used (23).…”