guanosine-triphosphate and dihydroneopterin-triphosphate

The biosynthesis of GTP derived metabolites such as tetrahydrofolate (THF), biopterin (BH(4)), and the modified tRNA nucleosides queuosine (Q) and archaeosine (G(+)) relies on several enzymes of the Tunnel-fold superfamily. A subset of these proteins includes the 6-pyruvoyltetrahydropterin (PTPS-II), PTPS-III, and PTPS-I homologues, all members of the COG0720 family that have been previously shown to transform 7,8-dihydroneopterin triphosphate (H(2)NTP) into different products. PTPS-II catalyzes the formation of 6-pyruvoyltetrahydropterin in the BH(4) pathway, PTPS-III catalyzes the formation of 6-hydroxylmethyl-7,8-dihydropterin in the THF pathway, and PTPS-I catalyzes the formation of 6-carboxy-5,6,7,8-tetrahydropterin in the Q pathway. Genes of these three enzyme families are often misannotated as they are difficult to differentiate by sequence similarity alone. Using a combination of physical clustering, signature motif, phylogenetic codistribution analyses, in vivo complementation studies, and in vitro enzymatic assays, a complete reannotation of the COG0720 family was performed in prokaryotes. Notably, this work identified and experimentally validated dual function PTPS-I/III enzymes involved in both THF and Q biosynthesis. Both in vivo and in vitro analyses showed that the PTPS-I family could tolerate a translation of the active site cysteine and was inherently promiscuous, catalyzing different reactions on the same substrate or the same reaction on different substrates. Finally, the analysis and experimental validation of several archaeal COG0720 members confirmed the role of PTPS-I in archaeosine biosynthesis and resulted in the identification of PTPS-III enzymes with variant signature sequences in Sulfolobus species. This study reveals an expanded versatility of the COG0720 family members and illustrates that for certain protein families extensive comparative genomic analysis beyond homology is required to correctly predict function.

Topics: Amino Acid Motifs; Archaeal Proteins; Biopterins; Genetic Complementation Test; Guanosine; Guanosine Triphosphate; Kinetics; Models, Molecular; Molecular Sequence Data; Neopterin; Nucleoside Q; Phosphorus-Oxygen Lyases; Phylogeny; Protein Structure, Tertiary; Recombinant Proteins; Sequence Homology, Amino Acid; Substrate Specificity; Sulfolobus; Tetrahydrofolates

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.

Topics: Archaeal Proteins; Cloning, Molecular; Escherichia coli; Evolution, Molecular; Genes, Archaeal; GTP Cyclohydrolase; Guanosine Triphosphate; Histidine; Iron; Methanococcaceae; Models, Chemical; Neopterin; Phylogeny; Pterins; Substrate Specificity

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.

Topics: Amino Acid Sequence; Animals; Binding Sites; Catalysis; Crystallization; Crystallography, X-Ray; Escherichia coli; Formamides; Formates; GTP Cyclohydrolase; Guanosine Triphosphate; Hydrogen Bonding; Hydrolysis; Kinetics; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Neopterin; Protein Conformation; Pteridines; Stereoisomerism; Zinc

GTP cyclohydrolase I catalyses the transformation of GTP into dihydroneopterin 3'-triphosphate, which is the first committed precursor of tetrahydrofolate and tetrahydrobiopterin. The kinetically competent reaction intermediate, 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone, was used as substrate for single turnover experiments monitored by multiwavelength photometry. The early reaction phase is characterized by the rapid appearance of an optical transient with an absorption maximum centred at 320. This species is likely to represent a Schiff base intermediate at the initial stage of the Amadori rearrangement of the carbohydrate side-chain. Deconvolution of the optical spectra suggested four linearly independent processes. A fifth reaction step was attributed to photodecomposition of the enzyme product. Pre-steady state experiments were also performed with the H179A mutant which can catalyse a reversible conversion of GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone but is unable to form the final product, dihydroneopterin triphosphate. Optical spectroscopy failed to detect any intermediate in the reversible reaction sequence catalysed by the mutant protein. The data obtained with the wild-type and mutant protein in conjunction with earlier quenched flow studies show that the enzyme-catalysed opening of the imidazole ring of GTP and the hydrolytic release of formate from the resulting formamide type intermediate are both rapid reactions by comparison with the subsequent rearrangement of the carbohydrate side-chain which precedes the formation of the dihydropyrazine ring of dihydroneopterin triphosphate.

Topics: Catalysis; Escherichia coli; Formates; GTP Cyclohydrolase; Guanosine Triphosphate; Hydrolysis; Kinetics; Mutation; Neopterin; Pteridines; Pyrimidine Nucleotides; Schiff Bases; Spectrophotometry, Ultraviolet; Stereoisomerism

GTP cyclohydrolase I catalyzes a mechanistically complex ring expansion affording dihydroneopterin triphosphate and formate from GTP. Single turnover quenched flow experiments were performed with the recombinant enzyme from Escherichia coli. The consumption of GTP and the formation of 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone triphosphate, formate, and dihydroneopterin triphosphate were determined by high pressure liquid chromatography analysis. A kinetic model comprising three consecutive unimolecular steps was used for interpretations where the first intermediate, 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone 5'-triphosphate, was formed in a reversible reaction. The rate constant k(1) for the reversible opening of the imidazole ring of GTP was 0.9 s(-1), the rate constant k(3) for the release of formate from 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone triphosphate was 2.0 s(-1), and the rate constant k(4) for the formation of dihydroneopterin triphosphate was 0.03 s(-1). Thus, the hydrolytic opening of the imidazole ring of GTP is rapid by comparison with the overall reaction.

Topics: Aldose-Ketose Isomerases; Escherichia coli; Flow Injection Analysis; Formates; GTP Cyclohydrolase; Guanosine Triphosphate; Models, Chemical; Neopterin; Pteridines; Pyrimidine Nucleotides

GTP cyclohydrolase I catalyzes the conversion of GTP to dihydroneopterin triphosphate. The replacement of histidine 179 by other amino acids affords mutant enzymes that do not catalyze the formation of dihydroneopterin triphosphate. However, some of these mutant proteins catalyze the conversion of GTP to 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone 5'-triphosphate as shown by multinuclear NMR analysis. The equilibrium constant for the reversible conversion of GTP to the ring-opened derivative is approximately 0.1. The wild-type enzyme converts the formylamino pyrimidine derivative to dihydroneopterin triphosphate; the rate is similar to that observed with GTP as substrate. The data support the conclusion that the formylamino pyrimidine derivative is an intermediate in the overall reaction catalyzed by GTP cyclohydrolase I.

Topics: Catalytic Domain; Escherichia coli; GTP Cyclohydrolase; Guanosine Triphosphate; Histidine; Models, Chemical; Mutation; Neopterin; Nuclear Magnetic Resonance, Biomolecular; Pteridines; Pyrimidine Nucleotides

Tetrahydrobiopterin serves as the cofactor for enzymes involved in neurotransmitter biosynthesis and as regulatory factor in immune cell proliferation and the biosynthesis of melanin. The biosynthetic pathway to tetrahydrobiopterin consists of three steps starting from GTP. The initial reaction is catalyzed by GTP cyclohdrolase I (GTP-CH-I) and involves the chemically complex transformation of the purine into the pterin ring system.. The crystal structure of the Escherichia coli GTP-CH-I was solved by single isomorphous replacement and molecular averaging at 3.0 A resolution. The functional enzyme is a homodecameric complex with D5 symmetry, forming a torus with dimensions 65 A x 100 A. The pentameric subunits are constructed via an unprecedented cyclic arrangement of the four-stranded antiparallel beta-sheets of the five monomers to form a 20-stranded antiparallel beta-barrel of 35 A diameter. Two pentamers are tightly associated by intercalation of two antiparallel helix pairs positioned close to the subunit N termini. The C-terminal domain of the GTP-CH-I monomer is topologically identical to a subunit of the homohexameric 6-pyruvoyl tetrahydropterin synthase, the enzyme catalyzing the second step in tetrahydrobiopterin biosynthesis.. The active site of GTP-CH-I is located at the interface of three subunits. It represents a novel GTP-binding site, distinct from the one found in G proteins, with a catalytic apparatus that suggest involvement of histidines and, possibly, a cystine in the unusual reaction mechanism. Despite the lack of significant sequence homology between GTP-CH-I and 6-pyruvoyl tetrahydropterin synthase, the two proteins, which catalyze consecutive steps in tetrahydrobiopterin biosynthesis, share a common subunit fold and oligomerization mode. In addition, the active centres have an identical acceptor site for the 2-amino-4-oxo pyrimidine moiety of their substrates which suggests an evolutionarily conserved protein fold designed for pterin biosynthesis.

Topics: Alcohol Oxidoreductases; Bacterial Proteins; Binding Sites; Biopterins; Catalysis; Crystallography, X-Ray; Escherichia coli; GTP Cyclohydrolase; Guanosine Triphosphate; Models, Molecular; Neopterin; Phosphorus-Oxygen Lyases; Protein Conformation; Pteridines

GTP cyclohydrolase I catalyses the first committing step in the biosynthesis of the pterin moiety of folic acid: conversion of GTP to dihydroneopterin triphosphate. GTP cyclohydrolase I of Bacillus subtilis was purified to homogeneity and shown to have a homo-octameric structure. The enzyme had an apparent Km for GTP of 4 microM and, in the absence of cations, a Vmax. of 80 nmol/min per mg of protein. K+ ions moderately increased its Vmax., whereas UTP and Ca2+ and Mg2+ ions drastically increased its Km for GTP. Dihydrofolate and other products of the folate and tetrahydrobiopterin pathways did not inhibit GTP cyclohydrolase I. In addition to their effect on the enzyme activity, Ca2+ and Mg2+ ions catalysed the chemical dephosphorylation of dihydroneopterin triphosphate to non-cyclic dihydroneopterin monophosphate, the substrate for the phosphomonoesterase reaction in folate biosynthesis. This dephosphorylation was specific and did not require the action of a phosphatase. We suggest a physiological role for Ca2+ ions and UTP in regulation of folate biosynthesis at the levels of GTP cyclohydrolase I and dephosphorylation of dihydroneopterin triphosphate.

Topics: Bacillus subtilis; Base Sequence; Binding, Competitive; Calcium; Folic Acid; Gene Expression; GTP Cyclohydrolase; Guanosine Triphosphate; Kinetics; Magnesium; Molecular Sequence Data; Neopterin; Operon; Phosphorylation; Plasmids; Potassium; Promoter Regions, Genetic; Pteridines; Recombinant Proteins; Uridine Triphosphate

Topics: Biopterins; GTP Cyclohydrolase; Guanosine Triphosphate; Humans; Neopterin; Pteridines

Using Escherichia coli guanosine triphosphate cyclohydrolase, dihydroneopterin triphosphate was synthesized from guanosine triphosphate and was compared with sepiapterin as a substrate for tetrahydrobiopterin formation in bovine adrenal medulla extracts. The dihydrofolate reductase inhibitor, methotrexate, blocks the formation of tetrahydrobiopterin from sepiapterin but not from dihydroneopterin triphosphate. Reduced nicotinamide adenine dinucleotide phosphate and a divalent metal ion are required in partially purified preparations (gel filtration of 40-60% ammonium sulfate fraction on Ultrogel ACA-34) for the biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate. Sepiapterin was converted only to dihydrobiopterin in the same fractions since dihydrofolate reductase was removed. The evidence indicates that both dihydroneopterin triphosphate and sepiapterin are good precursors of tetrahydrobiopterin but they are not on the same pathway, contrary to previous proposals.

Topics: Adrenal Medulla; Animals; Biopterins; Cattle; Chemical Phenomena; Chemistry; Guanosine Triphosphate; Methotrexate; Neopterin; Pteridines; Pterins