Conference Article| February 01 1996 Structure and mechanism of GTP cyclohydrolase I of Escherichia coli Herbert Nar; Herbert Nar ‡Max-Planck-Institut für Biochemie, Am Klopferspitz, D-82152 Martinsried, Federal Republic of Germany Search for other works by this author on: This Site PubMed Google Scholar Robert Huber; Robert Huber ‡Max-Planck-Institut für Biochemie, Am Klopferspitz, D-82152 Martinsried, Federal Republic of Germany Search for other works by this author on: This Site PubMed Google Scholar Winfried Meining; Winfried Meining #Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, D-85747 Garching, Federal Republic of Germany Search for other works by this author on: This Site PubMed Google Scholar Andreas Bracher; Andreas Bracher #Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, D-85747 Garching, Federal Republic of Germany Search for other works by this author on: This Site PubMed Google Scholar Markus Fischer; Markus Fischer #Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, D-85747 Garching, Federal Republic of Germany Search for other works by this author on: This Site PubMed Google Scholar Cornelia HÖSl; Cornelia HÖSl #Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, D-85747 Garching, Federal Republic of Germany Search for other works by this author on: This Site PubMed Google Scholar Harald Ritz; Harald Ritz #Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, D-85747 Garching, Federal Republic of Germany Search for other works by this author on: This Site PubMed Google Scholar Cornelia Schmid; Cornelia Schmid #Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, D-85747 Garching, Federal Republic of Germany Search for other works by this author on: This Site PubMed Google Scholar Sevil Weinkauf; Sevil Weinkauf #Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, D-85747 Garching, Federal Republic of Germany Search for other works by this author on: This Site PubMed Google Scholar Adelbert Bacher Adelbert Bacher #Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, D-85747 Garching, Federal Republic of Germany Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1996) 24 (1): 37S. https://doi.org/10.1042/bst024037s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation Herbert Nar, Robert Huber, Winfried Meining, Andreas Bracher, Markus Fischer, Cornelia HÖSl, Harald Ritz, Cornelia Schmid, Sevil Weinkauf, Adelbert Bacher; Structure and mechanism of GTP cyclohydrolase I of Escherichia coli. Biochem Soc Trans 1 February 1996; 24 (1): 37S. doi: https://doi.org/10.1042/bst024037s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1996 Biochemical Society1996 Article PDF first page preview Close Modal You do not currently have access to this content.

Background: Tetrahydrobiopterin serves as the cofactor for enzymes involved in neurotransmitter biosynthesis and as a 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 cyclohydrolase 1 (GTP-CH-I) and involves the chemically complex transformation of the purine into the pterin ring system.Results: The crystal structure of the Escherichia coli GTP-CH-I was solved by single isomorphous replacement and molecular averaging at 3.0 Angstrom resolution. The functional enzyme is a homodecameric complex with D-5 symmetry, forming a torus with dimensions 65 Angstrom x 100 Angstrom. 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 Angstrom 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.Conclusions: 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 suggests 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.

A monoclinic crystal modification of GTP cyclohydrolase I (space group P2(1), a = 204.2 A, b = 210.4 A, c = 71.8 A, alpha = gamma = 90 degrees, beta = 95.8 degrees) was studied by freeze-etching electron microscopy and by Patterson correlation techniques. The freeze-etched samples were either shadowed with Pt/C or decorated with monolayers of gold, silver or platinum. Correlation averaged electron micrographs of decoration replicas indicated 5-fold molecular symmetry. In conjunction with the molecular mass of the active GTP cyclohydrolase I enzyme complex of about 210,000 Da, which had been reported in the literature, and a molecular mass of the protomers of 24,700 Da, the electron microscopic observation suggests that the enzyme is a decamer with 5-fold symmetry. The processed images of decorated crystal surfaces also showed that the four protein multimers in the crystal unit cell are related by 4-fold pseudosymmetry. A Patterson analysis of the X-ray data showed two non-crystallographic 5-fold axes, inclined at 12 degrees to each other, thus confirming and extending the electron microscopic findings. Additionally, local 2-fold axes were found in planes perpendicular to the 5-fold particle axes. Thus, the combined X-ray and electron microscope data indicate that GTP cyclohydrolase I is a decamer with D5 symmetry. A procedure for hkl assignments of the crystal planes observed in electron micrographs was developed. On this basis, it was possible to determine the approximate molecular positions in the ab plane. Independent information on the crystal packing was obtained by single isomorphous replacement and electron density averaging. The 5-fold averaged 6 A electron density shows that the GTP cyclohydrolase I decamer is torus-shaped with an approximate diameter of 100 A and a thickness of 65 A. The study demonstrates that the combination of freeze-etching electron microscopy with Patterson analysis of X-ray data is a powerful approach for the solution of complex crystallographic problems. The procedure for this analysis as well as possible pitfalls are discussed in detail.

GTP cyclohydrolase I (EC 3.5.4.16) has been obtained from Escherichia coli wild type cells by affinity chromatography.1,2 The gene coding for the enzyme from E. coli has been cloned and sequenced3,4 and has been mapped at 2251 kb of the physical map of the E. coli chromosome.5 Strains carrying a plasmid with the gene under the control of its own promoter expressed about 100-fold increased enzyme levels. The protein has been crystallized from citrate buffer.6 GTP cyclohydrolase genes of rat,7 man,8,9 and Bacillus subtilis 10 have also been cloned, sequenced and expressed.

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.

SummaryIsotope-labeled dihydroneopterin 3'-triphosphate with 3H at positions C-1' and C-2', respectively, has been prepared from isotope-labeled glucose as starting material. Glucose was first converted enzymatically to ribose 5-phosphate. GMP was subsequently obtained by the action of phosphoribosylpyrophosphate synthetase and guanosine phosphoribosyl transferase. It was subsequently phosphorylated to GTP in two steps using adenylate kinase and guanylate kinase. Dihydroneopterin triphosphate was prepared from GTP by the action of recombinant GTP-cyclohydrolase I from Escherichia coli. The method allows the incorporation of 3H and 14C isotope labels into any desired position of dihydroneopterin triphosphate. Rapid purfication procedures for phosphoribosylpyrophosphate synthetase and guanosine phosphoribosyl transferase as well as HPLC assays for their determinations are described.

The induction of the enzymes in the tetrahydrobiopterin pathway by dimethyl sulfoxide (DMSO) was investigated in subclones F4N and B8/3 of the proerythroblastoid Friend erythroleukemia cell line (MEL). GTP-cyclohydrolase, the initial enzyme in the biosynthetic pathway, is virtually absent in both clones, but expression increases during 3 days of DMSO treatment. The final enzyme levels show 12-fold (subclone B8/3) and 40-fold (subclone F4N) increases compared to initial values. Enhancement of 6-pyruvoyl tetrahydropterin synthase activity is detectable 6 h after exposure to DMSO and continues to increase in the 3-day time period to 2.4-fold and 1.8-fold levels in subclones B8/3 and F4N, respectively. Sepiapterin reductase is present in unstimulated F4N cells and absent in B8/3 cells. The enzyme activity is not affected by DMSO treatment in either cell line. This explains why DMSO treatment causes accumulation of tetrahydrobiopterin in the MEL subclone F4N, but not in subclone B8/3. MEL cells are devoid of phenylalanine hydroxylase for which tetrahydrobiopterin serves as cofactor. In F4N, but not in B8/3, tetrahydrobiopterin modulates the rate of [3H]thymidine incorporation, thus being functionally linked with cell proliferation rather than with differentiation. In contrast to T lymphocytes, periods of tetrahydrobiopterin synthesis and of modulator function are uncoupled in MEL cells.

The putative gene coding for GTP cyclohydrolase I of Escherichia coli was isolated from a λgt11 expression vectori library by using antibodies as a probe and has been subcloned on a 3.8 kb BamHI fragment in the plasmid vector pUC13. E. coli cells carrying the recombinant plasmid designated pCYH express 100-fold increased levels of the enzyme. The protein formed under the control of the plasmid appears electrophoretically and immunochemically identical with the wild type enzyme.

Pyruvoyltetrahydropterin synthase catalyzes the release of tritiated water from [2'-3H]dihydroneopterin 3'-triphosphate. The tritiated water formed during the enzymatic reaction is separated from substrate by adsorption of the latter to activated charcoal. The sensitivity and specificity of the assay allows the determination of the enzyme in crude cell extract.

[1′-3H]- and [2′-3H]dihydroneopterin triphosphate (NH2TP) were prepared enzymatically from [4-3H]- and [5-3H]glucose and converted to tetrahydrobiopterin (BH4) by an extract from bovine adrenal medulla. The formation of BH4 from both [1′-3H]- and [2′-3H]-NH2TP proceeds with virtually complete loss of the respective tritium label. The breaking of the CHbond at C-1′ is characterized by a kinetic isotope effect of 2.6 ± 0.5. A smaller kinetic isotope effect of 1.5 ± 0.2 was found for the breaking of the CHbond at C-2′.