The unique identity of rat hepatoma phenylalanine hydroxylase.

Rat hepatoma H4-II-E-C3 culture phenylalanine hydroxylase is unique and differs from any of the three isozymes of liver or the one of kidney. Isoelectric focusing results in a single isozyme with an isoelectric point of 5.20 compared to 5.20, 5.30, 5.60 for the liver and 5.35 for the kidney. Hepatoma phenylalanine hydroxylase cross reacts with antibody # 2 of rat liver but differs from isozyme # 2 by a tenfold difference in the ratio of antigen to antibody at equivalence on immunotitration. Kidney phenylalanine hydroxylase which also cross reacts with antibody # 2 of liver can quantitatively (at 2 mU per mg immunoglobulin) absorb antibody against hepatoma enzyme, liver isozyme # 2 and itself without having any effect on antibody # 1 and # 3 of liver.     

Dictyostelium phenylalanine hydroxylase is activated by its substrate phenylalanine

We have studied the regulatory function of Dictyostelium discoideum Ax2 phenylalanine hydroxylase (dicPAH) via characterization of domain structures. Including the full-length protein, partial proteins truncated in regulatory, tetramerization, or both, were prepared from Escherichia coli as his-tag proteins and examined for oligomeric status and catalytic parameters for phenylalanine. The proteins were also expressed extrachromosomally in the dicPAH knockout strain to examine their in vivo compatibility. The results suggest that phenylalanine activates dicPAH, which is functional in vivo as a tetramer, although cooperativity was not observed. In addition, the results of kinetic study suggest that the regulatory domain of dicPAH may play a role different from that of the domain in mammalian PAH.

Structured summary of protein interactions

dicPAH and dicPAHbind by molecular sieving (View Interaction: 1, 2, 3, 4)     

Arabidopsis thaliana plants possess a specific farnesylcysteine lyase that is involved in detoxification and recycling of farnesylcysteine

In plants, prenylated proteins are involved in actin organization, calcium-mediated signal transduction, and many other biological processes. Arabidopsis thaliana mutants lacking functional protein prenyltransferase genes have also revealed roles for prenylated proteins in phytohormone signaling and meristem development. However, to date, the turnover of prenylated plant proteins and the fate of the prenylcysteine (PC) residue have not been described. We have detected an enzyme activity in Arabidopsis plants that metabolizes farnesylcysteine (FC) to farnesal, which is subsequently reduced to farnesol. Unlike its mammalian ortholog, Arabidopsis FC lyase exhibits specificity for FC over geranylgeranylcysteine (GGC), and recognizes N-acetyl-FC (AFC). FC lyase is encoded by a gene on chromosome 5 of the Arabidopsis genome (FCLY, At5g63910) and is ubiquitously expressed in Arabidopsis tissues and organs. Furthermore, T-DNA insertions into the FCLY gene cause significant decreases in FC lyase activity and an enhanced response to abscisic acid (ABA) in seed germination assays. The effects of FCLY mutations on ABA sensitivity are even greater in the presence of exogenous FC. These data suggest that plants possess a specific FC detoxification and recycling pathway.     

Direct evidence for a phenylalanine site in the regulatory domain of phenylalanine hydroxylase

The hydroxylation of phenylalanine to tyrosine by the liver enzyme phenylalanine hydroxylase is regulated by the level of phenylalanine. Whether there is a distinct allosteric binding site for phenylalanine outside of the active site has been unclear. The enzyme contains an N-terminal regulatory domain that extends through Thr117. The regulatory domain of rat phenylalanine hydroxylase was expressed in Escherichia coli. The purified protein behaves as a dimer on a gel filtration column. In the presence of phenylalanine, the protein elutes earlier from the column, consistent with a conformational change in the presence of the amino acid. No change in elution is seen in the presence of the non-activating amino acid proline. ¹H–¹⁵N HSQC NMR spectra were obtained of the ¹⁵N-labeled protein alone and in the presence of phenylalanine or proline. A subset of the peaks in the spectrum exhibits chemical shift perturbation in the presence of phenylalanine, consistent with binding of phenylalanine at a specific site. No change in the NMR spectrum is seen in the presence of proline. These results establish that the regulatory domain of phenylalanine hydroxylase can bind phenylalanine, consistent with the presence of an allosteric site for the amino acid.     

Evidence that bacterial cyanide oxygenase is a pterin-dependent hydroxylase

The soluble cell-free fraction (150,000g high-speed supernatants [HSS]) of Pseudomonas fluorescens NCIMB 11764 contains putative cyanide oxygenase (CNO) responsible for initiating cyanide oxidation and assimilation as a nitrogenous growth substrate. CNO activity, assayed either by cyanide-dependent O(2) or NADH uptake, or by conversion of radioactive K(14)CN to (14)CO(2), was detected at micromolar concentrations (apparent half-saturation constant, 4 microM). Results demonstrating that CNO requires a protein-enriched cell fraction and a low MW redox factor (<500 Da) for which reduced biopterin could substitute are presented. The properties of CNO are consistent with those of a pterin hydroxylase.     

Maize ZmFDR3 localized in chloroplasts is involved in iron transport

Iron is an essential nutrient for plant metabolism such that Fe-limited plants display chlorosis and suffer from reduced photosynthetic efficiency. Differential display previously identified genes whose expression was elevated in Fe-deficient maize roots. Here,we describe the functional characterization of one of the genes identified in the screen,ZmFDR3 (Zea maize Fe-deficiency-related). Heterologous functional complementation assays using a yeast iron uptake mutant showed that ZmFDR3 functions in iron tra...     

Pyrimidodiazepine, a ring-strained cofactor for phenylalanine hydroxylase

Homologues of 6-methyl-7,8-dihydropterin (6-Me-7,8-PH2) and 6-methyl-5,6,7,8-tetrahydropterin (6-Me-PH4), expanded in the pyrazine ring, were synthesized to determine the effect of increased strain on the chemical and enzymatic properties of the pyrimidodiazepine series. 2-Amino-4-keto-6-methyl-7,8-dihydro-3H,9H-pyrimido[4,5-b] [1,4]diazepine (6-Me-7,8-PDH2) was found to be more unstable in neutral solution than 6-Me-7,8-PH2. Its decomposition appears to proceed by hydrolytic ring opening of the 5,6-imine bond, followed by autooxidation. 6-Me-7,8-PDH2 can be reduced, either chemically or by dihydrofolate reductase (Km = 0.16 mM), to the 5,6,7,8-tetrahydro form (6-Me-PDH4). This can be oxidized with halogen to quinoid dihydropyrimidodiazepine (quinoid 6-Me-PDH2), which is a substrate for dihydropteridine reductase (Km = 33 microM). Whereas quinoid 6-methyldihydropterin was found to tautomerize to 6-Me-7,8-PH2 in 95% yield in 0.1 M tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 7.4, quinoid 6-Me-PDH2 gives only 53% 6-Me-7,8-PDH2, the remainder decomposing via an initial opening of the diazepine ring. Additional evidence for the extra strain in the pyrimidodiazepine system is the cyclization of quinoid 6-N-(2'-aminopropyl)divicine to quinoid 6-Me-PH2 in 57% yield in 0.1 M Tris-HCl, pH 7.4. By comparison, no quinoid 6-Me-PDH2 is formed from the homologue quinoid 6-N-(3'-aminobutyl)divicine. A small (2%) yield of 6-Me-PDH4 is found if the unstable C4a-carbinolamine intermediate is trapped by enzymatic dehydration and reduction. Although phenylalanine hydroxylase utilizes 6-Me-PDH4 (Km = 0.15 mM), the maximum velocity of tyrosine production is 20 times slower than that with 6-Me-PH4, indicating that a ring opening reaction is not a rate-limiting step in the hydroxylase pathway. Further, the maximum velocities of 2,5,6-triamino-4(3H)-pyrimidinone, 2,6-diamino-5-(methylamino)-4(3H)-pyrimidinone, and 2,6-diamino-5-(benzylamino)-4(3H)-pyrimidinone span a 35-fold range. These cofactors would theoretically form the same oxide of quinoid divicine if oxygen activation involves a carbonyl oxide intermediate. Thus, the limiting step is also not transfer of oxygen from this hypothetical intermediate to the phenylalanine substrate.     

An additional substrate binding site in a bacterial phenylalanine hydroxylase

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.     

Genetics of the mammalian phenylalanine hydroxylase system. IV. Evidence of phenylalanine hydroxylase in a cultured human hepatoma cell line

We report here the identification of a cultured human hepatoma cell line which possesses an active phenylalanine hydroxylase system. Phenylalanine hydroxylation was established by growth of cells in a tyrosine-free medium and by the ability of a cell-free extract to convert [¹⁴C]phenylalanine to [¹⁴C]tyrosine in an enzyme assay system. This enzyme activity was abolished by the presence in the assay system of p-chlorophenylalanine but no significant effect on the activity was observed with 3-iodotyrosine and 6-fluorotryptophan. Use of antisera against pure monkey or human liver phenylalanine hydroxylase has detected a cross-reacting material in this cell line which is antigenically identical to the human liver enzyme. Phenylalanine hydroxylase purified from this cell line by affinity chromatography revealed a multimeric molecular weight (estimated 275,000) and subunit molecular weights (estimated 50,000 and 49,000) which are similar to those of phenylalanine hydroxylase purified from a normal human liver. This cell line should be a useful tool for the study of the human phenylalanine hydroxylase system.     

Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site

Epsilon class glutathione transferases (GSTs) have been shown to contribute significantly to insecticide resistance. We report a new Epsilon class protein crystal structure from Drosophila melanogaster for the glutathione transferase DmGSTE6. The structure reveals a novel Epsilon clasp motif that is conserved across hundreds of millions of years of evolution of the insect Diptera order. This histidine-serine motif lies in the subunit interface and appears to contribute to quaternary stability as well as directly connecting the two glutathiones in the active sites of this dimeric enzyme.     

Natural phenylalanine hydroxylase variants that confer a mild phenotype affect the enzyme's conformational stability and oligomerization equilibrium

Hyperphenylalaninemias are genetic diseases prevalently caused by mutations in the phenylalanine hydroxylase (PAH) gene. The wild-type PAH enzyme is a homotetramer regulated by its substrate, cofactor and phosphorylation. We reproduced a full-length wild-type protein and seven natural full-length PAH variants, p.I65M, p.N223Y, p.R297L, p.F382L, p.K398N, p.A403V, and p.Q419R, and analyzed their biochemical and biophysical behavior. All mutants exhibited reduced enzymatic activity, namely from 38% to 69% of wild-type activity. Biophysical characterization was performed by size-exclusion chromatography, light scattering and circular dichroism. In the purified wild-type PAH, we identified the monomer in equilibrium with the dimer and tetramer. In most mutants, the equilibrium shifted toward the dimer and most tended to form aggregates. All PAH variants displayed different biophysical behaviors due to loss of secondary structure and thermal destabilization. Specifically, p.F382L was highly unstable at physiological temperature. Moreover, using confocal microscopy with the number and brightness technique, we studied the effect of BH4 addition directly in living human cells expressing wild-type PAH or p.A403V, a mild mutant associated with BH4 responsiveness in vivo. Our results demonstrate that BH4 addition promotes re-establishment of the oligomerization equilibrium, thus indicating that the dimer-to-tetramer shift in pA403V plays a key role in BH4 responsiveness. In conclusion, we show that the oligomerization process and conformational stability are altered by mutations that could affect the physiological behavior of the enzyme. This endorses the hypothesis that oligomerization and folding defects of PAH variants are the most common causes of HPAs, particularly as regards mild human phenotypes.     

The regulation of phenylalanine hydroxylase in rat tissues in vivo. Substrate- and cortisol-induced elevations in phenylalanine hydroxylase activity.

Injections of phenylalanine increased a 2.5-fold in 9 h the hepatic phenylalanine hydroxylase activity of 6-day-old or adult rats that had been pretreated (24h earlier) with p-chlorophenylalanine; without such pretreatment, phenylalanine did not raise the enzyme concentration. This difference is paralleled by the much greater extent to which the injected phenylalanine accumulated in livers of the pretreated compared with the normal animals. The hormonal induction of hepatic phenylalanine hydroxylase activity obeyed different rules: an injection of cortisol was without effect on adult livers but caused a threefold rise in phenylalanine hydroxylase activity of immature ones, both without and after pretreatment with p-chlorophenylalanine. In the latter instance, the effects of cortisol, and of phenylalanine were additive. Actinomycin inhibited the cortisol- but not the substrate-induced increase of phenylalanine hydroxylase, whereas puromycin inhibited both. The results indicate that substrate and hormone, two potential positive regulators of the amount of the hepatic (but not the renal) phenylalanine hydroxylase, act independently by two different mechanisms. The negative effector, p-chlorophenylalanine, also appears to interact with the synthetic (or degradative) machinery rather than with the existing phenylalanine hydroxylase molecules: 24h were required in vivo for an 85% decrease to ensue, and no inhibition occurred in vitro when incubating the enzyme with p-chlorophenylalanine or with liver extracts from p-chlorophenylalanine-treated rats.     

Development of phenylalanine hydroxylase activity in rat kidney

The developmental pattern of phenylalanine hydroxylase was studied in rat kidney and compared with that of liver from the same animal. Traces of activity were observed from 19 to 21 days of gestation in the liver and on Day 20 and 21 of gestation in the kidney. Significant amounts of activity were noticed on Day 22 of gestation. Kidney on Day 21 of gestation showed slightly higher values than that seen in corresponding liver. Fetal liver and kidney showed about 30% and 90% activity of newborn animals, respectively. Both synthetic cofactor and organic reductant were necessary for optimal activity of liver and kidney enzymes. pH studies showed an optimum at pH 7.0 in both liver and kidney. Storage at ?15 °C resulted in loss of activity in both liver and kidney to the same extent at a given time. No evidence of inhibition of either liver or kidney enzyme by phenylalanine was noticed up to a concentration of 4 μmoles per assay. Heat denaturation studies at 50 °C showed the kidney enzyme to be slightly less stable than the liver enzyme though a similar pattern was observed in both tissues at all ages studied.     

Processing of a chimeric protein in chloroplasts is different in transgenic maize and tobacco plants

Transgenic maize (Zea mays L.) and tobacco (Nicotiana tabacum Petit Havana SR1) plants have been generated, which overproduce a mitochondrial Nicotiana plumbaginifolia manganese superoxide dismutase (MnSOD) in chloroplasts. For this, the mature MnSOD-coding sequence was fused to a chloroplast transit peptide from a Pisum sativum ribulose-1,5-bisphosphate carboxylase (Rubisco) gene and expression of the chimeric gene was driven by the cauliflower mosaic virus (CaMV) 35S promoter. The transgenic MnSOD gene product was correctly targeted to the chloroplasts both in maize and tobacco. However, despite the use of the CaMV 35S promoter, the MnSOD was predominantly localized in the chloroplasts of the bundle sheath cells of maize. Furthermore, the transit peptide was cleaved off at a different position in maize and tobacco.     

Integron integrases possess a unique additional domain necessary for activity.

Integrons are genetic elements capable of integrating genes by a site-specific recombination system catalyzed by an integrase. Integron integrases are members of the tyrosine recombinase family and possess the four invariant residues (RHRY) and conserved motifs (boxes I and II and patches I, II, and III). An alignment of integron integrases compared to other tyrosine recombinases shows an additional group of residues around the patch III motif. We have analyzed the DNA binding and recombination properties of class I integron integrase (IntI1) variants carrying mutations at residues that are well conserved among all tyrosine recombinases and at some residues from the additional motif that are conserved among the integron integrases. The well-conserved residues studied were H277 from the conserved tetrad RHRY (about 90% conserved), E121 found in the patch I motif (about 80% conserved in prokaryotic recombinases), K171 from the patch II motif (near 100% conserved), W229 and F233 from the patch III motif, and G302 of box II (about 80% conserved in prokaryotic recombinases). Additional IntI1 mutated residues were K219 and a deletion of the sequence ALER215. We observed that E121, K171, and G302 play a role in the recombination activity but can be mutated without disturbing binding to DNA. W229, F233, and the conserved histidine (H277) may be implicated in protein folding or DNA binding. Some of the extra residues of IntI1 seem to play a role in DNA binding (K219) while others are implicated in the recombination activity (ALER215 deletion).     

Diagnosis of deficiency in cofactor of phenylalanine hydroxylase: a metabolic emergency

We report on two cases of children suffering from biopterin synthetase deficiency. Both were treated with the same treatment schedule with biopterin and neurotransmitters: 6-hydroxytryptophan and dihydrophenylalanine (DOPA). The only difference between the two cases is the time of diagnosis and therefore of treatment. The child who was treated early has a normal neurologic development. The other one has been treated since he was 7 months old and is mentally deficient (DQ = 0.60). This older child also suffers from dystonia probably secondary to Levodopa treatment. The authors emphasize the uncertainty of these patient's evolution owing to complications of the disease itself or those due to prolonged treatment by neurotransmitters.