7.8- Dihydroneopterin-3’-triphosphate

Determination of GTP cyclohydrolase and D-erythro-7,8-dihydroneopterin triphosphate synthetase... 

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. 

DIHYDROLIPOAMIDE DEHYDROGENASE DIHYDRONEOPTERIN ALDOLA.SE DIHYDRONEOPTERIN TRIPHOSPHATE... 

The putative E. coli gene of the 6-pyruvoyltetrahydropterin synthase (PTPS) was cloned and overexpressed in order to identify the enzymatic activity to synthesize 6-pyruvoyltetrahydropterin from dihydroneopterin triphosphate <2002MI234>. The protein was shown to have another new catalytic function to convert sepiapterin to 7,8-dihydroneopterin. 

GTPCH (EC 3.5.4.16) converts the substrate GTP to 7,8-dihydroneopterin triphosphate (H2NTP) and formate. GTPCH activity is determined by measuring neopterin, the completely oxidized and dephosphorylated H TP-product of the enzyme reaction. Conversion of H2NTP to neopterin is carried out after the enzymatic reaction in presence of iodine at pH 1.0, followed by dephosphorylation with alkaline phosphatase at pH 8.5-9.0. Neopterin is detected fluorimetrically at 350/440 nm upon HPLC separation. The assay is based with some modifications on the methods published by Viveros et al. and Hatakeyama and Yoneyama [15,16]. 

PTPS (EC 4.6.1.10) converts the substrate 7,8-dihydroneopterin triphosphate (H2NTP) in a manganese-dependent reaction to the highly unstable intermediate... 

GCH catalyses the hydrolytic release of formate from GTP (135) followed by cyclization to dihydroneopterin triphosphate (136) [139]. GCH is the rate-limiting enzyme for the biosynthesis of BH4 (43), and the cellular BH4 content is regulated mainly by the activity of this enzyme. 

D-e/yf/iro-7,8-Dihydroneopterin triphosphate synthetase, or GTP cyclohydrolase I (EC 3.5.4.16), catalyzes the formation of D-eryr/iro-dihydroneopterin triphosphate (NH2TP) from GTP. This activity is required for the synthesis of tetrahydrobiopterin. The HPLC assay developed for this activity involves the direct measurement of neopterin phosphates after separation from GTP and its other hydrolytic products. 

Pyrovoyl tetrahydropterin synthetase catalyzes the second step in the conversion of guanosine triphosphate into tetrahydrobiopterin. The substrate, 4,8-dihydroneopterin triphosphate, is converted to 6-pyruvoyl tetrahydropterin. 

The standard reaction mixture was composed of 5 /aL of Tris-HCl (pH 7.4), 5 /aL of 40 mM NADPH, 5 /aL of sepiapterin reductase (activity of 400 nmol/min/mL), and 65 /aL of cell extract (10-200 /Ag of protein). The reaction was started by the addition of 20 /aL of 0.4 mM 7,8-dihydroneopterin triphosphate. After 30 to 90 minutes of incubation in the dark at 37°C, the reaction was terminated by the addition of 50 /aL of a mixture of 0.2 M HC1 and 0.02 M KI-I2 (11, v/v). The resulting mixture was incubated for 1 hour in the dark to allow oxidation of tetrahydrobiopterin to biopterin. Excess iodine was destroyed by the addition of 50 /aL of 0.02 M ascorbic add. An aliquot of the mixture was applied to a solid phase cartridge (SCX from Analytichem) that had been preequilibrated with 0.1 M H3PO4. The sample was forced through the cartridge with air pressure. The cartridge was then washed with 0.5 mL of 0.1 M H3PO4. The eluates were used for HPLC analysis. Assays were linear with up to 150 fig of cellular protein and 90 minutes of incubation. 

The formation of biopterin involves dephosphorylation and reduction of the side chain of dihydroneopterin triphosphate, followed by inversion of the conformation of the two hydroxyl groups, by way of intermediate oxidation to (symmetrical) oxo-groups, catalyzed by sepiapterin reductase. 

Patients with a variety of cancers and some viral diseases excrete relatively large amounts of neopterin, formed by dephosphorylation and oxidation of dihydroneopterin triphosphate, an intermediate in biopterin synthesis. This reflects the induction of GTP cyclohydrolase by interferon-y and tumor necrosis factor-a in response to the increased requirement for tetrahydrobiopterin for nitric oxide synthesis (Section 10.4.2). It is thus a marker of ceU-mediated immune reactions and permits monitoring of disease progression (Werner et al., 1993,1998 Berdowska and Zwirska-Korczala, 2001). 

Figure 3 Coenzymes biosynthesized from GTP. 8, molybdopterin 22, GTP 23, 5-amino-6-ribitylamino-2,4(1 H,3H)-pyrimidinedione 24, riboflavin 25, FMN 26, 5,6-dimethylbenzimidazole 27, precursor Z 28, metal containing pterin 29, dihydroneopterin triphosphate 30, 6-pyruvoyl-tetrahydropterin 31, 6(R)-5,6,7,8-tetrahydrobiopterin 32, dihydroneopterin 33, 6(S)-5,6,7,8-tetrahydrofolate 34, 5,6,7,8-tetrahydromethanopterin 35, 5-deaza-7,8-didemethyl-8-hydroxyribo-flavin 36, coenzyme F42q-...

Branching of pathways is relevant in several cases. Thus, intermediates of the porphyrin biosynthetic pathway serve as precursors for chlorophyll (17, Fig. 2) and for the corrinoid ring systems of vitamin B12 (20, Fig. 2) (17). 1-Deoxy-D-xylulose 5-phosphate (43) serves as an intermediate for the biosynthesis of pyridoxal 5 -phosphate (39, Fig. 5), for the terpenoid precursor IPP (86) via the nonmevalonate pathway (Fig. 11), and for the thiazole moiety of thiamine pyrophosphate (46, Fig. 4). 7,8-Dihydroneopterin triphosphate (29, Fig. 3) serves as intermediate in the biosynthetic pathways of tetrahydrofolate (33) and tetrahydrobiopterin (31). The closely related compound 7,8-dihydroneopterin 2, 3 -cyclic phosphate is the precursor of the archaeal cofactor, tetrahydromethanopterin (34) (58). A common pyrimidine-type intermediate (23) serves as precursor for flavin and deazaflavin coenzymes. Various sulfur-containing coenzymes (thiamine (9), lipoic acid (7), biotin (6), Fig. 1) use a pyrosulfide protein precursor that is also used for the biosynthesis of inorganic sulfide as a precursor for iron/sulfur clusters (12). 

The insolubilization of NAD and AMP and the uses of these supports have already been described, as has the use of hydrazide-agarose for immobilization. Other insolubilized nucleotide affinity columns have also been described. For example, Olsen [101] isolated galactosyltransferase from whey using a UDP-Sepharose affinity column. GTP coupled to a hydrazide Sepharose derivative was used to isolate D-erythro-dihydroneopterin triphosphate synthetase, the first enzyme for folate biosynthesis in Lactobacillus plantarum [102]. ATP-agarose has been used in the purification of heavy meromysin, elution being effected with ATP [103]. 

In E. coh GTP cydohydrolase catalyzes the conversion of GTP (33) into 7,8-dihydroneopterin triphosphate (34) via a three-step sequence. Hydrolysis of the triphosphate group of (34) is achieved by a nonspecific pyrophosphatase to afford dihydroneopterin (35) (65). The free alcohol (36) is obtained by the removal of residual phosphate by an unknown phosphomonoesterase. The dihydroneopterin undeigoes a retro-aldol reaction with the elimination of a hydroxy acetaldehyde moiety. Addition of a pyrophosphate group affords hydroxymethyl-7,8-dihydropterin pyrophosphate (37). Dihydropteroate synthase catalyzes the condensation of hydroxymeth5i-7,8-dihydropteroate pyrophosphate with PABA to furnish 7,8-dihydropteroate (38). Finally, L-glutamic acid is condensed with 7,8-dihydropteroate in the presence of dihydrofolate synthetase. 

In the folate pathway, the ribosyl moiety of GTP supplies the carbon atoms required for the formation of a second heterocyclic ring, thus affording the first committed intermediate, dihydroneopterin triphosphate. A motif with close structural similarity to the dihydroneopterin motif is a structural part of molybdopterin the problem, however, is that the two carbon atoms required for the formation of the pyrazine ring plus the carbon atoms of the position 6 side chain add up to 6, and the ribosyl moiety of GTP is obviously insufficient to supply them all. 

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... 

BH4 biosynthesis proceeds in the de novo pathway in a Mg -, Zn -, and NADPH-dependent reaction from GTP through the two intermediates, 7,8-dihydroneopterin triphosphate (H2NTP, 7) and 6-pyruvoyl-5,6,7,8-tetrahydropterin (PTP, 42), which have been isolated although they are rather unstable. The three enzymes... 

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