Crystal structure of rat GTP cyclohydrolase I feedback regulatory protein, GFRP

Tetrahydrobiopterin, the cofactor required for hydroxylation of aromatic amino acids regulates its own synthesis in mammals through feedback inhibition of GTP cyclohydrolase I. This mechanism is mediated by a regulatory subunit called GTP cyclohydrolase I feedback regulatory protein (GFRP). The 2.6 A resolution crystal structure of rat GFRP shows that the protein forms a pentamer. This indicates a model for the interaction of mammalian GTP cyclohydrolase I with its regulator, GFRP. Kinetic investigations of human GTP cyclohydrolase I in complex with rat and human GFRP showed similar regulatory effects of both GFRP proteins.     

GTP cyclohydrolase I feedback regulatory protein-dependent and -independent inhibitors of GTP cyclohydrolase I

GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates the feedback inhibition of GTP cyclohydrolase I activity by (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) through protein complex formation. Since guanine and BH4 have a common pyrimidine ring structure, we examined the inhibitory effect of guanine and its analogs on the enzyme activity. Guanine, 8-hydroxyguanine, 8-methylguanine, and 8-bromoguanine inhibited the enzyme activity in a GFRP-dependent and pH-dependent manner and induced complex formation between GTP cyclohydrolase I and GFRP. The type of inhibition by this group is a mixed type. All these properties were shared with BH4. In striking contrast, inhibition by 8-azaguanine and 8-mercaptoguanine was GFRP-independent and pH-independent. The type of inhibition by 8-azaguanine and 8-mercaptoguanine was a competitive type. The two compounds did not induce complex formation between the enzyme and GFRP. These results demonstrate that guanine compounds of the first group bind to the BH4-binding site of the GTP cyclohydrolase I/GFRP complex, whereas 8-azaguanine and 8-mercaptoguanine bind to the active site of the enzyme. Finally, the possible implications in Lesch-Nyhan syndrome and Parkinson diseases of the inhibition of GTP cyclohydrolase I by guanine and 8-hydroxyguanine are discussed.     

Ligand binding to the inhibitory and stimulatory GTP cyclohydrolase I/GTP cyclohydrolase I feedback regulatory protein complexes

GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4), which is an essential cofactor for key enzymes producing catecholamines, serotonin, and nitric oxide as well as phenylalanine hydroxylase. GFRP also mediates feed-forward stimulation of GTP cyclohydrolase I activity by phenylalanine at subsaturating GTP levels. These ligands, BH4 and phenylalanine, induce complex formation between one molecule of GTP cyclohydrolase I and two molecules of GFRP. Here, we report the analysis of ligand binding using the gel filtration method of Hummel and Dreyer. BH4 binds to the GTP cyclohydrolase I/GFRP complex with a Kd of 4 microM, and phenylalanine binds to the protein complex with a Kd of 94 microM. The binding of BH4 is enhanced by dGTP. The binding stoichiometrics of BH4 and phenylalanine were estimated to be 10 molecules of each per protein complex, in other words, one molecule per subunit of protein, because GTP cyclohydrolase I is a decamer and GFRP is a pentamer. These findings were corroborated by data from equilibrium dialysis experiments. Regarding ligand binding to free proteins, BH4 binds weakly to GTP cyclohydrolase I but not to GFRP, and phenylalanine binds weakly to GFRP but not to GTP cyclohydrolase I. These results suggest that the overall structure of the protein complex contributes to binding of BH4 and phenylalanine but also that each binding site of BH4 and phenylalanine may be primarily composed of residues of GTP cyclohydrolase I and GFRP, respectively.     

The mechanism of potent GTP cyclohydrolase I inhibition by 2,4-diamino-6-hydroxypyrimidine: requirement of the GTP cyclohydrolase I feedback regulatory protein

Inhibition of GTP cyclohydrolase I (GTPCH) has been used as a selective tool to assess the role of de novo synthesis of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) in a biological system. Toward this end, 2,4-diamino-6-hydroxypyrimidine (DAHP) has been used as the prototypical GTPCH inhibitor. Using a novel real-time kinetic microplate assay for GTPCH activity and purified prokaryote-expressed recombinant proteins, we show that potent inhibition by DAHP is not the result of a direct interaction with GTPCH. Rather, inhibition by DAHP in phosphate buffer occurs via an indirect mechanism that requires the presence of GTPCH feedback regulatory protein (GFRP). Notably, GFRP was previously discovered as the essential factor that reconstitutes inhibition of pure recombinant GTPCH by the pathway end product BH4. Thus, DAHP inhibits GTPCH by engaging the endogenous feedback inhibitory system. We further demonstrate that L-Phe fully reverses the inhibition of GTPCH by DAHP/GFRP, which is also a feature in common with inhibition by BH4/GFRP. These findings suggest that DAHP is not an indiscriminate inhibitor of GTPCH in biological systems; instead, it is predicted to preferentially attenuate GTPCH activity in cells that most abundantly express GFRP and/or contain the lowest levels of L-Phe.     

Occurrence of GTP cyclohydrolase I in Bacillus stearothermophilus

A GTP cyclohydrolase which catalyzes the removal of carbon 8 of GTP as formic acid to yield a single pteridine compound occurs in an obligate thermophile Bacillus stearothermophilus ATCC 8005. The enzyme was purified 5.5-fold. Its molecular weight and Stoke's radius were estimated as 105,000 and 45.3 A, respectively. The Km for GTP was 0.98 microM. The temperature and pH optima for activity were 60-65 degrees C and 8.0-8.4, respectively. No divalent cation was required for the reaction. The pteridine product was 3'-triphosphate of 2-amino-4-hydroxy-6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropteridine (dihydroneopterin triphosphate), identified by isolating its immediate derivative, 2',3'-cyclic phosphate of 2-amino-4-hydroxy-6-(D-erythro-1',2',3'-trihydroxypropyl)pteridine (neopterin cyclic phosphate). The radioactive product from [8-14C]GTP agreed with 14C-formate. Molar ratio of formate release to pteridine formation was 1.0.     

Inhibition of GTP cyclohydrolase I by pterins

Pterins inhibit rat liver GTP cyclohydrolase I activity noncompetitively. Reduced pterins, such as 7,8-dihydro-D-neopterin, (6R,S)-5,6,7,8-tetrahydro-D-neopterin, 7,8-dihydro-L-biopterin, (6R)-5,6,7,8-tetrahydro-L-biopterin, L-sepiapterin, and DL-6-methyl-5,6,7,8-tetrahydropterin are approximately 12-times more potent as inhibitors than are oxidized pterins, such as D-neopterin, L-biopterin, and isoxanthopterin. They are also 12-times more potent than folates, such as folic acid, dihydrofolic acid, (+/-)-L-tetrahydrofolic acid, and aminopterin. The Ki values for 7,8-dihydro-D-neopterin, 7,8-dihydro-L-biopterin, and (6R)-5,6,7,8-tetrahydro-L-biopterin are 12.7 microM, 14.4 microM, and 15.7 microM, respectively. These results suggest that mammalian GTP cyclohydrolase I may be regulated by its metabolic end products.     

GTP cyclohydrolase I utilizes metal-free GTP as its substrate.

GTP cyclohydrolase I (GCH) is the rate-limiting enzyme for the synthesis of tetrahydrobiopterin and its activity is important in the regulation of monoamine neurotransmitters such as dopamine, norepinephrine and serotonin. We have studied the action of divalent cations on the enzyme activity of purified recombinant human GCH expressed in Escherichia coli. First, we showed that the enzyme activity is dependent on the concentration of Mg-free GTP. Inhibition of the enzyme activity by Mg2+, as well as by Mn2+, Co2+ or Zn2+, was due to the reduction of the availability of metal-free GTP substrate for the enzyme, when a divalent cation was present at a relatively high concentration with respect to GTP. We next examined the requirement of Zn2+ for enzyme activity by the use of a protein refolding assay, because the recombinant enzyme contained approximately one zinc atom per subunit of the decameric protein. Only when Zn2+ was present was the activity of the denatured enzyme effectively recovered by incubation with a chaperone protein. These are the first data demonstrating that GCH recognizes Mg-free GTP and requires Zn2+ for its catalytic activity. We suggest that the cellular concentration of divalent cations can modulate GCH activity, and thus tetrahydrobiopterin biosynthesis as well.     

A yeast 2-hybrid analysis of human GTP cyclohydrolase I protein interactions

The yeast 2-hybrid system was used to identify protein domains involved in the oligomerization of human guanosine 5'-triphosphate (GTP) Cyclohydrolase I (GCH1) and the interaction of GCH1 with its regulatory partner, GCH1 feedback regulatory protein (GFRP). When interpreted within the structural framework derived from crystallography, our results indicate that the GCH1 N-terminal alpha-helices are not the only domains involved in the formation of dimers from monomers and also suggest an important role for the C-terminal alpha-helix in the assembly of dimers to form decamers. Moreover, a previously unknown role of the extended N-terminal alpha-helix in the interaction of GCH1 and GFRP was revealed. To discover novel GCH1 protein binding partners, we used the yeast 2-hybrid system to screen a human brain library with GCH1 N-terminal amino acids 1-96 as prey. This protruding extension of GCH1 contains two canonical Type-I Src homology-3 (SH3) ligand domains located within amino acids 1-42. Our screen yielded seven unique clones that were subsequently shown to require amino acids 1-42 for binding to GCH1. The interaction of one of these clones, Activator of Heat Shock 90 kDa Protein (Aha1), with GCH1 was validated by glutathione-s-transferase (GST) pull-down assay. Although the physiological relevance of the Aha1-GCH1 interaction requires further study, Aha1 may recruit GCH1 into the endothelial nitric oxide synthase/heat shock protein (eNOS/Hsp90) complex to support changes in endothelial nitric oxide production through the local synthesis of BH4.     

Bacterial lipopolysaccharide down-regulates expression of GTP cyclohydrolase I feedback regulatory protein.

GTP cyclohydrolase I feedback regulatory protein (GFRP) is a 9.7-kDa protein regulating GTP cyclohydrolase I activity in dependence of tetrahydrobiopterin and phenylalanine concentrations, thus enabling stimulation of tetrahydrobiopterin biosynthesis by phenylalanine to ensure its efficient metabolism by phenylalanine hydroxylase. Here, we were interested in regulation of GFRP expression by proinflammatory cytokines and stimuli, which are known to induce GTP cyclohydrolase I expression. Recombinant human GFRP stimulated recombinant human GTP cyclohydrolase I in the presence of phenylalanine and mediated feedback inhibition by tetrahydrobiopterin. Levels of GFRP mRNA in human myelomonocytoma (THP-1) cells remained unaltered by treatment of cells with interferon-gamma or interleukin-1beta, but were significantly down-regulated by bacterial lipopolysaccharide (LPS, 1 microg/ml), without or with cotreatment by interferon-gamma, which strongly up-regulated GTP cyclohydrolase I expression and activity. GFRP expression was also suppressed in human umbilical vein endothelial cells treated with 1 microg/ml LPS, as well as in rat tissues 7 h post intraperitoneal injection of 10 mg/kg LPS. THP-1 cells stimulated with interferon-gamma alone showed increased pteridine synthesis by addition of phenylalanine to the culture medium. Cells stimulated with interferon-gamma plus LPS, in contrast, showed phenylalanine-independent pteridine synthesis. These results demonstrate that LPS down-regulates expression of GFRP, thus rendering pteridine synthesis independent of metabolic control by phenylalanine.     

Purification and characterization of rat liver GTP cyclohydrolase I. Cooperative binding of GTP to the enzyme

GTP cyclohydrolase I, an enzyme that catalyzes the first step in the biosynthetic pathway of tetrahydrobiopterin, has been purified about 38,000-fold to apparent homogeneity from rat liver extract with a yield of 5%. The molecular weight of the enzyme was estimated to be 300,000 by gel filtration on Ultrogel AcA 34. The purified enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at a position corresponding to a molecular weight of 30,000. N-terminal amino acid sequence analysis gave a single amino acid at every step of the Edman degradation up to residue 10. These results suggest that the enzyme is probably a homopolymer. The enzyme showed positive cooperativity with a Hill coefficient of 2.4 at a substrate (GTP) concentration of 10-50 microM. The Vmax value of the enzyme was 45 nmol/min.mg protein. The GTP concentration producing half-maximal velocity was 30 microM at a KCl concentration of 0.1 M. This value increased as the KCl concentration rose, without any change in Vmax or Hill number. Biosynthesis of tetrahydrobiopterin may be controlled by the intracellular level of GTP.     

Identification of proteins interacting with GTP cyclohydrolase I

GTP cyclohydrolase I (GCH-1) is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin, an essential cofactor for nitric oxide synthase and aromatic amino acid hydroxylase. To explore the interactome of GCH-1, we established a HEK 293 cell line stably expressing tetracycline-inducible FLAG-GCH-1. FLAG-GCH-1 and associated proteins were immunoprecipitated and analyzed by liquid chromatography/tandem mass spectrometry. Twenty-nine proteins, derived from different subcellular components such as cytosol, membranes, nucleus and mitochondria were identified to interact with GCH-1. Cell fractionation studies also showed that GCH-1 was present in the cytosol, membranes and nucleus. Gene ontology analysis revealed that GCH-1 interactome was involved in a variety of biological processes such as signal transduction, apoptosis, metabolism, transport and cell organization. To our knowledge, this study is the first to provide a comprehensive analysis of the GCH-1 interactome. Findings expand the number and diversity of proteins that are known to associate with GCH-1.     

Regulation of GTP cyclohydrolase I and dihydropteridine reductase in rat pheochromocytoma PC 12 cells

The addition of 8-bromo cyclic AMP, forskolin, theophylline, and 3-isobutyl-1-methylxanthine to the medium of PC 12 cells resulted in an increase in GTP cyclohydrolase I activity, but had no effect on dihydropteridine reductase activity, except theophylline which caused a decrease in dihydropteridine reductase activity at 96 h. GTP cyclohydrolase I activity peaked at 24 h and returned to normal 96 h after drug treatment. Cycloheximide decreased GTP cyclohydrolase I activity at 48 and 96 h, but had little effect on dihydropteridine reductase activity. The addition of reserpine selectively increased only GTP cyclohydrolase I activity. The addition of tetrahydrobiopterin and sepiapterin, however, coordinately inhibited both GTP cyclohydrolase I and dihydropteridine reductase activities. It appears that GTP cyclohydrolase I activity in PC 12 cells is regulated by cyclic AMP stimulation and by end-product inhibition, whereas dihydropteridine reductase activity is only subject to pterin inhibition.     

Biochemical characterization of oligomerization of Escherichia coli GTP cyclohydrolase I

GTP cyclohydrolase I (E.C. 3.5.4.16) is a homodecameric protein that catalyzes the conversion of GTP to 7,8- dihydroneopterin triphosphate (H(2)NTP), the initial step in the biosynthesis of pteridines. It was proposed that the enzyme complex could be composed of a dimer of two pentamers, or a pentamer of tightly associated dimers; then the active site of the enzyme was located at the interface of three monomers (Nar et al. 1995a, b). Using mutant enzymes that were made by site-directed mutagenesis, we showed that a decamer of GTP cyclohydrolase I should be composed of a pentamer of five dimers, and that the active site is located between dimers, as analyzed by a series of size exclusion chromatography and the reconstitution experiment. We also show that the residues Lys 136, Arg139, and Glu152 are of particular importance for the oligomerization of the enzyme complex from five dimers to a decamer.     

Biosynthesis of pteridines. Reaction mechanism of GTP cyclohydrolase I

GTP cyclohydrolase I catalyses the hydrolytic release of formate from GTP followed by cyclization to dihydroneopterin triphosphate. The enzymes from bacteria and animals are homodecamers containing one zinc ion per subunit. Replacement of Cys110, Cys181, His112 or His113 of the enzyme from Escherichia coli by serine affords catalytically inactive mutant proteins with reduced capacity to bind zinc. These mutant proteins are unable to convert GTP or the committed reaction intermediate, 2-amino-5-formylamino-6-(beta-ribosylamino)-4(3H)-pyrimidinone 5'-triphosphate, to dihydroneopterin triphosphate. The crystal structures of GTP complexes of the His113Ser, His112Ser and Cys181Ser mutant proteins determined at resolutions of 2.5A, 2.8A and 3.2A, respectively, revealed the conformation of substrate GTP in the active site cavity. The carboxylic group of the highly conserved residue Glu152 anchors the substrate GTP, by hydrogen bonding to N-3 and to the position 2 amino group. Several basic amino acid residues interact with the triphosphate moiety of the substrate. The structure of the His112Ser mutant in complex with an undefined mixture of nucleotides determined at a resolution of 2.1A afforded additional details of the peptide folding. Comparison between the wild-type and mutant enzyme structures indicates that the catalytically active zinc ion is directly coordinated to Cys110, Cys181 and His113. Moreover, the zinc ion is complexed to a water molecule, which is in close hydrogen bond contact to His112. In close analogy to zinc proteases, the zinc-coordinated water molecule is suggested to attack C-8 of the substrate affording a zinc-bound 8R hydrate of GTP. Opening of the hydrated imidazole ring affords a formamide derivative, which remains coordinated to zinc. The subsequent hydrolysis of the formamide motif has an absolute requirement for zinc ion catalysis. The hydrolysis of the formamide bond shows close mechanistic similarity with peptide hydrolysis by zinc proteases.     

Discovery of a new prokaryotic type I GTP cyclohydrolase family

GTP cyclohydrolase I (GCYH-I) is the first enzyme of the de novo tetrahydrofolate biosynthetic pathway present in bacteria, fungi, and plants, and encoded in Escherichia coli by the folE gene. It is also the first enzyme of the biopterin (BH4) pathway in Homo sapiens, where it is encoded by a homologous folE gene. A homology-based search of GCYH-I orthologs in all sequenced bacteria revealed a group of microbes, including several clinically important pathogens, that encoded all of the enzymes of the tetrahydrofolate biosynthesis pathway but GCYH-I, suggesting that an alternate family was present in these organisms. A prediction based on phylogenetic occurrence and physical clustering identified the COG1469 family as a potential candidate for this missing enzyme family. The GCYH-I activity of COG1469 family proteins from a variety of sources (Thermotoga maritima, Bacillus subtilis, Acinetobacter baylyi, and Neisseria gonorrhoeae) was experimentally verified in vivo and/or in vitro. Although there is no detectable sequence homology with the canonical GCYH-I, protein fold recognition based on sequence profiles, secondary structure, and solvation potential information suggests that, like GCYH-I proteins, COG1469 proteins are members of the tunnel-fold (T-fold) structural superfamily. This new GCYH-I family is found in approximately 20% of sequenced bacteria and is prevalent in Archaea, but the family is to this date absent in Eukarya.     

Allosteric characteristics of GTP cyclohydrolase I from Escherichia coli.

The kinetic and regulatory properties of GTP cyclohydrolase I were investigated using an improved enzyme assay and direct determination of the product, dihydroneopterin triphosphate. The enzyme was purified from Escherichia coli to absolute homogeneity as demonstrated by N-terminal sequencing of up to 50 amino acid residues. A 30-residue internal fragment showed 42% similarity with rat liver GTP cyclohydrolase I. The enzyme did not obey Michaelis-Menten kinetics or show a sigmoid reaction curve. The substrate saturation kinetics were found to be slow with low response to minor changes in GTP concentrations. GTP cyclohydrolase I has a relatively high apparent Km. The values are slightly different for enzyme purified by GTP-agarose (100 microM) and UTP-agarose (110 microM). Low turnover numbers of 12/min and 19/min were calculated for the respective enzyme preparations. GTP-cyclohydrolase-I activity was modulated in Vmax by K+, divalent cations, UTP and tetrahydrobiopterin. Divalent cations, such as Mg2+, had an activating effect with an optimum at 8 mM Mg2+. A different catalytic function and formation of a new, unidentified product by GTP cyclohydrolase I was observed in the presence of Ca2+. In the presence of 1 mM EDTA and Mg2+, GTP-cyclohydrolase-I activity was strongly inhibited by chelate complexes. UTP proved not to be a competitive inhibitor, but a positive modulator. The inhibition by chelate complexes was totally abolished by UTP. Tetrahydrobiopterin showed an inhibitory effect, with 50% inhibition at 100 microM tetrahydrobiopterin. UTP was able to reduce the inhibition by tetrahydrobiopterin. Using monoclonal antibody 1F11 (related to the GTP-binding site), and monoclonal antibody NS7 (mimicking tetrahydrobiopterin), different binding sites were demonstrated for GTP and tetrahydrobiopterin on each enzyme subunit. Western-blot competition analysis revealed a UTP-binding site different from the binding sites of GTP and tetrahydrobiopterin. Based on the kinetic behaviour and the kind of modulations observed we defined GTP cyclohydrolase I as an M-class allosteric enzyme.     

Interaction of human GTP cyclohydrolase I with its splice variants

Tetrahydrobiopterin is an essential cofactor for aromatic amino acid hydroxylases, ether lipid oxidase and nitric oxide synthases. Its biosynthesis in mammals is regulated by the activity of the homodecameric enzyme GCH (GTP cyclohydrolase I; EC 3.5.4.16). In previous work, catalytically inactive human GCH splice variants differing from the wild-type enzyme within the last 20 C-terminal amino acids were identified. In the present study, we searched for a possible role of these splice variants. Gel filtration profiles of purified recombinant proteins showed that variant GCHs form high-molecular-mass oligomers similar to the wild-type enzyme. Co-expression of splice variants together with wild-type GCH in mammalian cells revealed that GCH levels were reduced in the presence of splice variants. Commensurate with these findings, the GCH activity obtained for wild-type enzyme was reduced 2.5-fold through co-expression with GCH splice variants. Western blots of native gels suggest that splice variants form decamers despite C-terminal truncation. Therefore one possible explanation for the effect of GCH splice variants could be that inactive variants are incorporated into GCH heterodecamers, decreasing the enzyme stability and activity.     

Purification and characterization of GTP cyclohydrolase I from Drosophila melanogaster

The enzyme GTP cyclohydrolase I, which catalyzes the first step in the pteridine biosynthetic pathway, has been purified by at least 4400-fold from Drosophila melanogaster. The active complex has an apparent molecular mass of 575,000 daltons, as estimated from gel filtration. This high molecular mass complex appears to be composed of a number of 39,000-dalton subunits. A polyspecific antiserum generated against the active complex has been used to identify this polypeptide as being severely affected by mutations in Punch, the structural gene for GTP cyclohydrolase. Enzyme activity is inhibited by divalent cations and high ionic strength buffers. No cofactors have been demonstrated to be required for enzyme activity. The enzyme displays positive cooperativity in phosphate buffer, a Hill number of 2.1, but only slight cooperativity in Tris buffer, a Hill number of 1.2.