Structure of GTP cyclohydrolase I from Listeria monocytogenes, a potential anti-infective drug target.

A putative open reading frame encoding GTP cyclohydrolase I from Listeria monocytogenes was expressed in a recombinant Escherichia coli strain. The recombinant protein was purified and was confirmed to convert GTP to dihydroneopterin triphosphate (Km = 53 µM; vmax = 180 nmol mg-1 min-1). The protein was crystallized from 1.3 M sodium citrate pH 7.3 and the crystal structure was solved at a resolution of 2.4 Š(Rfree = 0.226) by molecular replacement using human GTP cyclohydrolase I as a template. The protein is a D5-symmetric decamer with ten topologically equivalent active sites. Screening a small library of about 9000 compounds afforded several inhibitors with IC50 values in the low-micromolar range. Several inhibitors had significant selectivity with regard to human GTP cyclohydrolase I. Hence, GTP cyclohydrolase I may be a potential target for novel drugs directed at microbial infections, including listeriosis, a rare disease with high mortality.

Characterization of an Fe(2+)-dependent archaeal-specific GTP cyclohydrolase, MptA, from Methanocaldococcus jannaschii.

The first step in the biosynthesis of pterins in bacteria and plants is the conversion of GTP to 7,8-dihydro-d-neopterin triphosphate catalyzed by GTP cyclohydrolase I (GTPCHI). Although GTP has been shown to be a precursor of pterins in archaea, homologues of GTPCHI have not been identified in most archaeal genomes. Here we report the identification of a new GTP cyclohydrolase that converts GTP to 7,8-dihydro-d-neopterin 2',3'-cyclic phosphate, the first intermediate in methanopterin biosynthesis in methanogenic archaea. The enzyme from Methanocaldococcus jannaschii is designated MptA to indicate that it catalyzes the first step in the biosynthesis of methanopterin. MptA is the archetype of a new class of GTP cyclohydrolases that catalyzes a series of reactions most similar to that seen with GTPCHI but unique in that the cyclic phosphate is the product. MptA was found to require Fe2+ for activity. Mutation of conserved histidine residues H200N, H293N, and H295N, expected to be involved in Fe2+ binding, resulted in reduced enzymatic activity but no reduction in the amount of bound iron.

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.

Cloning, expression, purification, and characterization of Nocardia sp. GTP cyclohydrolase I.

The sequence of the gene from Nocardia sp. NRRL 5646 encoding GTP cyclohydrolase I (GCH), gch, and its adjacent regions was determined. The open reading frame of Nocardia gch contains 684 nucleotides, and the deduced amino acid sequence represents a protein of 227 amino acid residues with a calculated molecular mass of 24,563Da. The uncommon start codon TTG was identified by matching the N-terminal amino acid sequence of purified Nocardia GCH with the deduced amino acid sequence. A likely ribosomal binding site was identified 9bp upstream of the translational start site. The 3' end flank region encodes a peptide that shares high homology with dihydropteroate synthases. Nocardia GCH has 73 and 60% identity to the proteins encoded by the putative gch of Mycobacterium tuberculosis and Streptomyces coelicolor, respectively. Nocardia GCH was highly expressed in Escherichia coli cells carrying a pHAT10 based expression vector, and moderately expressed in Mycobacterium smegmatis cells carrying a pSMT3 based expression vector. Enterokinase digestion of recombinant Nocardia GCH, and in-gel digestion of Nocardia GCH and recombinant GCH followed by MALDI-TOF-MS analysis, confirmed that the actual subunit size of the enzyme was 24.5kDa. Thus, we conclude that the active form of native Nocardia GCH is a decamer. Our earlier incorrect conclusion was that the native enzyme was an octamer derived from the anomalous SDS-PAGE migration of the subunit.

GTP cyclohydrolase I: purification, characterization, and effects of inhibition on nitric oxide synthase in nocardia species.

GTP cyclohydrolase I (GTPCH) catalyzes the first step in pteridine biosynthesis in Nocardia sp. strain NRRL 5646. This enzyme is important in the biosynthesis of tetrahydrobiopterin (BH4), a reducing cofactor required for nitric oxide synthase (NOS) and other enzyme systems in this organism. GTPCH was purified more than 5,000-fold to apparent homogeneity by a combination of ammonium sulfate fractionation, GTP-agarose, DEAE Sepharose, and Ultragel AcA 34 chromatography. The purified enzyme gave a single band for a protein estimated to be 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme was estimated to be 253 kDa by gel filtration, indicating that the active enzyme is a homo-octamer. The enzyme follows Michaelis-Menten kinetics, with a Km for GTP of 6.5 micromoles. Nocardia GTPCH possessed a unique N-terminal amino acid sequence. The pH and temperature optima for the enzyme were 7.8 and 56 degrees C, respectively. The enzyme was heat stable and slightly activated by potassium ion but was inhibited by calcium, copper, zinc, and mercury, but not magnesium. BH4 inhibited enzyme activity by 25% at a concentration of 100 micromoles. 2,4-Diamino-6-hydroxypyrimidine (DAHP) appeared to competitively inhibit the enzyme, with a Ki of 0.23 mM. With Nocardia cultures, DAHP decreased medium levels of NO2- plus NO3-. Results suggest that in Nocardia cells, NOS synthesis of nitric oxide is indirectly decreased by reducing the biosynthesis of an essential reducing cofactor, BH4.

A member of a new class of GTP cyclohydrolases produces formylaminopyrimidine nucleotide monophosphates.

The hyperthermophilic euryarchaeon Methanococcus jannaschii has no recognizable homologues of the canonical GTP cyclohydrolase enzymes that are required for riboflavin and pteridine biosyntheses. Instead, it uses a new type of thermostable GTP cyclohydrolase enzyme that produces 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone ribonucleotide monophosphate and inorganic phosphate. Whereas canonical GTP cyclohydrolases produce this formylamino-pyrimidine nucleotide as a reaction intermediate, this compound is shown to be an end product of the purified recombinant M.jannaschii enzyme. Unlike other enzymes that hydrolyze the alpha-beta phosphate anhydride bond of GTP, this new enzyme completely hydrolyzes pyrophosphate to inorganic phosphate. As a result, the enzyme has a steady-state turnover of 21 min(-)(1), which is much faster than those of canonical GTP cyclohydrolase enzymes. The effects of substrate analogues and inhibitors suggest that the GTP cyclohydrolase and pyrophosphate phosphohydrolase activities occur at independent sites, although both activities depend on Mg(2+).

Biosynthesis of vitamin b2 (riboflavin).

The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate as substrates. The imidazole ring of GTP is hydrolytically opened, yielding a 4, 5-diaminopyrimidine which is converted to 5-amino-6-ribitylamino-2, 4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction and dephosphorylation. Condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3, 4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate affords 6,7-dimethyl-8-ribityllumazine. Dismutation of the lumazine derivative yields riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is recycled in the biosynthetic pathway. The structure of the biosynthetic enzyme, 6,7-dimethyl-8-ribityllumazine synthase, has been studied in considerable detail.

Purification and characterization of GTP cyclohydrolase I from Streptomyces tubercidicus, a producer of tubercidin.

GTP cyclohydrolase I catalyzing the first reaction in the biosynthesis of pterin moiety of folic acid in bacteria, was purified from Streptomyces tubercidicus by at least 203-fold with a yield of 32% to apparent homogeneity, using ammonium sulfate fractionation, DEAE-cellulose, Sepharose CL-6B, and hydroxylapatite column chromatography. The molecular weight of the native enzyme was estimated to be 230,000 daltons by gel permeation chromatography. The purified enzyme gave a single band on sodium dodesyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was apparently 58,000 daltons. These results indicate that the enzyme consists of four subunits with the same molecular weight. The K(m) and Vmax values for GTP of the purified enzyme were determined to be 80 microM and 90 nmol/min (mg protein), respectively. The optimum pH and temperature for the enzyme reaction were pH 7.5-8.5 and 40-42 degrees C, respectively. Coenzyme or metal ion was not required for the enzyme activity. The enzyme activity was inhibited by most divalent cations, while it was slightly activated by potassium ion. In case of nucleotides, CTP, GMP, GDP, and UTP inhibited enzyme activity, among which GDP exhibited the strongest inhibitory effect.

Potential of Escherichia coli GTP cyclohydrolase II for hydrolyzing 8-oxo-dGTP, a mutagenic substrate for DNA synthesis.

MutT protein of Escherichia coli prevents the occurrence of A:T --> C:G transversion by hydrolyzing an oxidized form of dGTP, 8-oxo-7, 8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), which is produced by active oxygen species. In a search for mutT-related genes, we found that the ribA gene, encoding GTP cyclohydrolase II, is able to reduce the increased level of mutation frequency of the mutT strain to almost the normal level, provided that the gene product is overproduced. Purified preparations of Escherichia coli GTP cyclohydrolase II protein as well as the histidine hexamer-tagged recombinant GTP cyclohydrolase II protein efficiently hydrolyze 8-oxo-dGTP and 8-oxo-GTP, producing 8-oxo-dGMP and 8-oxo-GMP, respectively. dGTP was not hydrolyzed by these preparations. GTP cyclohydrolase II catalyzes conversion of GTP to 2, 5-diamino-6-hydroxy-4-(5-phosphoribosylamino)-pyrimidine, which constitutes the first step for riboflavin synthesis. The Km values for the three types of guanine nucleotides, GTP, 8-oxo-GTP, and 8-oxo-dGTP, were almost the same. In the mutT- background, ribA- cells showed higher spontaneous mutation frequencies as compared with that of ribA+ cells. Thus, GTP cyclohydrolase II, the ribA gene product, has a potential to protect genetic material from the untoward effects of endogenous oxygen radicals.

Purification and cloning of the GTP cyclohydrolase I feedback regulatory protein, GFRP.

The activity of GTP cyclohydrolase I, the initial enzyme of the de novo pathway for biosynthesis of tetrahydrobiopterin, the cofactor required for aromatic amino acid hydroxylations and nitric oxide synthesis, is sensitive to end-product feedback inhibition by tetrahydrobiopterin. This inhibition by tetrahydrobiopterin is mediated by the GTP cyclohydrolase I feedback regulatory protein GFRP, previously named p35 (Harada, T., Kagamiyama, H., and Hatakeyama, K. (1993) Science 260, 1507-1510), and -phenylalanine specifically reverses the tetrahydrobiopterin-dependent inhibition. As a first step in the investigation of the physiological role of this unique mechanism of regulation, a convenient procedure has been developed to co-purify to homogeneity both GTP cyclohydrolase I and GFRP from rat liver. GTP cyclohydrolase I and GFRP exist in a complex which can be bound to a GTP-affinity column from which GTP cyclohydrolase I and GFRP are separately and selectively eluted. GFRP is dissociated from the GTP agarose-bound complex with 0.2 NaCl, a concentration of salt which also effectively blocks the tetrahydrobiopterin-dependent inhibitory activity of GFRP. GTP cyclohydrolase I is then eluted from the GTP-agarose column with GTP. Both GFRP and GTP cyclohydrolase I were then purified separately to near homogeneity by sequential high performance anion exchange and gel filtration chromatography. GFRP was found to have a native molecular mass of 20 kDa and consist of a homodimer of 9.5-kDa subunits. Based on peptide sequences obtained from purified GFRP, oligonucleotides were synthesized and used to clone a cDNA from a rat liver cDNA library by polymerase chain reaction-based methods. The cDNA contained an open reading frame that encoded a novel protein of 84 amino acids (calculated molecular mass 9665 daltons). This protein when expressed in Escherichia coli as a thioredoxin fusion protein had tetrahydrobiopterin-dependent GTP cyclohydrolase I inhibitory activity. Northern blot analysis indicated the presence of an 0.8-kilobase GFRP mRNA in most rat tissues, the amounts generally correlating with levels of GTP cyclohydrolase I and tetrahydrobiopterin. Thus, mRNA levels were relatively high in liver and kidney and somewhat lower in testis, heart, brain, and lung. These results suggest that GFRP is widely expressed and may play a role in regulating not only phenylalanine metabolism in the liver, but also the production of biogenic amine neurotransmitters as well as nitric oxide synthesis.

Active site topology and reaction mechanism of GTP cyclohydrolase I.

GTP cyclohydrolase I of Escherichia coli is a torus-shaped homodecamer with D5 symmetry and catalyzes a complex ring expansion reaction conducive to the formation of dihydroneopterin triphosphate from GTP. The x-ray structure of a complex of the enzyme with the substrate analog, dGTP, bound at the active site was determined at a resolution of 3 A. In the decamer, 10 equivalent active sites are present, each of which contains a 10-A deep pocket formed by surface areas of 3 adjacent subunits. The substrate forms a complex hydrogen bond network with the protein. Active site residues were modified by site-directed mutagenesis, and enzyme activities of the mutant proteins were measured. On this basis, a mechanism of the enzyme-catalyzed reaction is proposed. Cleavage of the imidazole ring is initiated by protonation of N7 by His-179 followed by the attack of water at C8 of the purine system. Cystine Cys-110 Cys-181 may be involved in this reaction step. Opening of the imidazole ring may be in concert with cleavage of the furanose ring to generate a Schiff's base from the glycoside. The gamma-phosphate of GTP may be involved in the subsequent Amadori rearrangement of the carbohydrate side chain by activating the hydroxyl group of Ser-135.

Isolation of a full-length cDNA clone for human GTP cyclohydrolase I type 1 from pheochromocytoma.

Although the existence of three different cDNA forms of human GTP cyclohydrolase I (GCH I) have been reported (Togari et al., 1992), the full-length sequence of any human GCH I cDNA involving poly (A) tail has not yet been documented. In the present study, we first isolated a full-length cDNA clone encoding human GCH I type 1 from human pheochromocytoma cDNA library. The length of the cDNA insert was 2,921 base pairs including poly (A) tail. RNA blot analysis showed a single mRNA species of 4.0 kb in human pheochromocytoma tissue.

Identification of a rabphilin-3A-interacting protein as GTP cyclohydrolase I in PC12 cells.

Rabphilin-3A is a putative target protein for Rab3A small GTP-binding protein which is implicated in neurotransmitter release. To identify a Rabphilin-3A-interacting protein, proteins were immunoprecipitated by an anti-Rabphilin-3A polyclonal antibody from the lysate of PC12 cells and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by protein staining. Several proteins were coimmunoprecipitated with Rabphilin-3A and one of these proteins with a M(r) of about 30 KDa was phosphorylated in intact PC12 cells stimulated by high KCl. The amino acid sequence analysis of this 30 KDa protein revealed that it is GTP cyclohydrolase I.

Crystallization and preliminary crystallographic characterization of GTP cyclohydrolase I from Escherichia coli.

GTP cyclohydrolase I of Escherichia coli has been purified from a recombinant bacterial strain. The enzyme was crystallized from 0.6 M-sodium citrate and from 0.8 M-sodium/potassium phosphate, respectively. Crystals grown in citrate showed X-ray diffraction extending to a resolution better than 3 A. The space group was P2(1) with cell dimensions a = 204.8 A, b = 210.1 A, c = 72.2 A, alpha = gamma = 90 degrees and beta = 95.8 degrees.

[Purification and properties of GTP-cyclohydrolase from Bacillus subtilis]. / Ochistka i svoistva GTP-tsikogidrolazy Bacillus subtilis.

Highly purified GTP-cyclohydrolase was obtained by fractionation of cell extracts with ammonium sulfate, ion-exchange and hydrophobic chromatography. The N-terminal amino acid sequence and amino acid composition of the protein were determined. According to SDS-PAGE data, the molecular weight of the enzyme is 45 kDa. The active enzyme has several isoforms separable by native electrophoresis. The maximal enzyme activity is determined at 1.5 mM Mn2+; 70% of enzymatic activity is detected with Mg2+. The enzyme is inhibited by heavy metal ions and chelators and is inactive in the absence of thiol-reducing agents. The enzyme activity is detected in a broad range of pH with a maximum at pH 8.2. The pyrimidine product of the GTP-cyclohydrolase reaction. 2.5-diamino-6-hydroxy-4-ribosylaminopyrimidine-5'-phosphate was purified and identified. Another product of this reaction is pyrophosphate.

Isolation and characterization of GTP cyclohydrolase I from mouse liver. Comparison of normal and the hph-1 mutant.

GTP cyclohydrolase I, an enzyme that catalyzes the first reaction in the pathway for the biosynthesis of pterin compounds, was purified from of C3H mouse liver by 192-fold to apparent homogeneity, using Ultrogel AcA34, DEAE-Trisacryl, and GTP-agarose gels. Its native molecular weight was estimated at 362,000. When the enzyme was subjected to electrophoresis on a denaturing polyacrylamide gel, only one protein band was evident, and its molecular weight was estimated at 55,700. The NH2-terminal amino acid of this enzyme was serine. These results indicate the enzyme consists of six to eight subunits. No coenzyme or metal ion was required for activity. This enzyme activity was inhibited by most of divalent cations and was slightly activated by potassium ion. The Km value for GTP was determined to be 17.3 microM. The temperature and pH optima for the activity were 60 degrees C and pH 8.0-8.5, respectively. The expected products, a dihydroneopterin compound and formic acid, were found in a molar ratio of 1.01. A polyclonal antiserum generated against the purified enzyme was used to compare GTP cyclohydrolase I from the hph-1 mutant and normal mouse. The hph-1 mutant liver contained only 8% of normal specific activity, but a normal amount of GTP cyclohydrolase I antigen as compared with the C3H mouse. Subunit molecular weight and electrophoretic behavior of GTP cyclohydrolase I from hph-1 mutant were not different from those of the enzyme from C3H mouse. These results suggest that the hph-1 mutation may involve alteration of the catalytic site but does not detectably alter the whole enzyme structure.

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.

Human liver GTP cyclohydrolase I: purification and some properties.

Human liver guanosine triphosphate (GTP) cyclohydrolase I has been purified more than 1,700-fold to what appears to be homogeneity. The active enzyme complex has an estimated molecular weight of 453,000 +/- 11,500 by gel filtration chromatography. It consists of a polypeptide of 149,000 +/- 4,000 mol wt by SDS-polyacrylamide gel electrophoresis. The activity of the enzyme is heat stable and is inhibited by di- and trivalent cations. The enzyme has an optimum pH of 7.7 in sodium phosphate buffer. It uses GTP as a sole substrate, with a Km of 116 microM.

Purification of GTP cyclohydrolase I from human liver and production of specific monoclonal antibodies.

GTP cyclohydrolase I, the first enzyme in the de novo biosynthesis of tetrahydrobiopterin, was enriched more than 13,000-fold from human liver by preparative isoelectric focusing using Sephadex G-200 SF gels. The pI of the active enzyme was determined as 5.6 by analytical isoelectric focusing in the same matrix. The native enzyme has an apparent molecular mass of 440 kDa and appears to be composed of eight 50-kDa subunits as estimated from SDS/PAGE. The enriched enzyme preparation was used to produce specific monoclonal antibodies. From 11 monoclonal antibodies obtained, one was extensively characterized for further applications. This monoclonal antibody belongs to the IgM class and shows immunoreactivity with GTP cyclohydrolase I both from man and from Escherichia coli. It is capable of highly sensitive detection of GTP cyclohydrolase I by ELISA and by Western blot analysis. The monoclonal antibody was used for the immunoenzymatic localisation of GTP cyclohydrolase I in human peripheral blood mononuclear cells. Furthermore, it was possible to demonstrate the absence of immunoreactivity in cells with GTP cyclohydrolase I deficiency. The antibody's use as a tool either for differential diagnosis of atypical phenylketonuria due to GTP cyclohydrolase I deficiency or prenatal diagnosis of this severe inherited metabolic disease is now under investigation.