Biosynthesis Of Tetrahydrobiopterin Research Articles

A brain-specific decrease of the tyrosine hydroxylase protein in sepiapterin reductase-null mice—as a mouse model for Parkinson’s disease

Sepiapterin reductase (SPR) is an enzyme that acts in the third and final step of tetrahydrobiopterin (BH4) biosynthesis. The human Spr gene locates within the region of 2.5 MB mapped to PARK3, an autosomal dominant form of familial Parkinson’s diseases. In order to explore the role of SPR in the metabolism of BH4, we produced and analyzed Spr-deficient mice. Most of Spr-null mice survived beyond two weeks. Whereas the BH4 contents in the homozygous mutant mice were greatly decreased than those in wild-type and heterozygous mice, the substantial amounts of BH4 were remained even 17 days after delivery. Spr-null mice exhibited severe monoamine deficiencies and a tremor-like phenotype after weaning. The amount of TH protein in the brain of Spr-null mice was less than 10% of wild-type, while TH protein in the adrenal, phenylalanine hydroxylase protein in the liver, and nNOS in the brain were not altered. These data suggest an essential role of SPR in the biosynthesis of BH4, and that the SPR gene could be a candidate gene for PARK3.

Segawa's disease: dopa-responsive dystonia

The genetics and symptoms of Segawa's disease are described. The latter can show considerable variation, especially if the onset of the condition is delayed. It is usually of autosomal dominant inheritance, but a recessive form can occur. The dominant and recessive forms are caused by a gene mapped to chromosome 14. The fluctuating dystonia is typical, but is not essential for the diagnosis. Affected children can suffer from sleep disorders, such as excessive sleepiness and nightmares. In some children with this condition mutations in the GCH-1 gene coding for guanosine triphosphate cyclohydrolase 1 have been found. The enzyme catalyses the first step in the biosynthesis of tetrahydrobiopterin. A point mutation in the tyrosine hydrolase gene has been found in some of the recessive forms, and the gene mapped to chromosome 11, but this cannot be called Segawa's disease. The deficiency of guanosine triphosphate cyclohydrolase 1 causes a defect in serotonin metabolism and in the biosynthesis of tetrabiopterin and a disturbance of dopamine metabolism. This leads to a deficiency of dopamine in the striatum, and to the motor dysfunction of the syndrome. The diagnosis can be established by cerebrospinal fluid examination, and confirmed in some patients by genetic studies. Treatment is with levodopa, and the results are dramatic.

Regulation of Tetrahydrobiopterin Biosynthesis by Shear Stress

An essential cofactor for the endothelial NO synthase is tetrahydrobiopterin (H4B). In the present study, we show that in human endothelial cells, laminar shear stress dramatically increases H4B levels and enzymatic activity of GTP cyclohydrolase (GTPCH)-1, the first step of H4B biosynthesis. In contrast, protein levels of GTPCH-1 were not affected by shear. Shear did not change protein expression or activity of the downstream enzymes 6-pyruvoyl-tetrahydropterin synthase and sepiapterin reductase and decreased protein levels of the salvage enzyme dihydrofolate reductase. Oscillatory shear only modestly affected H4B levels and GPTCH-1 activity. We also demonstrate that laminar, but not oscillatory shear stress, stimulates phosphorylation of GTPCH-1 on serine 81 and that this is mediated by the alpha prime (alpha') subunit of casein kinase 2. The increase in H4B caused by shear is essential in allowing proper function of endothelial NO synthase because GPTCH-1 blockade with 2,4-diamino-6-hydroxypyrimidine during shear inhibited dimer formation of endothelial NO synthase, increased endothelial cell superoxide production, and prevented the increase in NO production caused by shear. Thus, shear stress not only increases endothelial NO synthase levels but also stimulates production of H4B by markedly enhancing GTPCH-1 activity via casein kinase 2-dependent phosphorylation on serine 81. These findings illustrate a new function of casein kinase 2 in the endothelium and provide insight into regulation of GTPCH-1 activity.

Widespread occurrence of glyceryl ether monooxygenase activity in rat tissues detected by a novel assay

An assay was set up for glyceryl ether monooxygenase activity in tissue samples using the novel substrate 1-O-pyrenedecyl-sn-glycerol and high-performance liquid chromatographic analysis of reaction mixtures with fluorescence detection, allowing robust detection of enzymatic activity in microgram amounts of tissue homogenates. The activity partially purified from rat liver strictly depended on the presence of a tetrahydropteridine. Tetrahydrobiopterin-dependent glyceryl ether monooxygenase activity was observed in all rat tissues tested except female heart, with highest activities in liver, intestine, and cerebellum. Activity was not uniformly distributed in brain: it was higher in cerebellum than in striatum or cortex. These data demonstrate that tetrahydrobiopterin-dependent glyceryl ether monooxygenase is found not only in liver and the gastrointestinal tract but also in brain and other organs of the rat and provide an additional goal for tetrahydrobiopterin biosynthesis in these organs.

Effect of peripherally administered lipopolysaccharide (LPS) on GTP cyclohydrolase I, tetrahydrobiopterin and norepinephrine in the locus coeruleus in mice

Lipopolysaccharide (LPS), an endotoxin released from the outer membranes of Gram-negative bacteria, triggers cells to synthesize and release inflammatory cytokines that may progress to septic shock in vivo. We found that LPS enhances tetrahydrobiopterin (BH4) biosynthesis by inducing the biosynthetic enzyme GTP cyclohydrolase I (GCH) in vitro in the mouse neuroblastoma cell line N1E-115. Furthermore, we observed that gene expression of GCH in the locus coeruleus (LC) in mice was enhanced by peripheral administration of LPS, resulting in increased concentrations of BH4, and norepinephrine, and its metabolite 4-hydroxy-3-methoxyphenylglycol (MHPG). These results suggest that tyrosine hydroxylase (TH) activity is increased by increased content of BH4 due to enhanced mRNA expression of GCH in the LC resulting in the increase in norepinephrine in the LC during endotoxemia. LPS in blood may act as a stressor to increase norepinephrine biosynthesis in the mouse LC.

The effect of neurogenin3 deficiency on pancreatic gene expression in embryonic mice

To understand the molecular mechanisms regulating pancreatic endocrine development and function, pancreatic gene expression was compared between Ngn3-deficient mice and littermate controls on embryonic days 13 and 15. Microarray analysis identified 504 genes with significant differences in expression. Fifty-two of these showed at least twofold reduction in Ngn3 knockouts compared to controls. Many of them were previously described to be involved in endocrine development and function. Among the genes not previously characterized were Rhomboid veinlet-like 4, genes involved in tetrahydrobiopterin biosynthesis and the Iroquois-type homeobox gene Irx1, the latter was selected for further investigation. In situ hybridisation demonstrated that two Iroquois genes, Irx1 and Irx2, were expressed in pancreatic endoderm of wild-type, but not Ngn3 mutant embryos. Furthermore, ectopic Ngn3 induced prominent Irx2 expression in chicken endoderm. Co-labelling established that Irx1 and Irx2 mRNA is located to glucagon-, but not insulin- or somatostatin-producing cells in mice and chicken. These data suggest that Irx1 and Irx2 serve an evolutionary conserved role in the regulation of alpha-cell-specific gene expression.

Phosphoinositide 3-Kinase in Nitric Oxide Synthesis in Macrophage: CRITICAL DIMERIZATION OF INDUCIBLE NITRIC-OXIDE SYNTHASE

Phosphoinositide 3-kinase (PI3K) has important functions in various biological systems, including immune response. Although the role of PI3K in signaling by antigen-specific receptors of the adaptive immune system has been extensively studied, less is known about the function of PI3K in innate immunity. In the present study, we demonstrate that macrophages deficient for PI3K (p85alpha regulatory subunit) are impaired in nitric oxide (NO) production upon lipopolysaccharide and interferon-gamma stimulation and thus vulnerable for intracellular bacterial infection such as Chlamydophila pneumoniae. Although expression of inducible nitric-oxide synthase (iNOS) is induced normally in PI3K-deficient macrophages, dimer formation of iNOS protein is significantly impaired. The amount of intracellular tetrahydrobiopterin, a critical stabilizing cofactor for iNOS dimerization, is decreased in the absence of PI3K. In addition, induction of GTP cyclohydrolase 1, a rate-limiting enzyme for biosynthesis of tetrahydrobiopterin, is greatly reduced. Our current results demonstrate a critical role of class IA type PI3K in the bactericidal activity of macrophages by regulating their NO production through GTP cyclohydrolase 1 induction.

Identification of Mutations Causing 6-Pyruvoyl- Tetrahydrobiopterin Synthase Deficiency in Polish Patients With Variant Hyperphenylalaninemia.

Background: 6-Pyruvoyl-tetrahydrobiopterin synthase (PTPS) is required for biosynthesis of tetrahydrobiopterin, the cofactor of various enzymes including the hepatic phenylalanine hydroxylase. Mutations in the PTS gene result in a variant type of hyperphenylalaninemia, requiring cofactor replacement therapy for treatment. Methods and Results: Four Polish patients with PTPS deficiency were screened for mutations in the PTS gene. Three novel mutations E35G, N36K, and F100V were identified. In one patient, a known mutation D136V was identified in both PTS alleles. Conclusions: Mutation D136V present in both alleles was proposed to be connected with a mild form of PTPS deficiency. The other three mutations were found in heterozygous patients with a central type of PTPS deficiency. D136V mutation is a common mutation in the Polish population.

Structure of Chlorobium tepidum Sepiapterin Reductase Complex Reveals the Novel Substrate Binding Mode for Stereospecific Production of l-threo-Tetrahydrobiopterin

Sepiapterin reductase (SR) is involved in the last step of tetrahydrobiopterin (BH(4)) biosynthesis by reducing the di-keto group of 6-pyruvoyl tetrahydropterin. Chlorobium tepidum SR (cSR) generates a distinct BH(4) product, L-threo-BH(4) (6R-(1'S,2'S)-5,6,7,8-BH(4)), whereas animal enzymes produce L-erythro-BH(4) (6R-(1'R,2'S)-5,6,7,8-BH(4)) although it has high amino acid sequence similarities to the other animal enzymes. To elucidate the structural basis for the different reaction stereospecificities, we have determined the three-dimensional structures of cSR alone and complexed with NADP and sepiapterin at 2.1 and 1.7 A resolution, respectively. The overall folding of the cSR, the binding site for the cofactor NADP(H), and the positions of active site residues were quite similar to the mouse and the human SR. However, significant differences were found in the substrate binding region of the cSR. In comparison to the mouse SR complex, the sepiapterin in the cSR is rotated about 180 degrees around the active site and bound between two aromatic side chains of Trp-196 and Phe-99 so that its pterin ring is shifted to the opposite side, but its side chain position is not changed. The swiveled sepiapterin binding results in the conversion of the side chain configuration, exposing the opposite face for hydride transfer from NADPH. The different sepiapterin binding mode within the conserved catalytic architecture presents a novel strategy of switching the reaction stereospecificities in the same protein fold.

Folic acid reverses endothelial dysfunction induced by inhibition of tetrahydrobiopterin biosynthesis

While folic acid has been shown to reverse endothelial dysfunction, the exact underlying mechanism remains elusive. Here, folic acid reversed both the endothelial dysfunction and increased production of superoxide following depletion of rabbit aortic ring tetrahydrobiopterin (BH 4) levels with 2,4-diamino-6-hydroxy-pyrimidine (DAHP) and N-acetyl-5-hydroxy-tryptamine (NAS). Incubation with l-nitroarginine methyl ester also attenuated the production of superoxide. DAHP and NAS reduced BH 4 concentrations in both aorta and cultured porcine aortic endothelial cells. Folic acid had no effect on BH 4 concentrations in either preparation. The superoxide anion scavenger Tiron but not folic acid reversed the endothelial dysfunction produced in aortic rings by inhibition of copper–zinc superoxide dismutase with diethyldithiocarbamic acid. Neither folic acid nor its metabolite 5-methyltetrahydrofolate prevented the in vitro oxidation of BH 4. This study demonstrates that folic acid reverses the endothelial dysfunction induced by BH 4 depletion independently of either the regeneration or stabilization of BH 4 or an antioxidant effect.

Corticotropin-releasing factor receptor antagonist attenuates LPS-induced increase of GTP cyclohydrolase I expression at murine locus coeruleus

It has been reported that corticotropin-releasing factor (CRF) is involved in the regulation of norepinephrine neuron responses to stress such as an immobilized stress. Furthermore, systemic lipopolysaccharide (LPS) injection upregulates the transcription of the genes encoding CRF and CRF type 1 receptor in the paraventricular nucleus of the hypothalamus. We have already reported that an increase in norepinephrine turnover within the murine locus coeruleus is accompanied by septic shock triggered by LPS intraperitoneal injection. We also elucidated that the expression levels of the enzymes involved in the catecholamine biosynthesis were altered by peripheral LPS injection. Collectively, the effects of CRF on the expression levels of the enzymes at murine locus coeruleus were investigated by peripherally injecting CP-154,526, a CRF receptor type 1 antagonist. Pretreatment with CP-154,526 attenuated the increase in expression levels of GTP cyclohydrolase I mRNA due to intraperitoneal LPS injection at 4 h after the injection. However, no effects on the expression level of tyrosine hydroxylase mRNA at the site were observed. Taken together with the fact that LPS injection enhances tetrahydrobiopterin biosynthesis at locus coeruleus, CP-154,526 may attenuate the increase of NE turnover by suppressing the enhanced GCH expression level at the site caused by peripheral LPS injection.

Neural tube closure depends on nitric oxide synthase activity

Neural tube (NT) closure is a multifactorial process that involves yet unresolved molecular mechanisms. It had been shown previously that high levels of nitric oxide (NO) block the process of NT closure in the chick embryo by inhibiting methionine synthase (MS). The MS inhibition and its effect on NT closure could be alleviated by folic acid, suggesting the involvement of the folate-methionine pathway in the process. Here we test the hypothesis that endogenous nitric oxide synthase (NOS) activity regulates the MS activity required in the process of NT closure. The experiments described here reveal that NOS activity per se, is indeed critical for NT closure in the chick embryo. Inhibition of NOS activity with either 2,4-diamino-6-hydroxypyrimidine (DAHP), which blocks biosynthesis of the NOS co-factor tetrahydrobiopterin (BH4), or with calmidazolium, which blocks calcium-calmodulin binding to NOS, resulted in reduced MS activity and consequently ablated NT closure. Addition of BH4 or the calcium ionophore A23187 restored NOS and MS activities, resulting in NT closure. The results described here imply that NOS and MS activities can serve as functional markers in this developmental process as they are essential in the process of NT closure.

GTP Cyclohydrolase II Structure and Mechanism

GTP cyclohydrolase II converts GTP to 2,5-diamino-6-beta-ribosyl-4(3H)-pyrimidinone 5'-phosphate, formate and pyrophosphate, the first step in riboflavin biosynthesis. The essential role of riboflavin in metabolism and the absence of GTP cyclohydrolase II in higher eukaryotes makes it a potential novel selective antimicrobial drug target. GTP cyclohydrolase II catalyzes a distinctive overall reaction from GTP cyclohydrolase I; the latter converts GTP to dihydroneopterin triphosphate, utilized in folate and tetrahydrobiopterin biosynthesis. The structure of GTP cyclohydrolase II determined at 1.54-A resolution reveals both a different protein fold to GTP cyclohydrolase I and distinctive molecular recognition determinants for GTP; although in both enzymes there is a bound catalytic zinc. The GTP cyclohydrolase II.GMPCPP complex structure shows Arg(128) interacting with the alpha-phosphonate, and thus in the case of GTP, Arg(128) is positioned to act as the nucleophile for pyrophosphate release and formation of the proposed covalent guanylyl-GTP cyclohydrolase II intermediate. Tyr(105) is identified as playing a key role in GTP ring opening; it is hydrogen-bonded to the zinc-activated water molecule, the latter being positioned for nucleophilic attack on the guanine C-8 atom. Although GTP cyclohydrolase I and GTP cyclohydrolase II both use a zinc ion for the GTP ring opening and formate release, different residues are utilized in each case to catalyze this reaction step.

Increased blood flow causes coordinated upregulation of arterial eNOS and biosynthesis of tetrahydrobiopterin

Shear stress, imposed on the vascular endothelium by circulating blood, critically sustains vascular synthesis of nitric oxide (NO). Endothelial NO synthase (eNOS) activity is determined by heat shock protein 90 (HSP90), caveolin-1, and the cofactor tetrahydrobiopterin (BH4). To determine whether increased blood flow concomitantly upregulates eNOS and GTP cyclohydrolase I (GTPCH I, the rate-limiting enzyme in BH4 biosynthesis), an aortocaval fistula model in the rat was employed wherein aortic blood flow is enhanced proximal but decreased distal to the fistula. Eight weeks after the creation of the aortocaval fistula, the proximal and distal aortic segments were harvested; sham-operated rats served as controls. Vasomotor function was assessed by isometric force recording. Expression of eNOS, HSP90, caveolin-1, Akt, phosphorylated eNOS (eNOS-Ser1177), and GTPCH I were determined by Western blot analysis. Biosynthesis of BH4 and GTPCH-I activity was examined by HPLC. In the aortic segments exposed to increased flow, contractions to KCl and phenylephrine were reduced, whereas endothelium-dependent relaxations were not affected compared with sham-operated or aortic segments with reduced blood flow. Expression of eNOS, caveolin-1, phosphorylated Akt, and eNOS-Ser1177 was enhanced in aortas exposed to increased blood flow. High flow augmented levels of cGMP and BH4 and increased expression of GTPCH I. In aggregate, these findings provide the first demonstration in vivo that coordinated vascular upregulation of eNOS, and GTPCH I accompanies increased blood flow. This induction of GTPCH I increases BH4 production, thereby optimizing the generation of NO by eNOS and thus the adaptive, vasorelaxant response required in sustaining increased blood flow.

Regulation of GTP cyclohydrolase I gene transcription by basic region leucine zipper transcription factors

Tetrahydrobiopterin is an essential cofactor for the phenylalanine, tyrosine and tryptophan hydroxylases, and the family of nitric oxide synthases. The initial and rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin is GTP cyclohydrolase I. The proximal promoter of the human GTP cyclohydrolase I gene contains the sequence motif 5'-TGACGCGA-3', resembling a cAMP response element (CRE). The objective of this study was to analyze the regulation of GTP cyclohydrolase I gene transcription by basic region leucine zipper (bZIP) transcription factors. A constitutively active mutant of the cAMP response element binding (CREB) protein strongly stimulated GTP cyclohydrolase I promoter activity, indicating that the CRE in the context of the GTP cyclohydrolase I gene is functional. Likewise, GTP cyclohydrolase I promoter/luciferase gene transcription was stimulated following nuclear expression of the catalytic subunit of cAMP-dependent protein kinase. Constitutively active mutants of activating transcription factor 2 (ATF2) and c-Jun additionally stimulated GTP cyclohydrolase I promoter activity, but to a lesser extent than the constitutively active CREB mutant. The fact that stress-activated protein kinases target the GTP cyclohydrolase I gene was corroborated by expression experiments involving p38 and MEKK1 protein kinases. We conclude that signaling pathways involving either the cAMP-dependent protein kinase or stress-activated protein kinases converge to the GTP cyclohydrolase I gene. Hence, enzymatic reactions that require tetrahydrobiopterin as cofactor are therefore indirectly controlled by signaling cascades involving the signal-responsive transcription factors CREB, c-Jun, and ATF2.

Genetically rescued tetrahydrobiopterin‐depleted mice survive with hyperphenylalaninemia and region‐specific monoaminergic abnormalities

One of the possibly mutated genes in DOPA-responsive dystonia (DRD, Segawa's disease) is the gene encoding GTP cyclohydrolase I, which is the rate-limiting enzyme for tetrahydrobiopterin (BH4) biosynthesis. Based on our findings on 6-pyruvoyltetrahydropterin synthase (PTS) gene-disrupted (Pts(-/-)) mice, we suggested that the amount of tyrosine hydroxylase (TH) protein in dopaminergic nerve terminals is regulated by the intracellular concentration of BH4. In this present work, we rescued Pts(-/-) mice by transgenic introduction of human PTS cDNA under the control of the dopamine beta-hydroxylase promoter to examine regional differences in the sensitivity of dopaminergic neurons to BH4-insufficiency. The DPS-rescued (Pts(-/-), DPS) mice showed severe hyperphenylalaninemia. Human PTS was efficiently expressed in noradrenergic regions but only in a small number of dopaminergic neurons. Biopterin and dopamine contents, and TH activity in the striatum were poorly restored compared with those in the midbrain. TH-immunoreactivity in the lateral region of the striatum was far weaker than that in the medial region or in the nucleus accumbens. We concluded that dopaminergic nerve terminals projecting to the lateral region of the striatum are the most sensitive to BH4-insufficiency. Biochemical and pathological changes in DPS-rescued mice were similar to those in human malignant hyperphenylalaninemia and DRD.

Antioxidant Effects of N‐Acetylserotonin

Abstract: This paper will review our recent data relevant to the antioxidant effects of N‐acetylserotonin (NAS), the immediate precursor of melatonin, the pineal gland indole. Mechanisms of the antioxidant effects of NAS might involve interaction with melatonin type 3 receptors and nonreceptor mechanisms such as stimulation of glutathione peroxidase, an antioxidant enzyme; inhibition of lipid peroxidation; suppression of phospholipase A2 activation; attenuation of tumor necrosis factor‐α production; prevention of pathological opening of the mitochondrial permeability transition pores; and inhibition of sepiapterin reductase, the key enzyme of biosynthesis of tetrahydrobiopterin, the essential cofactor of nitric oxide synthase. NAS actions on some of these enzymes might be receptor‐mediated. Protective effects of NAS against oxidative damage are independent from the effect of melatonin and, depending on the model, are 5 to 20 times stronger than that of melatonin. Antioxidant effect of NAS might underpin its cognition‐enhancing, antiaging, antidepressant, antihypertensive, and antitumor effects. NAS and its derivatives might be useful in protection against oxidative stress‐related disorders (cell death, mutagenesis, aging) and diseases (sepsis, cancer, postischemic trauma, Alzheimer's disease, parkinsonism).

Antioxidant Effects of N-Acetylserotonin: Possible Mechanisms and Clinical Implications

This paper will review our recent data relevant to the antioxidant effects of N-acetylserotonin (NAS), the immediate precursor of melatonin, the pineal gland indole. Mechanisms of the antioxidant effects of NAS might involve interaction with melatonin type 3 receptors and nonreceptor mechanisms such as stimulation of glutathione peroxidase, an antioxidant enzyme; inhibition of lipid peroxidation; suppression of phospholipase A2 activation; attenuation of tumor necrosis factor-alpha production; prevention of pathological opening of the mitochondrial permeability transition pores; and inhibition of sepiapterin reductase, the key enzyme of biosynthesis of tetrahydrobiopterin, the essential cofactor of nitric oxide synthase. NAS actions on some of these enzymes might be receptor-mediated. Protective effects of NAS against oxidative damage are independent from the effect of melatonin and, depending on the model, are 5 to 20 times stronger than that of melatonin. Antioxidant effect of NAS might underpin its cognition-enhancing, antiaging, antidepressant, antihypertensive, and antitumor effects. NAS and its derivatives might be useful in protection against oxidative stress-related disorders (cell death, mutagenesis, aging) and diseases (sepsis, cancer, postischemic trauma, Alzheimer's disease, parkinsonism).

Linkage and candidate gene analysis of 14q22-24 in bipolar disorder: support for GCHI as a novel susceptibility gene

Using a collection of Irish sib-pair nuclear families, we previously obtained modest evidence of linkage implicating 14q22-24 in bipolar disorder (BPD). To follow-up on this preliminary finding, an extended linkage analysis was performed which employed thirteen microsatellite markers, spanning a total distance of 85 cM on 14q. Effectively, P-values <0.05 were observed for a region extending over 41.88 cM, with the marker D14S281 displaying a peak multipoint non-parametric lod (NPL) score of 2.72 and an associated P-value of 0.003. Support for this finding was also obtained from flanking markers indicating excess allele sharing at 14q22-24 in Irish bipolar sib-pairs. A web-based candidate gene search of 14q22-24 resulted in the selection of GTP cyclohydrolase I (GCHI), located 200 kb 3' of D14S281, as the best plausible candidate gene for involvement in BPD. GCHI is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin (BH(4)), a natural cofactor for tyrosine and tryptophan hydroxylases. These enzymes play an essential role in the biosynthesis of various hormones and neurotransmitters such as dopamine, noradrenaline, adrenaline, and serotonin. Numerous studies have also suggested that the clinical symptoms of depression might be related to a deficiency of BH(4). An association study between BPD and a novel single nucleotide polymorphism (SNP) in GCHI (G to A at position -959 bp, upstream of the ATG codon), is also presented here. This study revealed that the variant A allele is preferentially transmitted to BPI probands (chi(2) = 4.54, P = 0.033) suggesting that variants within GCHI may contribute to BPD in the Irish population.

Structural Basis of Biopterin-induced Inhibition of GTP Cyclohydrolase I by GFRP, Its Feedback Regulatory Protein

GTP cyclohydrolase I (GTPCHI) is the rate-limiting enzyme involved in the biosynthesis of tetrahydrobiopterin, a key cofactor necessary for nitric oxide synthase and for the hydroxylases that are involved in the production of catecholamines and serotonin. In animals, the GTPCHI feedback regulatory protein (GFRP) binds GTPCHI to mediate feed-forward activation of GTPCHI activity in the presence of phenylalanine, whereas it induces feedback inhibition of enzyme activity in the presence of biopterin. Here, we have reported the crystal structure of the biopterin-induced inhibitory complex of GTPCHI and GFRP and compared it with the previously reported phenylalanine-induced stimulatory complex. The structure reveals five biopterin molecules located at each interface between GTPCHI and GFRP. Induced fitting structural changes by the biopterin binding expand large conformational changes in GTPCHI peptide segments forming the active site, resulting in inhibition of the activity. By locating 3,4-dihydroxy-phenylalanine-responsive dystonia mutations in the complex structure, we found mutations that may possibly disturb the GFRP-mediated regulation of GTPCHI.