Tetrahydrobiopterin (BH(4)) cofactor is essential for various processes, and is present in probably every cell or tissue of higher organisms. BH(4) is required for various enzyme activities, and for less defined functions at the cellular level. The pathway for the de novo biosynthesis of BH(4) from GTP involves GTP cyclohydrolase I, 6-pyruvoyl-tetrahydropterin synthase and sepiapterin reductase. Cofactor regeneration requires pterin-4a-carbinolamine dehydratase and dihydropteridine reductase. Based on gene cloning, recombinant expression, mutagenesis studies, structural analysis of crystals and NMR studies, reaction mechanisms for the biosynthetic and recycling enzymes were proposed. With regard to the regulation of cofactor biosynthesis, the major controlling point is GTP cyclohydrolase I, the expression of which may be under the control of cytokine induction. In the liver at least, activity is inhibited by BH(4), but stimulated by phenylalanine through the GTP cyclohydrolase I feedback regulatory protein. The enzymes that depend on BH(4) are the phenylalanine, tyrosine and tryptophan hydroxylases, the latter two being the rate-limiting enzymes for catecholamine and 5-hydroxytryptamine (serotonin) biosynthesis, all NO synthase isoforms and the glyceryl-ether mono-oxygenase. On a cellular level, BH(4) has been found to be a growth or proliferation factor for Crithidia fasciculata, haemopoietic cells and various mammalian cell lines. In the nervous system, BH(4) is a self-protecting factor for NO, or a general neuroprotecting factor via the NO synthase pathway, and has neurotransmitter-releasing function. With regard to human disease, BH(4) deficiency due to autosomal recessive mutations in all enzymes (except sepiapterin reductase) have been described as a cause of hyperphenylalaninaemia. Furthermore, several neurological diseases, including Dopa-responsive dystonia, but also Alzheimer's disease, Parkinson's disease, autism and depression, have been suggested to be a consequence of restricted cofactor availability.

The hinge-bending motion of the two domains upon closure of the structure, as seen in the Trypanosoma PGK structure, is confirmed. This closed conformation obviously occurs after binding of both substrates and is locked by the Arg62-Asp200 salt bridge. Re-orientations in the conserved active-site loop region around Thr374 also bring both domains into direct contact in the core region of the former inter-domain cleft, to form the complete catalytic site. Comparison of extremely thermostable TmPGK with less thermostable homologues reveals that its increased rigidity is achieved by a raised number of intramolecular interactions, such as an increased number of ion pairs and additional stabilization of alpha helix and loop regions. The covalent fusion with triosephosphate isomerase might represent an additional stabilization strategy.

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The crystal structure of recombinant human GTP cyclohydrolase I was solved by Patterson search methods by using the coordinates of the Escherichia coli enzyme as a model. The human as well as bacterial enzyme were shown to contain an essential zinc ion coordinated to a His side chain and two thiol groups in each active site of the homodecameric enzymes that had escaped detection during earlier studies of the E. coli enzyme. The zinc ion is proposed to generate a hydroxyl nucleophile for attack of imidazole ring carbon atom eight of the substrate, GTP. It may also be involved in the hydrolytic release of formate from the intermediate, 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5-triphosphate, and in the consecutive Amadori rearrangement of the ribosyl moiety.

(5), compound 4 is the biosynthetic precursor of tetrahydrobiopterin, which is involved in the formation of catecholamines and nitric oxide (6, 7). Tetrahydrobiopterin is also involved in the stimulation of T lymphocytes, although the details are still incompletely understood (8).CYH was originally detected in bacteria (9). More recently, the primary structure of CYH from a wide variety of organisms has been determined (10-16). The evolution of the protein has been relatively conservative. Thus, the C-terminal 120 residues of Escherichia coli and human enzymes are 60% identical. The crystal structure of E. coli CYH has recently been solved by single isomorphous replacement and averaging techniquesThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. (17). The knowledge of the crystal packing arrangement obtained by electron microscopy (18, 19) was helpful in the initial stages of structure determination. The enzyme complex, a decamer consisting of a pentamer of tightly associated dimers, is doughnut shaped with dimensions of 65 x 100 A.One CYH subunit folds into a a+f structure with a predominantly helical N terminus (Fig. 2). The N-terminal helix hl (residues 5-18) is followed by a helix pair, composed of helix h2 (residues 32-49) and helix h3 (residues 62-72), which are remote from the compact C-terminal domain of the molecule (residues 95-217). This domain has a central fourstranded antiparallel 13-sheet that is flanked on both sides by a-helices. The folding topology of this domain is identical to that of a subunit of the second enzyme in tetrahydrobiopterin biosynthesis pathway, 6-pyruvoyltetrahydropterin synthase (20).The association of two CYH monomers to dimers is driven by the formation of a four-helix bundle by helices h2 and h3 of each monomer. Apart from a large hydrophobic surface buried by this interaction, numerous hydrogen bonds and salt bridges serve to stabilize the dimer. The decamer is formed by a fivefold symmetric arrangement of the dimers. Its most striking structural feature is an unprecedented 20-stranded antiparallel ,B-barrel (Fig. 2). This paper describes the crystal structure of a complex of CYH with dGTP, the substrate analog.** By using the inforAbbreviation: CYH, GTP cyclohydrolase I.

The transformation of 4-hydroxyphenylpyruvate to homogentisate, catalyzed by 4-hydroxyphenylpyruvate dioxygenase (HPPD), plays an important role in degrading aromatic amino acids. As the reaction product homogentisate serves as aromatic precursor for prenylquinone synthesis in plants, the enzyme is an interesting target for herbicides. In this study we report the first x-ray structures of the plant HPPDs of Zea mays and Arabidopsis in their substrate-free form at 2.0 Å and 3.0 Å resolution, respectively. Previous biochemical characterizations have demonstrated that eukaryotic enzymes behave as homodimers in contrast to prokaryotic HPPDs, which are homotetramers. Plant and bacterial enzymes share the overall fold but use orthogonal surfaces for oligomerization. In addition, comparison of both structures provides direct evidence that the C-terminal helix gates substrate access to the active site around a nonheme ferrous iron center. In the Z. mays HPPD structure this helix packs into the active site, sequestering it completely from the solvent. In contrast, in the Arabidopsis structure this helix tilted by about 608 into the solvent and leaves the active site fully accessible. By elucidating the structure of plant HPPD enzymes we aim to provide a structural basis for the development of new herbicides.

Sepiapterin reductase catalyses the last steps in the biosynthesis of tetrahydrobiopterin, the essential cofactor of aromatic amino acid hydroxylases and nitric oxide synthases. We have determined the crystal structure of mouse sepiapterin reductase by multiple isomorphous replacement at a resolution of 1.25 Å in its ternary complex with oxaloacetate and NADP. The homodimeric structure reveals a single-domain α/β-fold with a central four-helix bundle connecting two seven-stranded parallel β-sheets, each sandwiched between two arrays of three helices. Ternary complexes with the substrate sepiapterin or the product tetrahydrobiopterin were studied. Each subunit contains a specific aspartate anchor (Asp258) for pterin-substrates, which positions the substrate side chain C1Ј-carbonyl group near Tyr171 OH and NADP C4ЈN. The catalytic mechanism of SR appears to consist of a NADPH-dependent proton transfer from Tyr171 to the substrate C1Ј and C2Ј carbonyl functions accompanied by stereospecific side chain isomerization. Complex structures with the inhibitor N-acetyl serotonin show the indoleamine bound such that both reductase and isomerase activity for pterins is inhibited, but reaction with a variety of carbonyl compounds is possible. The complex structure with N-acetyl serotonin suggests the possibility for a highly specific feedback regulatory mechanism between the formation of indoleamines and pteridines in vivo.

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.GTP cyclohydrolase I catalyzes the formation of dihydroneopterin triphosphate from GTP via a mechanistically complex ring expansion. In plants and micro-organisms, the enzyme product serves as the first committed intermediate in the biosynthesis of tetrahydrofolate (1). In animals, the enzyme product is converted to tetrahydrobiopterin, which serves as cofactor for the biosynthesis of catecholamines and of nitric oxide (2-4). Genetic defects of GTP cyclohydrolase I result in severe neurological impairment (5-8).GTP cyclohydrolase I of Escherichia coli is a 247-kDa homodecamer (9, 10). The structure of the protein has been studied by x-ray structure analysis at a resolution of 2.6 Å (11). The torus-shaped protein obeys D 5 symmetry. Each of the 10 equivalent active sites is located at the interface of three adjacent subunits.Brown, Shiota, and their co-workers (12-14) could show the reaction sequence catalyzed by GTP cyclohydrolase I to involve the opening of the imidazole ring of GTP (compound 1, Fig. 1) with release of formate. Carbon atoms 1Ј and 2Ј of the ribose moiety of GTP are then used to form the dihydropyrazine ring of dihydroneopterin triphosphate (compound 5, Fig. 1). However, the mechanistic details of the highly complex enzymecatalyzed reactions are incompletely understood.The catalytic activity of GTP cyclohydrolase is highly sensitive to the replacement of amino acid residues at the active site cavity (11 13 C]formate, phosphoryl chloride, trimethyl phosphate, N,N-dimethylformamide, tri-n-butylamine, and pyrophosphoric acid were purchased from Sigma-Aldrich. All other chemicals were reagent grade.Enzyme Assays-Assay mixtures contained 100 mM Tris hydrochloride, pH 8.5, 100 mM KCl, 2.5 mM EDTA, 1 mM GTP, and protein in a total volume of 450 l. The mixtures were incubated at 37°C, and 100-l aliquots were retrieved at intervals. The formation of dihydroneopterin triphosphate and 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone 5Ј-phosphate was monitored as follows.Assay of Dihydroneopterin Triphosphate-Aliquots of enzyme assay mixture were mixed with 30 l of a solution containing 1% iodine and 2% KI in 1 M HCl. After incubation for 30 min at ambient temp...