Pterin-4a-carbinolamine Dehydratase Research Articles

Analysis of Catecholamines and Pterins in Inborn Errors of Monoamine Neurotransmitter Metabolism-From Past to Future.

Inborn errors of monoamine neurotransmitter biosynthesis and degradation belong to the rare inborn errors of metabolism. They are caused by monogenic variants in the genes encoding the proteins involved in (1) neurotransmitter biosynthesis (like tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC)), (2) in tetrahydrobiopterin (BH4) cofactor biosynthesis (GTP cyclohydrolase 1 (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), sepiapterin reductase (SPR)) and recycling (pterin-4a-carbinolamine dehydratase (PCD), dihydropteridine reductase (DHPR)), or (3) in co-chaperones (DNAJC12). Clinically, they present early during childhood with a lack of monoamine neurotransmitters, especially dopamine and its products norepinephrine and epinephrine. Classical symptoms include autonomous dysregulations, hypotonia, movement disorders, and developmental delay. Therapy is predominantly based on supplementation of missing cofactors or neurotransmitter precursors. However, diagnosis is difficult and is predominantly based on quantitative detection of neurotransmitters, cofactors, and precursors in cerebrospinal fluid (CSF), urine, and blood. This review aims at summarizing the diverse analytical tools routinely used for diagnosis to determine quantitatively the amounts of neurotransmitters and cofactors in the different types of samples used to identify patients suffering from these rare diseases.

Four Years of Diagnostic Challenges with Tetrahydrobiopterin Deficiencies in Iranian Patients.

Hyperphenylalaninemia (HPA) is a condition caused by tetrahydrobiopterin (BH4) and phenylalanine hydroxylase (PAH) deficiencies. It is essential that differential diagnosis be conducted to distinguish these two causes of HPA, because BH4 deficiency is a more severe disease involving progressive neurologic deterioration. Based on the biological findings, HPA is defined as a plasma phenylalanine level of >2.0mg/dl (>120μmol/l). The National Biochemistry Reference Laboratory at the Pasteur Institute of Iran initiated BH4 deficiency screening tests for the first time during the implementation of a nationwide phenylketonuria (PKU) screening program. Measurement of blood phenylalanine and urinary neopterin and biopterin was conducted by high-performance liquid chromatography in 617 patients with HPA. Dihydropteridine reductase (DHPR) activity was measured in all patients by kinetic spectrophotometry. Differential diagnosis was conducted for PKU, transient HPA, and BH4 deficiencies.Our results indicated that out of 76 cases involving BH4 deficiencies, 37 had 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency, 35 had DHPR deficiency, 1 case had pterin-4a-carbinolamine dehydratase (PCD) deficiency, and 3 cases had GTP cyclohydrolase I (GTPCH) deficiency. In this study, 1 novel deletion mutation and 18 novel missense mutations were reported in addition to mutations that had previously been identified and registered in BIOMDB. At present, the screening program for PKU in Iran includes tests that detect different forms of BH4 deficiency presenting with HPA. Newborns that are BH4-deficient benefit from the availability of the tests because they can receive necessary care before being clinically affected.

Molecular and enzymatic characterization of two enzymes BmPCD and BmDHPR involving in the regeneration pathway of tetrahydrobiopterin from the silkworm Bombyx mori.

Tetrahydrobiopterin (BH4) is an essential cofactor of aromatic amino acid hydroxylases and nitric oxide synthase so that BH4 plays a key role in many biological processes. BH4 deficiency is associated with numerous metabolic syndromes and neuropsychological disorders. BH4 concentration in mammals is maintained through a de novo synthesis pathway and a regeneration pathway. Previous studies showed that the de novo pathway of BH4 is similar between insects and mammals. However, knowledge about the regeneration pathway of BH4 (RPB) is very limited in insects. Several mutants in the silkworm Bombyx mori have been approved to be associated with BH4 deficiency, which are good models to research on the RPB in insects. In this study, homologous genes encoding two enzymes, pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) involving in RPB have been cloned and identified from B. mori. Enzymatic activity of DHPR was found in the fat body of wild type silkworm larvae. Together with the transcription profiles, it was indicated that BmPcd and BmDhpr might normally act in the RPB of B. mori and the expression of BmDhpr was activated in the brain and sexual glands while BmPcd was expressed in a wider special pattern when the de novo pathway of BH4 was lacked in lemon. Biochemical analyses showed that the recombinant BmDHPR exhibited high enzymatic activity and more suitable parameters to the coenzyme of NADH in vitro. The results in this report give new information about the RPB in B. mori and help in better understanding insect BH4 biosynthetic networks.

Interactions with the Bifunctional Interface of the Transcriptional Coactivator DCoH1 Are Kinetically Regulated

Pterin-4a-carbinolamine dehydratase (PCD) is a highly conserved enzyme that evolved a second, unrelated function in mammals, as a transcriptional coactivator. As a coactivator, PCD is known as DCoH or dimerization cofactor of the transcription factor HNF-1. These two activities are associated with a change in oligomeric state: from two dimers interacting as an enzyme in the cytoplasm to a dimer interacting with a dimer of HNF-1 in the nucleus. The same interface of DCoH forms both complexes. To determine how DCoH partitions between its two functions, we studied the folding and stability of the DCoH homotetramer. We show that the DCoH1 homotetramer is kinetically trapped, meaning once it forms it will not dissociate to interact with HNF-1. In contrast, DCoH2, a paralog of DCoH1, unfolds within hours. A simple mutation in the interface of DCoH2 from Ser-51 to Thr, as found in DCoH1, increases the kinetic stability by 9 orders of magnitude, to τ(½) ∼ 2 million years. This suggests that the DCoH1·HNF-1 complex must co-fold to interact. We conclude that simple mutations can dramatically affect the dissociation kinetics of a complex. Residue 51 represents a "kinetic hot spot" instead of a "thermodynamic hot spot." Kinetic regulation allows PCD to adopt two distinct functions. Mutations in DCoH1 associated with diabetes affect both functions of DCoH1, perhaps by disrupting the balance between the two DCoH complexes.

An international survey of patients with tetrahydrobiopterin deficiencies presenting with hyperphenylalaninaemia

The present study summarizes clinical and biochemical findings, current treatment strategies and follow-up in patients with tetrahydrobiopterin (BH(4)) deficiencies. We analyzed the clinical, biochemical and treatment data of 626 patients with BH(4) deficiencies [355 with 6-pyruvoyl-tetrahydropterin synthase (PTPS), 217 with dihydropteridine reductase (DHPR), 31 with autosomal recessive GTP cyclohydrolase I (GTPCH), and 23 with pterin-4a-carbinolamine dehydratase (PCD) deficiencies] from the BIODEF Database. Patients with autosomal dominant GTPCH and SR deficiencies will not be discussed in detail. Up to 57 % of neonates with BH(4) deficiencies are already clinically symptomatic. During infancy and childhood, the predominant symptoms are muscular hypotonia, mental retardation and age-dependent movement disorders, including dystonia. The laboratory diagnosis of BH(4) deficiency is based on a positive newborn screening (NBS) for phenylketonuria (PKU), characteristic profiles of urinary or dried blood spot pterins (biopterin, neopterin, and primapterin), and the measurement of DHPR activity in blood. Some patients with autosomal recessive GTPCH deficiency and all with sepiapterin reductase deficiency may be diagnosed late due to normal blood phenylalanine in NBS. L-dopa, 5-hydroxytryptophan, and BH(4) are supplemented in PTPS and GTPCH-deficient patients, whereas L-dopa, 5-hydroxytryptophan, folinic acid and diet are used in DHPR-deficient patients. Medication doses vary widely among patients, and our understanding of the effects of dopamine agonists and monoamine catabolism inhibitors are limited. BH(4) deficiencies are a group of treatable pediatric neurotransmitter disorders that are characterized by motor dysfunction, mental retardation, impaired muscle tone, movement disorders and epileptic seizures. Although the outcomes of BH(4) deficiencies are highly variable, early diagnosis and treatment result in improved outcomes.

Kinetic Stability May Determine the Interaction Dynamics of the Bifunctional Protein DCoH1, the Dimerization Cofactor of the Transcription Factor HNF-1α,

The two disparate functions of DCoH1 (dimerization cofactor of HNF-1)/PCD (pterin-4a-carbinolamine dehydratase) are associated with a change in oligomeric state. DCoH dimers enhance the activity of the diabetes-associated transcription factor HNF-1α (hepatocyte nuclear factor-1α), while the PCD activity of DCoH1 homotetramers aids in aromatic amino acid metabolism. These complexes compete for the same interface of the DCoH dimer. Formation of the DCoH1/HNF-1α complex requires cofolding. The homotetramer of the DCoH1 paralogue, DCoH2, interacts with HNF-1α through simple mixing. To further investigate regulation of DCoH/HNF-1α complex formation, we measured the stability of the DCoH1 homotetramer through unfolding studies by intrinsic tryptophan fluorescence. DCoH2 unfolding is reversible. Surprisingly, the DCoH1 homotetramer is resistant to guanidine unfolding but refolds at a much lower guanidine concentration. We show that a point mutation at the DCoH1 tetramer interface, Thr 51 Ser, overcomes the dissociation barrier of the homotetramer and increases the interaction with HNF-1α. The 1.8 Ǻ resolution crystal structure of DCoH1 T51S shows the presence of an ordered water molecule at the tetramer interface, as in DCoH2, which may destabilize the homotetramer. The equilibrium unfolding data were fit to a two-state model with no apparent intermediate. Folding intermediates were detectable by size exclusion chromatography. For wild-type DCoH1 the intermediates changed with time, suggesting a kinetic origin for the unfolding barrier of the homotetramer. We propose an unfolding pathway in which the tetramer unfolds slowly, but the dimer folds reversibly. Implications for regulation of DCoH1/HNF-1α complex formation are discussed.

FolX and FolM Are Essential for Tetrahydromonapterin Synthesis in Escherichia coli and Pseudomonas aeruginosa

Tetrahydromonapterin is a major pterin in Escherichia coli and is hypothesized to be the cofactor for phenylalanine hydroxylase (PhhA) in Pseudomonas aeruginosa, but neither its biosynthetic origin nor its cofactor role has been clearly demonstrated. A comparative genomics analysis implicated the enigmatic folX and folM genes in tetrahydromonapterin synthesis via their phyletic distribution and chromosomal clustering patterns. folX encodes dihydroneopterin triphosphate epimerase, which interconverts dihydroneopterin triphosphate and dihydromonapterin triphosphate. folM encodes an unusual short-chain dehydrogenase/reductase known to have dihydrofolate and dihydrobiopterin reductase activity. The roles of FolX and FolM were tested experimentally first in E. coli, which lacks PhhA and in which the expression of P. aeruginosa PhhA plus the recycling enzyme pterin 4a-carbinolamine dehydratase, PhhB, rescues tyrosine auxotrophy. This rescue was abrogated by deleting folX or folM and restored by expressing the deleted gene from a plasmid. The folX deletion selectively eliminated tetrahydromonapterin production, which far exceeded folate production. Purified FolM showed high, NADPH-dependent dihydromonapterin reductase activity. These results were substantiated in P. aeruginosa by deleting tyrA (making PhhA the sole source of tyrosine) and folX. The DeltatyrA strain was, as expected, prototrophic for tyrosine, whereas the DeltatyrA DeltafolX strain was auxotrophic. As in E. coli, the folX deletant lacked tetrahydromonapterin. Collectively, these data establish that tetrahydromonapterin formation requires both FolX and FolM, that tetrahydromonapterin is the physiological cofactor for PhhA, and that tetrahydromonapterin can outrank folate as an end product of pterin biosynthesis.

Exhaustive analysis of BH4 and dopamine biosynthesis genes in patients with Dopa-responsive dystonia

Dopa-responsive dystonia is a childhood-onset dystonic disorder, characterized by a dramatic response to low dose of L-Dopa. Dopa-responsive dystonia is mostly caused by autosomal dominant mutations in the GCH1 gene (GTP cyclohydrolase1) and more rarely by autosomal recessive mutations in the TH (tyrosine hydroxylase) or SPR (sepiapterin reductase) genes. In addition, mutations in the PARK2 gene (parkin) which causes autosomal recessive juvenile parkinsonism may present as Dopa-responsive dystonia. In order to evaluate the relative frequency of the mutations in these genes, but also in the genes involved in the biosynthesis and recycling of BH4, and to evaluate the associated clinical spectrum, we have studied a large series of index patients (n = 64) with Dopa-responsive dystonia, in whom dystonia improved by at least 50% after L-Dopa treatment. Fifty seven of these patients were classified as pure Dopa-responsive dystonia and seven as Dopa-responsive dystonia-plus syndromes. All patients were screened for point mutations and large rearrangements in the GCH1 gene, followed by sequencing of the TH and SPR genes, then PTS (pyruvoyl tetrahydropterin synthase), PCBD (pterin-4a-carbinolamine dehydratase), QDPR (dihydropteridin reductase) and PARK2 (parkin) genes. We identified 34 different heterozygous point mutations in 40 patients, and six different large deletions in seven patients in the GCH1 gene. Except for one patient with mental retardation and a large deletion of 2.3 Mb encompassing 10 genes, all patients had stereotyped clinical features, characterized by pure Dopa-responsive dystonia with onset in the lower limbs and an excellent response to low doses of L-Dopa. Dystonia started in the first decade of life in 40 patients (85%) and before the age of 1 year in one patient (2.2%). Three of the 17 negative GCH1 patients had mutations in the TH gene, two in the SPR gene and one in the PARK2 gene. No mutations in the three genes involved in the biosynthesis and recycling of BH4 were identified. The clinical presentations of patients with mutations in TH and SPR genes were strikingly more complex, characterized by mental retardation, oculogyric crises and parkinsonism and they were all classified as Dopa-responsive dystonia-plus syndromes. Patient with mutation in the PARK2 gene had Dopa-responsive dystonia with a good improvement with L-Dopa, similar to Dopa-responsive dystonia secondary to GCH1 mutations. Although the yield of mutations exceeds 80% in pure Dopa-responsive dystonia and Dopa-responsive dystonia-plus syndromes groups, the genes involved are clearly different: GCH1 in the former and TH and SPR in the later.

Crystal structures of Toxoplasma gondii pterin-4a-carbinolamine dehydratase and comparisons with mammalian and parasite orthologues

The enzyme pterin-4a-carbinolamine dehydratase (PCD) is important for the recycling of pterins within eukaryotic cells. A recombinant expression system for PCD from the apicomplexan parasite Toxoplasma gondii has been prepared, the protein purified and crystallised. Single crystal X-ray diffraction methods have produced a high-resolution structure (1.6 Å) of the apo-enzyme and a low-resolution structure (3.1 Å) of a complex with a substrate-like ligand dihydrobiopterin (BH 2). Analysis of the hydrogen bonding interactions that contribute to binding BH 2 suggest that the ligand is present in an enol tautomeric state, which makes it more similar to the physiological substrate. The enzyme can process ( R)- and ( S)-forms of pterin-4a-carbinolamine and the ligand complex suggests that His61 and His79 are placed to act independently as general bases for catalysis of the individual enantiomers. Comparisons with orthologues from other protozoan parasites ( Plasmodium falciparum and Leishmania major) and with rat PCD, for which the structure is known, indicate a high degree of sequence and structure conservation of this enzyme. The molecular determinants of ligand recognition and PCD reactivity are therefore highly conserved across species.

Structure of Phenylalanine Hydroxylase from Colwellia psychrerythraea 34H, a Monomeric Cold Active Enzyme with Local Flexibility around the Active Site and High Overall Stability

The characteristic of cold-adapted enzymes, high catalytic efficiency at low temperatures, is often associated with low thermostability and high flexibility. In this context, we analyzed the catalytic properties and solved the crystal structure of phenylalanine hydroxylase from the psychrophilic bacterium Colwellia psychrerythraea 34H (CpPAH). CpPAH displays highest activity with tetrahydrobiopterin (BH(4)) as cofactor and at 25 degrees C (15 degrees C above the optimal growth temperature). Although the enzyme is monomeric with a single L-Phe-binding site, the substrate binds cooperatively. In comparison with PAH from mesophilic bacteria and mammalian organisms, CpPAH shows elevated [S(0.5)](L-Phe) (= 1.1 +/- 0.1 mm) and K(m)(BH(4))(= 0.3 +/- 0.1 mm), as well as high catalytic efficiency at 10 degrees C. However, the half-inactivation and denaturation temperature is only slightly lowered (T(m) approximately 52 degrees C; where T(m) is half-denaturation temperature), in contrast to other cold-adapted enzymes. The crystal structure shows regions of local flexibility close to the highly solvent accessible binding sites for BH(4) (Gly(87)/Phe(88)/Gly(89)) and l-Phe (Tyr(114)-Pro(118)). Normal mode and COREX analysis also detect these and other areas with high flexibility. Greater mobility around the active site and disrupted hydrogen bonding abilities for the cofactor appear to represent cold-adaptive properties that do not markedly affect the thermostability of CpPAH.

Specific interaction of the diastereomers 7(R)‐ and 7(S)‐tetrahydrobiopterin with phenylalanine hydroxylase: implications for understanding primapterinuria and vitiligo

Pterin-4a-carbinolamine dehydratase (PCD) is an essential component of the phenylalanine hydroxylase (PAH) system, catalyzing the regeneration of the essential cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin [6(R)BH4]. Mutations in PCD or its deactivation by hydrogen peroxide result in the generation of 7(R,S)BH4, which is a potent inhibitor of PAH that has been implicated in primapterinuria, a variant form of phenylketonuria, and in the skin depigmentation disorder vitiligo. We have synthesized and separated the 7(R) and 7(S) diastereomers confirming their structure by NMR. Both 7(R)- and 7(S)BH4 function as poor cofactors for PAH, whereas only 7(S)BH4 acts as a potent competitive inhibitor vs. 6(R)BH4 (Ki=2.3-4.9 microM). Kinetic and binding studies, as well as characterization of the pterin-enzyme complexes by fluorescence spectroscopy, revealed that the inhibitory effects of 7(R,S)BH4 on PAH are in fact specifically based on 7(S)BH4 binding. The molecular dynamics simulated structures of the pterin-PAH complexes indicate that 7(S)BH4 inhibition is due to its interaction with the polar region at the pterin binding site close to Ser-251, whereas its low efficiency as cofactor is related to a suboptimal positioning toward the catalytic iron. 7(S)BH4 is not an inhibitor for tyrosine hydroxylase (TH) in the physiological range, presumably due to the replacement of Ser-251 by the corresponding Ala297. Taken together, our results identified structural determinants for the specific regulation of PAH and TH by 7(S)BH4, which in turn aid in the understanding of primapterinuria and acute vitiligo.

The Repair Enzyme Peptide Methionine-S-Sulfoxide Reductase Is Expressed in Human Epidermis and Upregulated by UVA Radiation

Recently, we reported that photoaging correlates well with the amount of oxidized protein accumulated in the upper dermis, while protein oxidation levels in the viable epidermis are very low. We hypothesized that this might be due to epidermal expression of the repair enzymes methionine sulfoxide reductases (MSRs). The expression of human methionine sulfoxide reductase A (MSRA) was investigated in HaCaT cells, primary human keratinocytes, and in human skin. High MSRA mRNA and protein levels as well as MSR activity were found in cultured human keratinocytes. MSRA was expressed in human epidermis, as shown by immunohistochemistry in healthy human skin. Repetitive in vivo exposure of human skin to solar-simulated light on 10 consecutive days (n=10 subjects) significantly increased epidermal MSRA expression. To further assess the functional relevance of the enzyme, its expression in response to UVB, UVA, and H(2)O(2) was investigated in HaCaT cells. While UVB lowered protein expression of MSRA, an upregulation was observed in response to low doses of UVA and H(2)O(2). In summary, MSRA represents the only enzyme so far identified in human skin that is capable of repairing oxidative protein damage. In addition to melanogenesis and DNA repair systems, a wavelength-specific activation of epidermal MSRA may be involved in epidermal photoprotection.

Functional identification of orthologous genes encoding pterin recycling activity in Plasmodium falciparum and Toxoplasma gondii

Functional identification of orthologous genes encoding pterin recycling activity in Plasmodium falciparum and Toxoplasma gondii

Human epidermal acetylcholinesterase (AchE) is regulated by hydrogen peroxide (H2O2)

Previously it has been demonstrated that the human epidermis synthesizes and degrades acetylcholine and expresses both muscarinic and nicotinic receptors. These cholinergic systems have been implicated in the development of the epidermal calcium gradient and differentiation in normal healthy skin. In vitiligo severe oxidative stress occurs in the epidermis of these patients with accumulation of H2O2 in the 10−3M range together with a decrease in catalase expression/activity due to deactivation of the enzyme active site. It was also shown that the entire recycling of the essential cofactor (6R)‐L‐erytho 5, 6, 7, 8 tetrahydrobiopterin via pterin‐4a‐carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) is affected by H2O2 oxidation of Trp/Met residues in the enzyme structure leading to deactivation of these proteins. Using fluorescence immunohistochemistry we now show that epidermal H2O2 in vitiligo patients yields also almost absent epidermal acetylcholinesterase (AchE) in association with accumulation of epidermal acetylcholine. This result was confirmed by Fluorescence excitation spectroscopy following the Trp fluorescence at λmax 280 nm. A kinetic analysis using pure recombinant human AchE revealed that low concentrations of H2O2(10−6M) activate this enzyme by increasing the Vmax > 2 fold, meanwhile high concentrations of H2O2(10−3M) deactivate the enzyme with a significant decrease in Vmax. Molecular modelling based on the established 3D structure of human AchE supported that H2O2‐mediated oxidation of Trp432, Trp435 and Met436 moves and disorients the active site His440 of the enzyme, thus explaining the deactivation of the protein. To our knowledge these results identified for the first time H2O2 regulation of AchE. Moreover, it was shown that H2O2‐mediated oxidation of AchE contributes significantly to the well established oxidative stress in vitiligo.

Evidence for epidermal acetylcholine accumulation after oxidative stress by H2O2 and possible implications

Acetylcholine (Ach) has been shown to be synthesized de novo and degraded in the human skin (1), while the presence of acetylcholinesterase (AchE), the degrading enzyme, was first mentioned in 1989 (2). It is now accepted that H2O2‐related oxidative stress is a major player in the development/acceleration of vitiligo (3). This oxidative stress affects the recycling of the essential cofactor (6R)‐l‐erythro 5,6,7,8 tetrahydrobiopterin (6BH4) via H2O2‐mediated oxidation of Trp and Met residues in the structure of pterin‐4a carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) (3). Only very recently, it was recognized that AchE is also affected and deactivated by 10−3 m H2O2 causing the accumulation of Ach in the epidermis of patients with vitiligo (4). One of the implications of oxidative stress in vitiligo includes pruritus, which was for a long time attributed to the presence of epidermal H2O2 in the 10−3 m range. In this context, a role for Ach has been suggested in association with pruritus, and therefore, we would like to suggest that Ach accumulation caused by H2O2‐mediated deactivation of AchE may well initiate pruritus (5). The Ach accumulation can also explain the earlier documented decreased sweating in patients with vitiligo (6).

Activation/deactivation of acetylcholinesterase by H 2O 2: more evidence for oxidative stress in vitiligo

Previously it has been demonstrated that the human epidermis synthesises and degrades acetylcholine and expresses both muscarinic and nicotinic receptors. These cholinergic systems have been implicated in the development of the epidermal calcium gradient and differentiation in normal healthy skin. In vitiligo severe oxidative stress occurs in the epidermis of these patients with accumulation of H 2O 2 in the 10 −3 M range together with a decrease in catalase expression/activity due to deactivation of the enzyme active site. It was also shown that the entire recycling of the essential cofactor (6 R)- l-erythro-5,6,7,8-tetrahydrobiopterin via pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) is affected by H 2O 2 oxidation of Trp/Met residues in the enzyme structure leading to deactivation of these proteins. Using fluorescence immunohistochemistry we now show that epidermal H 2O 2 in vitiligo patients yields also almost absent epidermal acetylcholinesterase (AchE). A kinetic analysis using pure recombinant human AchE revealed that low concentrations of H 2O 2 (10 −6 M) activate this enzyme by increasing the V max>2-fold, meanwhile high concentrations of H 2O 2 (10 −3 M) inhibit the enzyme with a significant decrease in V max. This result was confirmed by fluorescence excitation spectroscopy following the Trp fluorescence at λ max 280 nm. Molecular modelling based on the established 3D structure of human AchE supported that H 2O 2-mediated oxidation of Trp 432, Trp 435, and Met 436 moves and disorients the active site His 440 of the enzyme, leading to deactivation of the protein. To our knowledge these results identified for the first time H 2O 2 regulation of AchE. Moreover, it was shown that H 2O 2-mediated oxidation of AchE contributes significantly to the well-established oxidative stress in vitiligo.

Molecular evidence that halo in Sutton's naevus is not vitiligo.

Both halo naevus and vitiligo are acquired leucodermas of unknown aetiology. To date a significant contribution of oxidative stress through accumulation of hydrogen peroxide (H2O2) has been documented in the pathomechanism of vitiligo but not in halo naevus. Both epidermal pterin-4a-carbinolamine dehydratase (PCD) and catalase are sensitive markers to follow H2O2 concentration-dependent deactivation of these proteins. In situ protein expression of PCD and catalase was examined in full-skin biopsies from skin phototype-matched controls (n=5), untreated and treated vitiligo patients (n=5) and patients with untreated halo naevus in association with vitiligo (n=3). Vitiligo was treated with pseudocatalase (PC-KUS) only. Catalase levels were determined in epidermal suction blister extracts using fast protein liquid chromatography (FPLC). In addition, epidermal H2O2 levels were followed in vivo by Fourier-transform Raman spectroscopy. The results of this study ruled out a contribution of H2O2 in the millimolar range in the depigmentation process of halo naevus as previously documented in vitiligo. Therefore, it can be concluded that both leucodermas exercise distinct concentration-dependent H2O2 signalling in their pathomechanisms.

Effects of dietary glycerol on the expression of pterin carbinolamine dehydratase in the rat.

Earlier studies have shown that the abundance of hepatic phenyl-alanine hydroxylase (PAH) diminishes to 60% of control values in rats fed with a diet composed of 40% (w/w) glycerol [Guerin, Walsh, Donlon and Kaufman (1998) Int. J. Biochem. Cell Biol. 30, 1047-1054]. In this experimental model, there are corresponding decreases in the hepatic concentrations of both the hydroxylase cofactor, tetrahydrobiopterin, and the nucleotide guanosine triphosphate. We now show that the cytoplasmic activities of hepatic pterin-4a-carbinolamine dehydratase (PCD) are also lower in these animals, by approx. 50% compared with control values. Immunoblotting confirmed a diminution of protein abundance in vivo. PCD also functions as a dimerization cofactor (DCoH) for the hepatocyte nuclear factor 1alpha (HNF1alpha) and the relative abundance of PCD/DCoH in the nucleus is also decreased. There is a small reduction in the mRNA levels for PAH and for PCD/DCoH in the glycerol-fed animals. In the kidney, there is also a diminution in the abundance of both PAH and PCD proteins. Hepatic GTP cyclohydrolase I activity was not altered and the abundance of hepatic HNF1alpha remained unchanged. HNF1alpha is required for the expression of PAH in the liver and our results support a role for PCD/DCoH, through its interaction with HNF1alpha, in regulating the expression of PAH.

In situ and in vitro evidence for DCoH/HNF-1α transcription of tyrosinase in human skin melanocytes

Human epidermal melanocytes hold the full capacity for autocrine de novo synthesis/regulation/recycling of the essential cofactor 6-tetrahydrobiopterin (6BH 4) for conversion of l-phenylalanine via phenylalanine hydroxylase to l-tyrosine and for production of l-Dopa via tyrosine hydroxylase to initiate both pigmentation and catecholamine synthesis in these neural crest-derived cells. Earlier we have demonstrated pterin-4a-carbinolamine dehydratase (PCD) mRNA and enzyme activities in epidermal melanocytes and keratinocytes. This protein dimerises also the transcription factor hepatocyte nuclear factor 1 (HNF-1), leading to activation of multiple genes. This study demonstrates for the first time DCoH/HNF-1α expression and transcriptional activity in human epidermal melanocytes in vitro and in situ and identified tyrosinase, the key enzyme for pigmentation, as a new transcriptional target. Specific binding of DCoH/HNF-1 complex to the human tyrosinase promoter was confirmed by gel shift analysis. These results provide a novel mechanism in the regulation of skin pigmentation.

Disorders of tetrahydrobiopterin metabolism and their treatment.

Tetrahydrobiopterin (BH4) deficiencies are disorders affecting phenylalanine metabolism in liver and neurotransmitters biosynthesis in brain. BH4 is the essential cofactor in the enzymatic hydroxylation of 3 aromatic amino acids (phenylalanine, tyrosine, and tryptophan). BH4 is synthesized from guanosine triphosphate (GTP) catalyzed by GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase, and sepiapterin reductase (SPR), and in aromatic amino acids hydoxylating system is regenerated by pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR). To date, 4 enzyme deficiencies (GTPCH, PTPS, DHPR, PCD) have been reported and they all follow an autosomal recessive mode of inheritance. The incidence of BH4 deficiency is at 1 in 1,000,000, except that in Taiwanese (much higher than in Japanese and Caucasians). BH4 deficiency has been diagnosed in patients with hyperphenylalaninemia (HPA) by neonatal mass-screening based on BH4 oral loading tests, analysis of urinary or serum pteridines, and measurement of dihydropterindine reductase (DHPR) activity in blood from a Guthrie card. BH4 deficiency without treatment causes combined symptoms of HPA and neurotransmitter (dopamine, norepinephrine, epinephrine, and serotonin) deficiency, such as red hair, psychomotor retardation, and progressive neurological deterioration. Treatment of BH4 deficiencies consists of BH4 supplementation (2-20 mg/kg per day) or diet to control blood phenylalanine concentration and replacement therapy with neurotransmitters precausers (L-dopa/CarbiDOPA and 5-hydroxytryptophan), and supplements of folinic acid in DHPR deficiency.