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The incorporation of [15N]glycine and [U-methyl-2H]methionine into methanopterin by growing cells of a methanogenic bacterium was measured to establish the biosynthetic route of the methylated pterin in the structure. The tetrahydromethanopterin produced by the cells was oxidatively cleaved to produce 7-methylpterin, and the amount of label incorporated into this pterin was measured by gas chromatography-mass spectrometry of the ditrimethylsilyl derivative of this compound. Approximately 27% of the 7-methylpterin and the guanine present in the cell was derived from the fed [15N]glycine. [U-methyl-2H]methionine was incorporated with the initial retention of all three deuteriums. These results are consistent with the biosynthesis of the pterin of methanopterin originating from GTP and its 7-methyl group arising from the methyl group of methionine.

In a recent publication, evidence was presented that cellular immune responses are associated with increased in vivo and in vitro excretion of neopterin. Our study aimed at investigating the biosynthesis of unconjugated pterins in highly purified human macrophages and T lymphocytes before and during stimulation with supernatants of activated T cells or with recombinant human interferon-gamma (IFN-gamma) by monitoring the following parameters: substrate concentration (GTP, guanosine triphosphate), activity of the enzyme initiating the biosynthesis of pterins (GTP-cyclohydrolase I) and product concentrations of total neopterin, biopterin, and pterin. In contrast to T cells and other tissues, macrophages were unable to produce tetrahydrobiopterin. This was indicated by our failure to detect biopterin and pterin. Instead, products of the first biosynthetic step accumulated, which were measured as total neopterin. We concluded that in macrophages the other enzymes required for biosynthesis of tetrahydrobiopterin are limiting. GTP concentration correlated with GTP cyclohydrolase I activity. An increase in both was induced by IFN-gamma and suppressed by neutralization of T-cell supernatants with monoclonal antibodies having specificity for IFN-gamma. Addition of tetrahydrobiopterin to the culture medium only led to a suppressed increase in GTP cyclohydrolase I activity and neopterin, but not in GTP concentration. Thus, it appears that IFN-gamma selectively stimulates the early steps of pterin biosynthesis in macrophages, thereby leading to accumulation and excretion of dihydroneopterin and neopterin. Although the physiological role of this phenomenon remains obscure, the fact that it seems to reflect endogenous release of IFN-gamma deserves particular attention.

The structure of dyspropterin, a new name given to an intermediate which is formed from dihydroneopterin triphosphate in the biosynthetic pathway of tetrahydrobiopterin, has been studied. Sepiapterin reductase (EC 1.1.1.153) was found to reduce dyspropterin to tetrahydrobiopterin in the presence of NADPH. Several lines of evidence showing the formation of tetrahydrobiopterin have been presented. Stoichiometric analysis revealed that there is a 1:2 relationship between the production of biopterin and the oxidation of NADPH during the reductase-catalyzed reduction of dyspropterin. The tetrahydrobiopterin production from dyspropterin was enhanced by dihydropteridine reductase (EC 1.6.99.7). Dyspropterin could also serve as a cofactor in phenylalanine hydroxylase (EC 1.14.16.1) system. These results are consistent with the view that dyspropterin is 6-(1,2-dioxopropyl)-5,6,7,8-tetrahydropterin. Based on our findings, the biosynthetic pathway of tetrahydrobiopterin from dihydroneopterin triphosphate has been discussed.

The enzyme guanosine triphosphate cyclohydrolase (GTP cyclohydrolase), which in bacteria is known to be the first enzyme in the biosynthetic pathway for the synthesis of pteridines, has been discovered in extracts of Drosophila melanogaster. Most of the enzyme (80%) is located in the head of the adult fly. An analysis of enzyme activity during development in Drosophila has revealed the presence of a relatively small peak of activity at pupariation and a much larger peak that appears at about the time of eclosion. Enzyme activity declines radidly as the fly ages. Analysis for the production of the typical pteridine pigments of Drosophila have indicated that the small peak of GTP cyclohydrolase activity evident at pupariation coincides with the appearance of isoxanthopterin, sepiapterin, and pterin, and the larger peak at eclosion roughly corresponds to the accumulation of drospterin as well as to the appearence in larger amounts of pterin and sepiaterin. These observations strongly suggest that in Drosophila, like bacteria, GTP cyclohydrolase is involved in the biosynthesis of pteridines. Analyses of a variety of zeste mutants of Drosophila melanogaster have shown that these mutants all contain GTP cyclohydrolase equal approximately to the amount found in the wild-type fly. These observations do not support the suggestions made by Rasmusson et al. (1973) that zeste is the strucural locus for GTP cyclohydrolase.

The properties, regularities of biosynthesis and biochemical functions are considered of GTP-cyclohydrolases of microorganisms. The existence of two groups of these enzymes is established. The first group enzymes convert GTP into 7,8-dihydroneopterin-triphosphate and formiate. They participate in biosynthesis of tetrahydrofolic acid, tetrahydrobiopterin, molybdenic cofactor, pyrrolopyrimidine antibiotics and in a series of pigments. Representatives of the second group of cyclohydrolases convert GTP into 2,5-diamino-4-oxy-6-ribosylaminopyrimidine-5'-phosphate, formiate and pyrophosphate. They catalyze the first stages of formation of 6,7-dimethyl-8-ribityllumazine, flavins and their derivatives, toxoflavin (azapteridine antibiotics). The regulation of biosynthesis and activity of GTP-cyclohydrolases is studied only for individual enzymes of this group.

Lectin stimulation of human peripheral blood mononuclear cells causes an increase in neopterin, biopterin, 6-hydroxymethylpterin and 6-formylpterin, as was determined by HPLC after iodine oxidation of the acid extract. After 72 h, pteridines peak at levels 5-10 fold as compared to resting cells. Levels decline to initial values during the following 24 h. Changes in pteridine proportions indicate that the synthesis of tetrahydrobiopterin proceeding from dihydroneopterin triphosphate is controlled during the process of lymphocyte activation. The release of both cellular neopterin and biopterin, but not of 6-hydroxymethylpterin and its aldehyde, is controlled by interferon-gamma.

Plasmodium falciparum was shown to synthesize pteroylpolyglutamate de novo from guanosine 5'-triphosphate (GTP), p-aminobenzoate (PABA), and L-glutamate (L-Glu). The parasite also had the capacity to synthesize pteroylpolyglutamate from both intact and degradation moieties (p-aminobenzoylglutamate and pterin-aldehyde) of exogenous folate added into the growth medium. The major product was identified as 5-methyl-tetrahydroteroylpentaglutamate following exposure to pteroylpolyglutamate hydrolase and oxidative degradation of the C9-N10 bond in the molecule and identification of products by reversed-phase high performance liquid chromatography. Inhibition of pteroylpentaglutamate synthesis from the radiolabelled metabolic precursors (GTP, PABA, L-Glu) and folate by the antifolate antimalarials, pyrimethamine and sulfadoxine at therapeutic concentrations, may suggest the existence of a unique biosynthetic pathway in the malaria parasite.