Enzyme GTP cyclohydrolase

This interesting conversion of a five- into a six-membered heterocyclic ring was proven by the isolation of the enzyme GTP-cyclohydrolase from E. coli (71MI21600) and a similar one from Lactobacillus platarum (B-71MI21601) which catalyzes the reaction (300)(303). Dephosphorylation leads to 7,8-dihydro-D-neopterin (304), which is then cleaved in the side-chain to 6-hydroxymethyl-7,8-dihydropterin (305), the direct precursor of 7,8-dihy-dropteroic acid and 7,8-dihydrofolic acid (224). The alcohol (305) requires ATP and Mg " for the condensation with p-aminobenzoic and p-aminobenzoylglutamic acid, indicating pyrophosphate formation to (306) prior to the substitution step. 

Neopterin. Neopterin is a low-molecular-weight substance derived from guanosine triphosphate (GTP) via the enzyme GTP-cyclohydrolase 1. Numerous investigators have demonstrated that neopterin, a product of human macrophages stimulated by y interferon and other cytokines, is a useful in vivo marker of the activation of cellular immunity (U4, Wl). Increased values of neopterin in body fluids have been reported in patients with malignancy, infections, and several inflammatory states. 

The present information on the riboflavin biosynthetic pathway is summarized in Figure 1. Briefly, the pathway starts from GTP (1), which is converted into the first committed intermediate 2 by the hydrolytic release of pyrophosphate and of C-8 of the imidazole ring that are both catalyzed by a single enzyme, GTP cyclohydrolase II (reaction I). In Archaea and in fungi, that compound is transformed into 5-amino-6-ribitylamino-2,4(li/,3f/)-pyrimidinedione phosphate (5) by a reduction (reaction IV) that transforms the ribosyl side chain into the ribityl side chain (4) and by subsequent deamination (reaction V) of the pyrimidine ring yielding compound 5. In plants and in eubacteria (reactions II and III), these reaction steps occur in inverse order via the ribosylaminopyrimidine derivative 3. 

FIGURE 40-2 The phenylalanine hydroxylase (PAH) pathway. Phenylketonuria usually is caused by a congenital deficiency of PAH (reaction 1), but it also can result from defects in the metabolism of biopterin, which is a cofactor for the hydroxylase. Enzymes (1) Phenylalanine hydroxylase (2) Dihydropteridine reductase (3) GTP cyclohydrolase (4) 6-pyruvoyltetrahydrobiopterin synthase. BH4, tetrahydrobiopterin DEDT, o-erythro-dihydroneopterin triphosphate QH2, dihydrobiopterin. 

These patients suffer from a genetic defect of dopamine synthesis, caused by reduced GTP cyclohydrolase activity. This enzyme is rate-limiting in the biosynthesis of tetra-hydrobiopterin, a cofactor of the dopamine-synthesizing enzyme tyrosine hydroxylase (see Fig. 40-2). 

The enzymes in the zebra fish pathway are presumably very similar to those of other vertebrates. However a completely different type of GTP cyclohydrolase has been identified in the hyperthermophilic euryarchaeon, Methanococcus jannashii <2002B15074>. This enzyme, in purified recombinant form, produced as a stable end product 2-amino-5-formylamino-6-ribofuranosylamino-4(3//)-pyrimidinone monophosphate, a compound that is an intermediate in the action of normal GTP cyclohydrolases. The biosynthesis of the incorporation of the pterin into methanopterin in Methanobacterium thermoautotrophicum has been proposed to occur via substitution of 7,8-dihydro-6-hydroxymethylpterin diphosphate 227 (Scheme 44) <1998BBA257>. 

An important discovery is that procaryotes contain a different GTP cyclohydrolase 1 family distinct from the well studied canonical eukaryotic enzyme. Potentially, this enzyme is a target for new antibacterial drugs <2006JBC37586>. 

To prepare lysates from (nonstimulated) fibroblasts, cells from one confluent 78-cm2 plate are suspended in 0.15 ml lysis buffer (see below) and lysed by freezing and thawing six times and subsequent centrifugation at 13,000 x for 5 min. An aliquot of 0.05 ml of the supernatant is directly used for the enzyme assay. The preparation of tissue homogenate is described in section 6.1.4.1. GTP cyclohydrolase I, subheading Specimen . 

Most of hyperphenylalaninemia are caused by a mutation in the PAH gene. About 5% of hyperphenylalaninemia is caused by genetic defects in the BH4-metabolizing enzymes, and called malignant-type or atypical hyperphenylalaninemia. Patients with malignant-type hyperphenylalaninemia develop neurological symptoms due to deficiency of catecholamines and serotonin, as well as hyperphenylalaninemia. For example, patients with GTP cyclohydrolase deficiency show severe retardation of development, severe muscular hypotonia of the trunk and hypertonia of extremities, convulsions, and frequent episodes of hyperthermis without infection [160,161]. 

Specifically, the deflciency of certain enzymes of tetrahydrobiopterin biosynthesis (GTP cyclohydrolase 1, pymvoyltetrahy-drobiopterin synthase. Fig. 3) result in severe neurological and developmental deflcits designated as atypical phenylketonuria caused by the ensuing deflciency in catecholamine type neurotransmitter biosynthesis. The condition can be treated with some success by the oral application of synthetic tetrahydrobiopterin in large amounts. More recently, tetrahydrobiopterin therapy has also been advocated for certain patients with classic phenylketonuria that results from mutations of phenylalanine hydroxylase... 

Tetrahydrobiopterin is synthesized starting from GTP and requires at least three enzymes. The first committed step is GTP-cyclohydrolase, which converts GTP to dihy-droneopterin triphosphate. 6-Pyruvoyltetrahydrobiopterin synthase transforms dihydroneopterin triphosphate into 6-pyruvoyltetrahydrobiopterin. The latter is reduced to tetrahydrobiopterin by NADPH-dependent sepi-apterin reductase. Deficiency of GTP-cyclohydrolase and... 

A. The patient, despite being put on a low-Phe diet, exhibits neurologic problems resulting from an inability to synthesize catecholamine and indoleamine neurotransmitters. This is caused by a deficiency in dihydropteridine reductase (DHPR). DHPR regenerates tetrahydro-biopterin (BH ), which is oxidized to dihydrobiopterin by phenylalanine hydroxylase, as well as tyrosine hydroxylase and tryptophan hydroxylase (tryptophan 5-monooxygenase). If phenylalanine hydroxylase were deficient, a diet low in Phe would alleviate the effects. Since the urinary biopterin concentration is elevated, a deficiency in GTP cyclohydrolase I is eliminated because that is an enzyme in the biosynthetic pathway of BH. Phe hydroxylase, Tyr hydroxylase, and Tip hydroxylase activities are low because of a lack of BH. ... 

The crystal structure of CJueF from B. suhtilis was solved to 2.25 A, the protein exhibited a quaternary structure consisting of a dimer of hexamers (a dodecamer) belonging to the tunnel-fold superfamily, where substrate binding is believed to be at the subunit interface. To confirm the role of the enzyme in vitro, the genes from B. suhtilis and E. coli were cloned and expressed. After determining that the predicted GTP cyclohydrolase activity was not exhibited by CJiieF from these two species, alternative roles were explored. 

GTP cyclohydrolase II (reaction I) catalyzing the first committed step in the biosynthesis of riboflavin was discovered in the wake of work on GTP cyclohydrolase I (Figure 2), the enzyme catalyzing the first reaction step in the biosynthesis of tetrahydrofolate. ... 

The GTP cyclohydrolase involved in folate biosynthesis was subsequently designated GTP cyclohydrolase I, whereas enzymes involved in the biosynthetic pathways of riboflavin and methanopterin were designated GTP cyclohydrolase II and III, respectively. 

The classical type I GTP cyclohydrolases all carry one essential Ln ion per subunit (see, however, below for a different class of type I cyclohydrolases). In the decameric enzymes from microorganisms and animals, the metal ion is complexed by two cysteine and one histidine residues. The enzyme from E. coli or H. sapiens can lose the metal relatively easily. 

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