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Tetrahydrobiopterin (BH 4 ) deficiency is caused by genetic variants in the three genes involved in de novo cofactor biosynthesis, GTP cyclohydrolase I (GTPCH/GCH1), 6-pyruvoyl-tetrahydropterin synthase (PTPS/PTS), sepiapterin reductase (SR/SPR), and the two genes involved in cofactor recycling, carbinolamine-4α-dehydratase (PCD/PCBD1) and dihydropteridine reductase (DHPR/QDPR). Dysfunction in BH 4 metabolism leads to reduced cofactor levels and may result in systemic hyperphenylalaninemia and/or neurological sequelae due to secondary deficiency in monoamine neurotransmitters in the central nervous system. More than 1100 patients with BH 4 deficiency and 800 different allelic variants distributed throughout the individual genes are tabulated in database of pediatric neurotransmitter disorders PNDdb. Here we provide an update on the molecular-genetic analysis and structural considerations of these variants, including the clinical courses of the genotypes. From a total of 324 alleles, 11 are associated with the autosomal recessive form of GTPCH deficiency presenting with hyperphenylalaninemia (HPA) and neurotransmitter deficiency, 295 GCH1 variant alleles are detected in the dominant form of L-dopa-responsive dystonia (DRD or Segawa disease) while phenotypes of 18 alleles remained undefined. Autosomal recessive variants observed in the PTS (199 variants), PCBD1 (32 variants), and QDPR (141 variants) genes lead to HPA concomitant with central monoamine neurotransmitter deficiency, while SPR deficiency (104 variants) presents without hyperphenylalaninemia. The clinical impact of reported variants is essential for genetic counseling and important for development of precision medicine., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Introduction: 6-Pyruvoyl-tetrahydropterin synthase deficiency (PTPSd) is a rare autosomal recessive disorder of synthesis of biogenic amines, which is characterized by variable neurological impairment and hyperphenylalaninemia. We aimed to assess the long-term clinical outcome of this disorder and the factors affecting it., Methods: At total of 28 PTPSd patients (aged 19.9 ± 10.9 years) underwent clinical (neurological and psychiatric) and neuropsychological assessment (BRIEF, VABS-II, and IQ). Based on CSF homovanillic (HVA) and 5-hydroxyindolacetic acid (5-HIAA) and pterin concentrations at diagnosis, patients were classified as having either a severe [SF; low level of CSF, HVA, and 5-HIAA with altered neopterin/biopterin (Neo/Bio)] or mild form (MF; normal HVA and 5-HIAA with altered Neo/Bio) of PTPSd., Results: Approximately 36% of patients had MF PTPSd. At the last examination, 43% of patients had movement disorders (2 MF, 10 SF), 43% of patients had variable degrees of intellectual disability (SF only), 39% met the criteria for a psychiatric disorder (3 MF, 9 SF). Applying a linear regression model, we found that HVA and phenylalanine levels at birth had a significant influence on IQ, BRIEF, and VABS-II variability. Lastly, 5-HIAA further contributed to VABS-II variability. The disease showed a self-limiting clinical course and its treatment, although delayed, is effective in improving the neurological status., Conclusions: Neurodevelopmental impairment due to PTPSd shows a self-limiting course. A continuous improvement in the neurological condition has been observed in patients receiving treatment, even when delayed. The severity of brain biogenic amine depletion at diagnosis predicts neurological and psychiatric outcomes., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Tetrahydrobiopterin is an essential cofactor for the hydroxylation of aromatic amino acids phenylalanine, tyrosine, and tryptophan. Therefore, tetrahydrobiopterin deficiency results in hyperphenylalaninemia as well as dopamine and serotonin depletion in the central nervous system. The enzyme 6-pyruvoyltetrahydropterin synthase catalyzes the second step of de novo synthesis of tetrahydrobiopterin, and its deficiency is the most frequent cause of tetrahydrobiopterin metabolism disorders., Method: We conducted a retrospective chart review of 28 subjects from 24 families with molecularly confirmed 6-pyruvoyltetrahydropterin synthase deficiency from six centers in three Arab countries. We reviewed clinical, biochemical, and molecular data. We also reviewed previously published cohorts of subjects with 6-pyruvoyltetrahydropterin synthase deficiency., Results: Similar to previous observations, we show that early treatment (less than two months) is associated with better outcome. We identify eight PTS variants in 24 independent families. The most common variant is (c.238A>G; p.M80V) with an allele count of 33%. We also identify one novel variant (c.2T>G; p.?)., Conclusion: The deficiency of 6-pyruvoyltetrahydropterin synthase is relatively common in the Arab population and should be considered in individuals with hyperphenylalaninemia. More natural history studies with comprehensive biochemical and molecular genetics data are needed for a robust base for the development of future therapy., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Genetic defects on 6-pyruvoyl-tetrahydropterin synthase (PTPS) are the most prevalent cause of hyperphenylalaninaemia not due to phenylalanine hydrolyase deficiency (phenylketonuria). PTPS catalyses the second step of tetrahydrobiopterin (BH 4 ) cofactor biosynthesis, and its deficiency represents the most common form of BH 4 deficiency. Untreated PTPS deficiency results in depletion of the neurotransmitters dopamine, catecholamine and serotonin causing neurological symptoms. We archived reported missense variants of the PTS gene. Common in silico algorithms were used to predict the effects of such variants, and substantial proportions (up to 19%) of the variants were falsely classified as benign or uncertain. We have determined the crystal structure of the human PTPS hexamer, allowing another level of interpretation to understand the potential deleterious consequences of the variants from a structural perspective. The in silico and structure approaches appear to be complimentary and may provide new insights that are not available from each alone. Information from the protein structure suggested that the variants affecting amino acid residues required for interaction between monomeric subunits of the PTPS hexamer were those misclassified as benign by in silico algorithms. Our findings illustrate the important utility of 3D protein structure in interpretation of variants and also current limitations of in silico prediction algorithms. However, software to analyse mutation in the perspective of 3D protein structure is far less readily available than other in silico prediction tools., (Copyright © 2019 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Background: Tetrahydrobiopterin (BH4) is the cofactor for 6-pyruvoyl-tetrahydropterin synthase (PTPS); it is involved in BH4 biosynthesis and is encoded by PTS gene. Its deficiency (PTPSD) is characterized by hyperphenylalaninemia (HPA) and deficit in central monoamine neurotransmitters. We describe the clinical and mutational spectrum of five patients with PTPSD, from four unrelated Mexican families. All patients had symptomatic diagnosis and presented severe early neurological manifestations and HPA., Methods: Clinical and biochemical data from studied patients were recorded. Responsible PTPSD genotypes was determined by direct and bidirectional Sanger DNA sequencing of the six PTS coding exons and their exon-intron borders, and these were directly searched in the available relatives. The novel PTS missense variant [NM_3000317.2:331G > T, p.(Ala111Ser)] was subjected to in silico, to predict a possible deleterious effect., Results: Diminished fetal movements were perceived as a uniform characteristic in the studied group. DNA sequencing showed two known p.(Arg25∗) and p.(Val132TyrFs∗19) and the novel missense p.(Ala111Ser) PTS variants, the latter representing potentially a frequent PTPSD-responsible allele (50%, 4/8) in Mexican patients. In silico protein modeling analysis of the p.(Ala111Ser) variant revealed loss of hydrophobic interactions between the alanine and neighboring valines, suggesting that these changes in polarity may be detrimental for enzyme function, structure and/or stability., Conclusions: This work contributes to the knowledge of PTPS molecular spectrum. The delayed diagnosis of these patients emphasizes the importance of considering BH4 metabolism defects in the differential diagnosis of HPA, especially for countries that are beginning their HPA newborn screening programs., (Copyright © 2018 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Tetrahydrobiopterin (BH4) is an essential cofactor for the aromatic amino acid hydroxylases, alkylglycerol monooxygenase, and nitric oxide synthases (NOS). Inborn errors of BH4 metabolism lead to severe insufficiency of brain monoamine neurotransmitters while augmentation of BH4 by supplementation or stimulation of its biosynthesis is thought to ameliorate endothelial NOS (eNOS) dysfunction, to protect from (cardio-) vascular disease and/or prevent obesity and development of the metabolic syndrome. We have previously reported that homozygous knock-out mice for the 6-pyruvolytetrahydropterin synthase (PTPS; Pts-ko/ko) mice with no BH4 biosynthesis die after birth. Here we generated a Pts-knock-in (Pts-ki) allele expressing the murine PTPS-p.Arg15Cys with low residual activity (15% of wild-type in vitro) and investigated homozygous (Pts-ki/ki) and compound heterozygous (Pts-ki/ko) mutants. All mice showed normal viability and depending on the severity of the Pts alleles exhibited up to 90% reduction of PTPS activity concomitant with neopterin elevation and mild reduction of total biopterin while blood L-phenylalanine and brain monoamine neurotransmitters were unaffected. Yet, adult mutant mice with compromised PTPS activity (i.e., Pts-ki/ko, Pts-ki/ki or Pts-ko/wt) had increased body weight and elevated intra-abdominal fat. Comprehensive phenotyping of Pts-ki/ki mice revealed alterations in energy metabolism with proportionally higher fat content but lower lean mass, and increased blood glucose and cholesterol. Transcriptome analysis indicated changes in glucose and lipid metabolism. Furthermore, differentially expressed genes associated with obesity, weight loss, hepatic steatosis, and insulin sensitivity were consistent with the observed phenotypic alterations. We conclude that reduced PTPS activity concomitant with mildly compromised BH4-biosynthesis leads to abnormal body fat distribution and abdominal obesity at least in mice. This study associates a novel single gene mutation with monogenic forms of obesity.

This study aimed to investigate the clinical variability and factors implied in the outcome of 6-pyruvoyl-tetrahydropterin synthase deficiency (PTPSd). Biochemical and clinical phenotype, treatment variables, and 6-pyruvoyl-tetrahydropterin synthase (PTS) genotype, were explored retrospectively in 19 Italian patients (12 males and 7 females, aged 4 months to 33 years). According to the level of biogenic amines in cerebrospinal fluid (CSF) at the diagnosis, the patients were classified as mild (6) (normal level) or severe (13) (abnormal low level) form (MF and SF, respectively). Blood Phe ranged from 151 to 1053 micromol/l in MF (mean +/- SD: 698 +/- 403) and 342-2120 micromol/l in SF (mean +/- SD: 1175 +/- 517) (p = 0.063). Patients with MF showed a normal neurological development (a transient dystonia was detected in one), while all SF patients except one presented with severe neurological impairment and only four had a normal neurological development. The outcome of the SF was influenced by the precocity of the treatment. Serial CSF examinations revealed a decline of 5-hydroxyindolacetic acid in MFs and an incomplete restoration of neurotransmitters in SFs: neither obviously affected the prognosis. PTS gene analysis detected 17 different mutations (seven so far unreported) (only one affected allele was identified in three subjects). A good correlation was found between genotype and clinical and biochemical phenotype. The occurrence of brain neurotransmitter deficiency and its early correction (by the therapy) are the main prognostic factors in PTPSd.

Tetrahydrobiopterin (BH(4)) deficiency comprises heterogeneous disorders resulting in hyperphenylalaninaemia (HPA) and lack of monoamine neurotransmitters. Among these, 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency is the most common disorder. We report a female Thai patient with PTPS deficiency who was initially detected by newborn screening for HPA, and later treated by supplements of BH(4), L-dopa/carbidopa, and 5-hydroxytryptophan. Monitoring of serum prolactin representing dopamine sufficiency is used for optimizing the dosage of L-dopa. She showed a remarkable progress of development despite delayed treatment at 5 months of age. Mutation analysis revealed two heterozygous missense mutations of the PTS gene: c.259C>T (p.P87S) inherited from the father; and c.147T>G (p.H49Q) inherited from the mother. The latter is a novel mutation that affects the pterin-binding site of the PTPS enzyme. This novel mutation expands the mutation spectrum of PTPS deficiency. Notably, some PTS mutations have been reported in both Thai and Chinese patients. Whether these common mutations are the result of a founder effect with common ancestors of Thai and Chinese people or intermarriage between Thai and Chinese descents in Thailand remain unclear. In conclusion, severe neurological impairment from BH(4) deficiency could be prevented by newborn screening for HPA and proper metabolic management. However, pterin analysis for early diagnosis of BH(4) deficiency is still not available in most developing countries.

We describe the treatment, the clinical, and biochemical findings and the outcome of 26 patients with 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency and 10 patients with dihydropteridine reductase (DHPR) deficiency. These are the two most common forms of the autosomal-recessively inherited tetrahydrobiopterin (BH4) deficiency. Time of diagnosis, dosage of BH4 and neurotransmitter precursors, folinic acid substitution, and levels of 5-hydroxyindoleacetic acid (5HIAA) and homovanillic acid (HVA) in cerebrospinal fluid (CSF) are essential parameters in the follow-up of patients. Unfortunately, treatment protocols vary greatly among patients and clinical centers, and CSF investigations and outcome assessments are not always available. Seventeen patients with PTPS deficiency and four patients with DHPR deficiency were diagnosed within 2 months after birth. In 14 patients with PTPS deficiency (54%; 9 early and 5 late diagnosed) and 2 patients with DHPR deficiency (20%; all early diagnosed) no developmental delay is observed, while in 10 patients with PTPS deficiency (38%; 6 early and 4 late diagnosed) and 8 patients with DHPR deficiency (80%; 2 early and 6 late diagnosed) development was delayed. Two PTPS-deficient patients died in the newborn period. DHPR deficiency seems to be more severe than PTPS deficiency and it is clearly the onset of treatment that determines the outcome. Our data suggest that diagnosis within the first month of life is essential for a good outcome and that low CSF5 HIAA and HVA values in CSF could be an indicator for the ongoing developmental impairment

Tetrahydrobiopterin (BH(4)) deficiencies are a highly heterogeneous group of disorders with several hundred patients, and so far a total of 193 different mutant alleles or molecular lesions identified in the GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), sepiapterin reductase (SR), carbinolamine-4a-dehydratase (PCD), or dihydropteridine reductase (DHPR) genes. The spectrum of mutations causing a reduction in one of the three biosynthetic (GTPCH, PTPS, and SR) or the two regenerating enzymes (PCD and DHPR) is tabulated and reviewed. Furthermore, current genomic variations or SNPs are also compiled. Mutations in GCH1 are scattered over the entire gene, and only 5 out of 104 mutant alleles, present in a homozygous state, are reported to cause the autosomal recessive form of inheritable hyperphenylalaninemia (HPA) associated with monoamine neurotransmitter deficiency. Almost all other 99 different mutant alleles in GCH1 are observed together with a wild-type allele and cause Dopa-responsive dystonia (DRD, Segawa disease) in a dominant fashion with reduced penetrance. Compound heterozygous or homozygous mutations are spread over the entire genes for PTS with 44 mutant alleles, for PCBD with nine mutant alleles, and for QDPR with 29 mutant alleles. These mutations cause an autosomal recessive inherited form of HPA, mostly accompanied by a deficiency of the neurotransmitters dopamine and serotonin. Lack of sepiapterin reductase activity, an autosomal recessive variant of BH(4) deficiency presenting without HPA, was diagnosed in patients with seven different mutant alleles in the SPR gene in exons 2 or 3 or in intron 2. Details on all mutations presented here are constantly updated in the BIOMDB database (www.bh4.org)., ((c) 2006 Wiley-Liss, Inc.)

Little information is available on the long-term course and adult outcome of patients with 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency. We describe the course of a 32-year-old woman with hypotonia, dystonia, choreoathetosis, mental retardation, behavioral disturbances, and incomplete puberty due to PTPS deficiency. From the age of 6 months she developed progressive hypotonia and choreoathtetoid movements despite good control of hyperphenylalaninemia. Tetrahydrobiopterin deficiency was diagnosed at age 3 years. She had a dramatic response to L-dopa, which persisted at a stable dose for 29 years. Reducing the L-dopa dose led to severe axial hypotonia and limb dystonia, and increasing it led to florid abnormal movements and behavioral disorders. This report illustrates the role of dopamine modulation in motor, psychiatric, and endocrine functions., (Copyright (c) 2005 Movement Disorder Society.)

Severe 6-pyruvoyl-tetrahydrobiopterin synthase deficiency is a tetrahydrobiopterin deficiency disorder that presents in infancy with developmental delay, seizures, and abnormal movements associated with hyperphenylalaninemia usually detectable by neonatal phenylketonuria screening programs. We describe an 8-year-old girl with delay, seizures, and dystonia with mild hyperphenylalaninemia detected in late childhood. The diagnosis of 6-pyruvoyl-tetrahydrobiopterin synthase deficiency was made by analysis of pterins in urine, pterins and neurotransmitters in cerebrospinal fluid, and enzyme assay. The patient improved clinically taking oral tetrahydrobiopterin, levodopa/carbidopa, and 5-hydroxytryptophan. This treatable condition may not always be detected by routine population screening for hyperphenylalaninemia.

Objective: Synthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthases, is strongly induced on immunostimulation in vascular endothelial cells (VECs). Expression of GTP cyclohydrolase I (GTPCH), the first enzyme in BH4 biosynthesis, is regulated by cytokines and considered rate-limiting. Herein we investigated the molecular mechanism and relevance of cytokine-dependent regulation of 6-pyruvoyltetrahydropterin synthase (PTPS), the second enzyme in BH4 synthesis, in human coronary artery endothelial cells (HCAECs)., Methods and Results: Real-time polymerase chain reaction revealed a 4-fold induction of PTPS and a 300-fold induction of GTPCH expression by interleukin (IL)-1beta/tumor necrosis factor-alpha/interferon-gamma, mainly through de novo transcription. On immunostimulation, PTPS became rate-limiting. Importantly, IL-1beta induced PTPS rather than GTPCH. As a result, IL-1beta contributed significantly to the amount of BH4 produced (+40%) but concomitantly reduced the accumulation of the GTPCH intermediate, 7,8-dihydroneopterin triphosphate (-50%)., Conclusions: Our data show that PTPS induction is necessary for optimized BH4 synthesis in cytokine-stimulated HCAECs and point to IL-1beta as a leading cytokine in this process.

The tetrahydrobiopterin (BH4) cofactor is essential for the biosynthesis of catecholamines and serotonin and for nitric-oxide synthase (NOS). Alterations in BH4 metabolism are observed in various neurological and psychiatric diseases, and mutations in one of the human metabolic genes causes hyperphenylalaninemia and/or monoamine neurotransmitter deficiency. We report on a knockout mouse for the Pts gene, which codes for a BH4-biosynthetic enzyme. Homozygous Pts-/- mice developed with normal morphology but died after birth. Upon daily oral administration of BH4 and neurotransmitter precursors the Pts-/- mice eventually survived. However, at sexual maturity (6 weeks) the mice had only one-third of the normal body weight and were sexually immature. Biochemical analysis revealed no hyperphenylalaninemia, normal brain NOS activity, and almost normal serotonin levels, but brain dopamine was 3% of normal. Low dopamine leads to impaired food consumption as reflected by the severe growth deficiency and a 7-fold reduced serum insulin-like growth factor-1 (IGF-1). This is the first link shown between 6-pyruvoyltetrahydropterin synthase- or BH4-biosynthetic activity and IGF-1.

Unlabelled: The outcome of 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency, the most common form of tetrahydrobiopterin (BH4) deficiency, depends on factors such as severity of the disease, type of mutation, time of diagnosis, and mode of treatment. We investigated five patients from four different families, four of them presenting with the severe form of PTPS deficiency and one with the mild peripheral form. In this study, missense (L26F, T67M, P87L, V124L, D136G, D136V) and nonsense (R15-16ins) mutations were detected by reverse transcriptase polymerase chain reaction and sequence analysis. Two patients with the severe form were compound heterozygotes (T67M/P87L and D136G/R15-16ins), two siblings were homozygous for the D136V mutation, and in the patient with the mild form, heterozygous L26F/V124L mutations were present. Two patients are on combined therapy with L-dopa/carbidopa/5-hydroxytryptophan plus BH4, the siblings are on monotherapy with BH4, and the patient with the mild form is now off treatment, presenting with normal plasma phenylalanine levels., Conclusion: Long-term follow-up shows that the outcome of 6-pyruvoyl-tetrahydropterin synthase deficiency benefits from treatment started in the first months of life and that the phenotype may change with age. Additionally, depending on the type of mutations, prenatal damage to the fetus may multiply the clinical abnormalities and thus worsen the prognosis of the disease. In patients initially diagnosed with the mild peripheral form of the disease, therapy with tetrahydrobiopterin should be stopped after some time to test whether hyperphenylalaninaemia was only a transient condition.

Tetrahydrobiopterin (BH(4)) cofactor is essential for various processes, and is present in probably every cell or tissue of higher organisms. BH(4) is required for various enzyme activities, and for less defined functions at the cellular level. The pathway for the de novo biosynthesis of BH(4) from GTP involves GTP cyclohydrolase I, 6-pyruvoyl-tetrahydropterin synthase and sepiapterin reductase. Cofactor regeneration requires pterin-4a-carbinolamine dehydratase and dihydropteridine reductase. Based on gene cloning, recombinant expression, mutagenesis studies, structural analysis of crystals and NMR studies, reaction mechanisms for the biosynthetic and recycling enzymes were proposed. With regard to the regulation of cofactor biosynthesis, the major controlling point is GTP cyclohydrolase I, the expression of which may be under the control of cytokine induction. In the liver at least, activity is inhibited by BH(4), but stimulated by phenylalanine through the GTP cyclohydrolase I feedback regulatory protein. The enzymes that depend on BH(4) are the phenylalanine, tyrosine and tryptophan hydroxylases, the latter two being the rate-limiting enzymes for catecholamine and 5-hydroxytryptamine (serotonin) biosynthesis, all NO synthase isoforms and the glyceryl-ether mono-oxygenase. On a cellular level, BH(4) has been found to be a growth or proliferation factor for Crithidia fasciculata, haemopoietic cells and various mammalian cell lines. In the nervous system, BH(4) is a self-protecting factor for NO, or a general neuroprotecting factor via the NO synthase pathway, and has neurotransmitter-releasing function. With regard to human disease, BH(4) deficiency due to autosomal recessive mutations in all enzymes (except sepiapterin reductase) have been described as a cause of hyperphenylalaninaemia. Furthermore, several neurological diseases, including Dopa-responsive dystonia, but also Alzheimer's disease, Parkinson's disease, autism and depression, have been suggested to be a consequence of restricted cofactor availability.

6-Pyruvoyl-tetrahydropterin synthase (PTS or PTPS) is involved in tetrahydrobiopterin (BH(4)) biosynthesis, the cofactor for various enzymes including the aromatic amino acid hydroxylases. Inherited PTPS deficiency is a heterogeneous disease with different phenotypes leading to BH(4) depletion. The severe form of PTPS deficiency causes hyperphenylalaninemia and monoamine neurotransmitter deficiency, whereas the mild form gives rise to hyperphenylalaninemia only. From 228 patients with PTPS deficiency at least 32 different mutant alleles have been identified on its corresponding gene, located on chromosome 11q22.3-q23.3. Here we describe a new allele from a child with PTPS deficiency who exhibited a mild but transient form of hyperphenylalaninemia, yet was deficient in CSF monoamines. The patient was found to carry, on her genomic DNA and cDNA, a homozygous A>G transition, leading to PTPS codon alteration Tyr99 to Cys (Y99C). The mother and several members of the maternal family were carriers of the Y99C allele, also verified by the reduced PTPS enzyme activity in erythrocytes. By cytogenetic, molecular, and FISH analyses, a de novo deletion spanning from 11q14 to 11q23.3 on the patient's paternal chromosome was mapped, establishing hemizygosity of the Y99C allele. The PTPS mutation observed in this patient generates a novel phenotype with an apparently isolated central form of BH(4) deficiency., (Copyright 2000 Wiley-Liss, Inc.)

Background: Tetrahydrobiopterin (BH4) is an essential cofactor for catecholamine and serotonin neurotransmitter biosynthesis. BH4 biosynthesis is carried out in a three-enzyme pathway involving GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS) and sepiapterin reductase (SR). Treatment of genetic defects leading to BH4 deficiency requires neurotransmitter replacement since synthetic cofactor does not efficiently penetrate the blood-brain barrier. Autologous fibroblasts transplanted into the brain as depository cells for drug delivery might offer an alternative. However, normal fibroblasts do not express GTPCH, and fibroblasts from PTPS patients lack two biosynthetic enzymes for BH4 production., Methods: We engineered primary fibroblasts by the use of triple-cistronic, retroviral vectors for cofactor production., Results: Constitutive SR activity in these cells enabled BH4 biosynthesis by transducing GTPCH and PTPS cDNAs together with a selective marker coupled in a single transcript with two IRES-elements in tandem. Upon reaching a critical concentration (> 400 pmol/mg protein) of intracellular BH4, the fibroblasts efficiently released cofactor even under non-dividing conditions., Conclusion: The use of triple-cistronic vectors for single transduction to reconstitute metabolic pathways or to treat multi-genetic diseases may be useful for engineering, for instance, depository cells for various organs, including the nervous system.

Background: Deficiency of 6-pyruvoyltetrahydropterin synthase (PTPS) is a recessively inherited disorder that leads to depletion of 5,6,7, 8-tetrahydrobiopterin, the obligatory cofactor for hydroxylation of phenylalanine, tyrosine, and tryptophan. A marker for neonatal detection of PTPS deficiency is hyperphenylalaninemia (HPA). Molecular analysis would provide a simple and reliable means for distinguishing PTPS deficiency from other potential causes of HPA., Methods: We developed a method based on PCR in combination with denaturing gradient gel electrophoresis (DGGE) that rapidly scans the six coding sequences and all splice sites of the PTPS gene (PTS) for mutations. This method was used to examine the status of the PTS gene in control samples with known PTS mutations and in five patients with PTPS deficiency., Results: Two features of the PTS gene posed particular problems in relation to DGGE analysis: the very high GC content of exon 1, and a 15-bp poly(dT) stretch in the acceptor splice site of intron 1. Both problems were solved by special design of amplification primers. PCR and DGGE conditions were adjusted to allow simultaneous analysis of all six regions of the PTS gene. Using this one-step approach, all control mutations were readily resolved. Among the five PTPS patients, four mutations were identified, including IVS1-3C-->G, IVS2-7T-->A, V57del, and V97M (289G-->A). The IVS1-3C-->G mutation was shown by reverse transcription-PCR analysis to produce multiple splice variants., Conclusions: We have established a fast and reliable screening method for detection of mutations and small deletions/insertions in the PTS gene. This method should be useful for rapid diagnosis of PTPS deficiency in newborns with HPA.

6-Pyruvoyltetrahydropterin synthase (PTPS) participates in tetrahydrobiopterin cofactor biosynthesis. We previously identified in a PTPS-deficient patient an inactive PTPS allele with an Arg(16) to Cys codon mutation. Arg(16) is located in the protein surface exposed phosphorylation motif Arg(16)-Arg-Ile-Ser, with Ser(19) as the putative phosphorylation site for serine-threonine protein kinases. Purification of recombinant PTPS-S19A from bacterial cells resulted in an active enzyme (k(cat)/K(m) = 6.4 x 10(3) M(-1) s(-1)), which was similar to wild-type PTPS (k(cat)/K(m) = 4.1 x 10(3) M(-1) s(-1)). In assays with purified enzymes, wild-type but not PTPS-S19A was a specific substrate for the cGMP-dependent protein kinase (cGK) type I and II. Upon expression in COS-1 cells, PTPS-S19A was stable but not phosphorylated and had a reduced activity of approximately 33% in comparison to wild-type PTPS. Extracts from several human cell lines, including brain, contained a kinase that bound to and phosphorylated immobilized wild-type, but not mutant PTPS. Addition of cGMP stimulated phosphotransferase activity 2-fold. Extracts from transfected COS-1 cells overexpressing cGKII stimulated Ser(19) phosphorylation more than 100-fold, but only 4-fold from cGKI overexpressing cells. Moreover, fibroblast extracts from mice lacking cGKII exhibited significantly reduced phosphorylation of PTPS. These results suggest that Ser(19) of human PTPS may be a substrate for cGKII phosphorylation also in vivo, a modification that is essential for normal activity.

Mutations in the 6-pyruvoyltetrahydropterin synthase (PTPS) gene result in persistent hyperphenylalaninemia and severe catecholamine and serotonin deficiencies. We investigated at the DNA level a family with a PTPS-deficient child presenting with an unusual form of transient hyperphenylalaninemia. The patient exhibited compound heterozygosity for the PTPS-mutant alleles N47D and D116G. Transfection studies with single PTPS alleles in COS-1 cells showed that the N47D allele was inactive, while D116G had around 66% of the wild-type activity. Upon co-transfection of two PTPS alleles into COS-1 cells, the N47D allele had a dominant negative effect on both the wild-type PTPS and the D116G mutant with relative reduction to about 20% of control values. Whereas the mother and the father had reduced enzyme activity in red blood cells (34.7% and 51.7%, respectively) and skin fibroblasts (2.8% and 15.4%, respectively), the clinically normal patient had in these cells activities at the detection limits, although PTPS-cross-reactive material was present in the fibroblasts. The specifically low PTPS activity in the mother's cells corroborated the evidence of a dominant negative effect of the maternal N47D allele on wild-type PTPS.

The 6-pyruvoyl-tetrahydropterin synthase (PTPS) is the second enzyme in the biosynthetic pathway from GTP to tetrahydrobiopterin (BH4). BH4 is an essential cofactor of NO synthases and aromatic amino acid hydroxylases, the latter being responsible for hepatic phenylalanine degradation and monoamine neurotransmitter biosynthesis. BH4 deficiency due to autosomal recessive mutations in the human gene for PTPS leads to a broad range of phenotypes ranging from mild hyperphenylalaninemia to high phenylalanine levels concomitant with neurotransmitter depletion. An animal model to study PTPS deficiency is thus desired to investigate the molecular basis of the disease and its variability. Here, we report on the isolation and recombinant expression of the mouse PTPS gene, Pts. It is located on chromosome 9C-D and contains six exons with an open reading frame of 144 codons. The derived protein monomer has a molecular mass of 16187 Da and shows 82% and 93% identity to its human and rat counterparts, respectively. The mouse PTPS was expressed in bacterial cells and purified to homogeneity. The kinetic properties of the recombinant protein, apparent Km of approximately 10 microM and k(cat) of 0.27 s(-1), were similar to the native mouse enzyme in liver and brain extracts, and to the corresponding human and rat PTPS.

Background: The nitric oxide synthase cofactor tetrahydrobiopterin (BH4) is involved in the regulation of endothelium-dependent vascular functions mediated by nitric oxide. Vascular endothelial cells synthesize and secrete large amounts of BH4 on cytokine activation. There is scant knowledge about molecular mechanisms of cytokine-triggered BH4 production in endothelial cells., Methods and Results: Pteridine production, mRNA expression of GTP cyclohydrolase (GTPCH) and 6-pyruvoyltetrahydropterin synthase (PTPS) (both key enzymes of BH4 biosynthesis), and PTPS activity were studied in human umbilical vein endothelial cells (HUVECs) exposed to inflammatory cytokines. BH4 levels were

The tetrahydrobiopterin (BH4) cofactor is essential for the aromatic amino acid hydroxylases that are involved in phenylalanine degradation and catecholamine and serotonin biosynthesis. Furthermore, BH4 is an essential and limiting cofactor for all types of nitric oxide synthases. BH4 deficiency results in hyperphenylalaninemia and monoamine neurotransmitter depletion associated with progressive mental retardation and is most commonly due to autosomal recessive mutations in 6-pyruvoyltetrahydropterin synthase (PTPS), the second enzyme for cofactor biosynthesis. Due to the relatively poor blood-brain barrier penetration of the cofactor, conventional therapy requires, besides oral doses of synthetic BH4, administration of neurotransmitter precursors and an aromatic amino acid decarboxylase inhibitor. The outcome of this therapy is not always beneficial. In this study we transduced into primary patient fibroblasts the human cDNAs for the BH4 biosynthetic enzymes GTP cyclohydrolase I and PTPS, expressed from different retroviral vectors. This allowed BH4 biosynthesis in originally PTPS-deficient cells. Moreover, the double-transduced fibroblasts released between 200 and 800 pmol of BH4/10(6) cells/day. Such engineered fibroblasts may be grafted into the central nervous system and used as depository cells for constitutive delivery of BH4.

Severe tetrahydrobiopterin (BH4) deficiency is a naturally occurring model of cerebral catecholamine and serotonin shortage. Examination of the stimulated release and physiologic secretion pattern of several hormones in affected individuals permits certain conclusions concerning the involvement of these neurotransmitters in hormone regulation. Treatment, moreover, permits the ranking of the quality of the therapeutic regimens in use according to the degree of hormonal alteration. The 24-h secretion pattern of prolactin, GH, cortisol, and melatonin and the stimulated release of prolactin, GH, TSH, and gonadotropins were studied in an affected girl. Severe hyperprolactinemia with disruption of the pulsatile and circadian secretion pattern was the prevailing feature. The GH physiologic secretion pattern was not affected, but its stimulation was impaired. Melatonin displayed a normal circadian secretion pattern; the rhythm, however, was advanced by several hours. Conventional treatment of BH4 deficiency, i.e. BH4, 5-hydroxytryptophan, and L-DOPA/carbidopa (the last named given in three doses per day), suppresses prolactin levels merely for a few hours. L-DOPA/carbidopa given at shorter intervals or, even better, as a slow release preparation, is more effective in suppressing prolactin levels. Our data indicate immense hyperprolactinemia but few other hormonal disturbances in severe BH4 deficiency. Prolactin secretion may serve as an extremely sensitive marker for the hypothalamic dopamine content under different therapeutic regimens. Treatment with an L-DOPA/carbidopa slow release preparation produces virtually normal prolactin levels.

6-Pyruvoyl-tetrahydrobiopterin synthase (PTPS) is involved in tetrahydrobiopterin (BH4) biosynthesis, the cofactor for various enzymes including the hepatic phenylalanine hydroxylase. Inherited PTPS deficiency leads to BH4 depletion, causes hyperphenylalaninemia, and requires cofactor replacement therapy for treatment. We previously isolated the human PTPS cDNA and recently characterized its corresponding gene, PTS. Here we developed PCR-based mutation analysis with newly designed primers to detect genomic alterations and describe five mutations, four of which are novel, in the PTS gene of four Italian families with affected individuals. The mutant alleles found included three missense mutations (T67M, K129E, D136V), a previously described triplet deletion (delta V57), and a single c-3-->g transversion in the 3'-acceptor splice site of intron 1, leading to cryptic splice site usage that resulted in a 12 bp deletion (mutant allele delta (K29-S32)). Except for K129E, all mutant alleles were inactive and/or unstable proteins, as shown by recombinant expression and Western blot analysis of patients' fibroblasts. The PTPS-deficient patient with the homozygous K129E allele had transient hyperphenylalaninemia, did not depend on BH4 replacement therapy, and showed normal PTPS immunoreactivity, but no enzyme activity in primary fibroblasts and red blood cells. In contrast to its inactivity in these cells, the K129E mutant was 2-3 fold more active than wild-type PTPS when transfected into COS-1 or the human hepatoma cell line Hep G2. K129E appears thus as a mutant PTPS whose activity depends on the cell type.

Tetrahydrobiopterin deficiencies are highly heterogeneous disorders, with more than 30 molecular lesions identified in the past 2 years in the GTP cyclohydrolase I and 6-pyruvoyl-tetrahydropterin synthase genes. The spectrum of mutations causing a reduction of these two biosynthetic enzymes is reviewed. Only three mutations, two present homozygously, are reported in the GTP cyclohydrolase I gene to cause the rare autosomal recessively inherited form of hyperphenylalaninemia. Most of the other mutations, which are scattered over the entire coding region for the six exon-containing GTP cyclohydrolase I gene, are observed in a heterozygous state with the wild-type allele and are associated with the dominant DOPA-responsive dystonia. Compound heterozygous or homozygous mutations spread over all six exons encoding the 6-pyruvoyl-tetrahydropterin synthase cause an autosomal recessively inherited variant of hyperphenylalaninemia, mostly accompanied by a deficiency of dopamine and serotonin.

Autosomal recessive mutations in the 6-pyruvoyltetrahydropterin synthase (PTPS) gene are the most common reason for hyperphenylalaninemia due to tetrahydrobiopterin deficiency. We used the previously isolated PTPS cDNA as a probe and identified the human gene, PTS, located on chromosome 11q22.3-q23.3, and a retropseudogene, PTS-P1, assigned to 9p12-p13 (symbols approved by the human genome nomenclature committee). PTS-P1 has 74% similarity to the 3' portion of PTPS cDNA. The PTS gene spans about 8 kb and consists of 6 exons, as revealed by DNA-sequence analysis. This gene structure differs from that published previously which was reported to contain only two exons [Ashida, A., Owada, M. & Hatakeyama, K. (1994) Genomics 24,408-410]. By means of intron-specific primers, we amplified exon 3 from genomic DNA of a PTPS-deficient patient and found a mutation in the 3' acceptor splice site, which is responsible for skipping of exon 3.

Tetrahydrobiopterin (BH4) deficiency, a variant form of hyperphenylalaninemia with progressive neurological dysfunction, is primarily caused by autosomal recessive mutations in the gene encoding the 6-pyruvoyl-tetrahydropterin synthase (PTPS). PTPS is a biosynthetic enzyme for the BH4 co-factor, and its deficiency is associated with a malfunction of the phenylalanine catabolism in the liver and a lack of biogenic amine neurotransmitters dopamine and serotonin in the brain. We have previously isolated the wild-type PTPS cDNA and identified several mutations responsible for a decreased enzyme in patients. This study reports the in vitro correction of BH4 deficiency by using retrovirus mediated transfer of the PTPS cDNA into primary fibroblast cultures established from different patients. The Bing packaging cell line was used for amphotropic virus production. Following PTPS gene transfer, stimulation with cytokines restored biosynthesis of BH4 in originally defective cells to values comparable to those of heterozygous fibroblasts from clinically healthy subjects. These results not only provide a direct proof that the mutations in PTPS were causative for the mutant phenotype, but they are also the first step toward gene therapy as a potential alternative approach to treat BH4 deficiency.

Four naturally occurring mutants with single amino acid alterations in human 6-pyruvoyltetrahydropterin synthase (PTPS) were overexpressed and characterized in vitro. The corresponding DNA mutations were found in patients with hyperphenylalaninemia and monoamine neurotransmitter insufficiency due to lack of the tetrahydrobiopterin biosynthetic enzyme PTPS. To predict the structure of the mutant enzymes, computer modeling was performed based on the solved three-dimensional structure of the homohexameric rat enzyme. One mutant (delta V57) is incorrectly folded and thus unstable in vitro and in vivo, while a second mutant (P87L) has substantial activity but enhanced sensitivity to local unfolding. Two other mutants, R16C and R25Q, form stable homomultimers and exhibit significant activity in vitro but no activity in COS-1 cells. In vivo 32P labeling showed that wild-type PTPS, P87L, and R25Q are phosphorylated, while R16C is not modified. This strongly suggests that the serine 19 within the consensus sequence for various kinases, RXXS, is the site of modification. Our results demonstrate that PTPS undergoes protein phosphorylation and requires additional, not yet identified post-translational modification(s) for its in vivo function.

A variant type of hyperphenylalaninemia is caused by a deficiency of tetrahydrobiopterin (BH4), the obligatory cofactor for phenylalanine hydroxylase. The most frequent form of this cofactor deficiency is due to lack of 6-pyruvoyl-tetrahydropterin synthase (PTPS) activity, the second enzyme in the biosynthetic pathway for BH4. The human liver cDNA for PTPS was previously isolated, and the recombinant protein was found to be active when expressed in Escherichia coli. We now have investigated two patients for their molecular nature of this autosomal recessive disorder. Both patients were diagnosed as PTPS deficient, one with the central and one with the peripheral form, on the basis of an elevated serum phenylalanine concentration concomitant with lowered levels of urinary biopterin and PTPS activity in erythrocytes. Molecular analysis was performed on the patients' cultured primary skin fibroblasts. PTPS activities were found in vitro to be reduced to background activity. Direct cDNA sequence analysis using reverse transcriptase-PCR technology showed for the patient with the central from a homozygous G-to-A transition at codon 25, causing the replacement of an arginine by glutamine (R25Q). Expression of this mutant allele in E. coli revealed 14% activity when compared with the wild-type enzyme. The patient with the peripheral form exhibited compound heterozygosity, having on one allele a C-to-T transition resulting in the substitution of arginine 16 for cysteine (R16C) in the enzyme and having on the second allele a 14-bp deletion (delta 14bp), leading to a frameshift at lysine 120 and a premature stop codon (K120-->Stop). Heterologous expression of the enzyme with the single-amino-acid exchange R16C revealed only 7% enzyme activity, whereas expression of the deletion allele delta 14bp exhibited no detectable activity. All three mutations, R25Q, R16C, and K120-->Stop, affect evolutionarily conserved residues in PTPS, result in reduced enzymatic activity when reconstituted in E. coli, and are thus believed to be the molecular cause for the BH4 deficiency. This is the first report describing mutations in PTPS that lead to BH4 deficiency.

Prenatal diagnosis of tetrahydrobiopterin (BH4) deficiency was undertaken by evaluating the pterin patterns in amniotic fluid and the specific enzyme activities in fetal or extrafetal tissues. This allowed the prenatal diagnosis in 19 pregnancies at risk. In 8 families with a child already affected by dihydropteridine reductase deficiency 4 fetuses were diagnosed as homozygotes and 4 as heterozygotes for the defect. In 11 families with a child affected by 6-pyruvoyl tetrahydropterin synthase deficiency 4 fetuses were homozygous, 4 heterozygous and 3 normal. This study also advanced our knowledge of tetrahydrobiopterin metabolism during fetal development. The key enzymes involved in the biosynthesis of BH4 are expressed early and allow the fetus to be autotrophous for its cofactor requirement. In a twin pregnancy, both fetuses were diagnosed to be heterozygotes for dihydropteridine reductase deficiency and primapterin (7-biopterin) in amniotic fluid was increased. This indicates that pterin-4 alpha-carbinolamine dehydratase activity seems to be differently expressed during fetal life. As a consequence, pterins detected in amniotic fluid are of fetal origin and 6- and 7-substituted pterins can be present in amniotic fluid in higher proportions when compared with other body fluids.

6-Pyruvoyl-tetrahydropterin synthase is the rate-limiting enzyme in the synthesis of human tetrahydrobiopterin, a cofactor for several hydroxylases involved in catecholamine and serotonin biosynthesis. The human and rat liver cDNAs encoding the 16-kDa subunit of 6-pyruvoyl tetrahydropterin synthase were expressed as maltose-binding-6-pyruvoyl-tetrahydropterin-synthase fusion proteins. After cleavage from the fusion protein, the human and rat enzymes were purified to homogeneity. Apparent Km for the substrate dihydroneopterin triphosphate (8.5 microM for the human and 8.0 microM for the rat enzyme), pI (4.6 and 4.8) and heat stability of the recombinant enzymes were similar to the native enzymes. The specific activity of the enzymes was enhanced up to fourfold in the presence of dithiothreitol during purification. The modification of the only cysteine residue in rat 6-pyruvoyl tetrahydropterin synthase, which is conserved in the human enzyme, inhibited its activity up to 80%. Modification under non-reducing conditions of both cysteine residues of the human enzyme by N-ethylpyridine resulted in a 95% loss of enzyme activity. This demonstrates that the two cysteines are not linked by disulfide bridges but rather involved in catalysis. Cross-linking experiments and analysis by gel electrophoresis showed predominantly trimeric and hexameric forms of the recombinant enzymes from both species suggesting that the native form is a homohexamer of 98 kDa, for the human, and 95 kDa, for the rat enzyme, composed of two trimeric subunits.

We describe a new fully reliable method for the differential diagnosis of tetrahydrobiopterin-dependent hyperphenylalaninaemia (HPA). The method comprises the combined phenylalanine (Phe) plus tetrahydrobiopterin (BH4) oral loading test and enables the selective screening of BH4 deficiency when pterin analysis is not available or when a clear diagnosis has not been previously made. It should be performed together with the measurement of dihydropteridine reductase (DHPR) activity in blood. The new combined loading test was performed in nine patients with primary HPA, three with classical phenylketonuria (PKU), three with DHPR deficiency, and three with 6-pyruvoyl tetrahydropterin synthase (PTPS) deficiency. Three hours after oral Phe loading (100 mg/kg body weight), synthetic BH4 was administered orally at doses of either 7.5 or 20 mg/kg body weight. Amino acid (Phe and tyrosine) and pterin (neopterin and biopterin) metabolism and kinetics were analysed. By exploiting the decrease in serum Phe 4 and 8 h after administration, a clear response was obtained with the higher BH4 dose (20 mg/kg body weight), allowing detection of all cases of BH4 deficiency, as well as differentiation of BH4 synthesis from regeneration defects. Since DHPR deficient patients who were previously shown to be non-responsive to the simple BH4 loading test gave a positive response, the combined Phe plus BH4 loading test can be used as a more reliable tool for the differential diagnosis of HPA in these patients. Moreover, it takes advantage of being performed while patients are on a Phe-restricted diet.

The relationship between blood phenylalanine concentrations and serum and erythrocyte biopterin and neopterin concentrations was investigated in 20 phenylketonuric patients with different dietary compliance. At serum phenylalanine concentrations ranging from 43 to 1004 mumol/l, a good correlation was found with serum biopterin (r = 0.76, P < 0.001) and with red blood cell biopterin (r = 0.62, P < 0.001). A similar correlation was found between serum neopterin and phenylalanine (r = 0.60, P < 0.001). The correlation between red blood cell neopterin and serum phenylalanine was less evident, however (r = 0.47, P < 0.005). After oral loading with phenylalanine (100 mg/kg body weight), serum and red blood cell biopterin concentrations increased in patients with classical phenylketonuria as well as in one patient with dihydropteridine reductase deficiency in response to the induced acute hyperphenylalaninemia. One patient suffering from 6-pyruvoyl tetrahydropterin synthase deficiency was loaded orally with tetrahydrobiopterin (20 mg/kg body weight). The kinetics of administered cofactor confirmed its rapid absorption, with early increase of serum concentrations followed by its transport into the red blood cells. The half-life of biopterin was approximately 7 h in serum and 15 h in red blood cells. Because both values are less than the half-life of phenylalanine (20-30 h) in serum, biopterin measurement offers no advantage in monitoring dietary control in hyperphenylalaninemic patients.

A new immunization method has been developed for the production of monoclonal antibodies. This technique uses small amounts of partially purified and weak immunogenic antigen, bound to membranes after blotting from SDS-PAGE. For this purpose two different membranes have been tested. Immobilon-P polyvinylidene difluoride (PVDF) membranes were less mitogenic than nitrocellulose membranes, and were therefore selected for the in vitro immunization using 6-pyruvoyl tetrahydropterin synthase as antigen. The in vitro immunization method was then used for the production of monoclonal antibodies against 6-pyruvoyl tetrahydropterin synthase, one of the key enzymes on the biosynthetic pathway of tetrahydrobiopterin, the natural cofactor of the mammalian aromatic amino acid hydroxylases. The antibodies obtained were mainly of the IgM type.

The most frequent variant of atypical phenylketonuria, an inborn error of metabolism, is characterized by a low activity of the 6-pyruvoyl tetrahydropterin synthase. We purified and characterized this enzyme from salmon liver known to contain high levels. After digestion, peptides were sequenced by tandem mass spectrometry and/or automated Edman microsequence analysis. Both a free amine terminus and an N-acetylated amine terminus were found, indicating the presence of two isoforms. The peptide sequences determined here have a high degree of homology with the protein sequence deduced from cDNA for rat 6-pyruvoyl tetrahydropterin synthase (1), however, the amine termini of these proteins differ significantly.

Monoclonal antibodies were produced against human pituitary gland 6-pyruvoyl tetrahydropterin synthase, one of the key enzymes in the biosynthesis of tetrahydrobiopterin, by in vitro immunization with the antigen directly blotted from SDS-PAGE to polyvinylidene difluoride membranes. The antibodies produced show crossreactivity in the enzyme linked immunosorbent assay, not only with the human 6-pyruvoyl tetrahydropterin synthase but some also with the same enzyme isolated from salmon liver. 6-Pyruvoyl tetrahydropterin synthase was localized immuno-enzymatically in peripheral blood smears and in skin fibroblasts by the use of these monoclonal antibodies and the alkaline phosphatase monoclonal anti-alkaline phosphatase labeling technique.

6-Pyruvoyl tetrahydropterin synthase, the enzyme that catalyses the conversion of 7,8-dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, was purified 3,330-fold from human pituitary gland with an overall recovery of 30%. The native enzyme has a molecular mass of 68 kD and consists of four identical subunits of 16.5 kD. The pH optimum of the enzyme in Tris/HCl buffer is 7.5. The enzyme is dependent on Mg2+ and NADPH and has a Michaelis-Menten constant of 10 microM for its natural substrate, 7,8-dihydroneopterin triphosphate. The isoelectric point of the human enzyme is 4.3-4.6. The human pituitary gland enzyme is heat instable in contrast to the enzymes from human, rat and salmon liver, and Drosophila head. The amino acid composition showed remarkably high content of acidic amino acids Asp and Glu. The N-terminus was found to be blocked.

We describe the clinical, neurologic, and biochemical findings in 10 patients with 6-pyruvoyl tetrahydropterin synthase (6-PTS) deficiency from seven families, all of whom originate from one large tribe in Saudi Arabia. This deficiency presents with severe, early onset of failure to thrive, neurologic deterioration, and morbidity and mortality secondary to repeated episodes of bronchopneumonia or cardiorespiratory abnormalities. The urinary pterin excretion pattern indicates deficient activity of 6-PTS, which has been confirmed by direct enzyme assay in red blood cells of three patients. We treated our patients with combined use of tetrahydrobiopterin 20 mg/kg/d, L-dihydroxyphenylalanine 15 mg/kg/d, carbidopa 3.75 mg/kg/d, and L-5-hydroxytryptophan 5 mg/kg/d. Neurologic findings improved significantly in all after 5 to 24 months. Although head circumference and weight returned to the lower limit of normal in four, height normalized only in one of seven patients. Despite an unrestricted diet during combined therapy, blood phenylalanine and urinary excretion of neopterin and biopterin returned to normal.