Biosynthesis of tetrahydrobiopterin: possible involvement of tetrahydropterin intermediates

Abstract

-The biosynthetic pathway of tetrahydrobiopterin (BH 4 ) from dihydroneopterin triphosphate (NH 2 P 3 ) was studied in fresh as well as heat-treated human liver extracts. The question of NAD(P)H dependency for the formation of sepiapterin was examined. NH 2 P 3 was converted by fresh extracts to sepiapterin in low quantities (2% conversion) in the absence of exogenously added NADPH as well as under conditions that ensured the destruction of endogenous, free NAD(P)H. The addition of NADPH to the fresh liver extracts stimulated the synthesis of BH 4 to a much higher yield (17% conversion), and the amount of sepiapterin formed was reduced to barely detectable levels. In contrast, the heat-treated extract (enzyme A2 fraction) formed sepiapterin (1.3% conversion) only in the presence and not in the absence of NADPH. These results indicate that sepiapterin may not be an intermediate on the pathway leading to BH 4 biosynthesis under normal~vivo conditions. Rather, sepiapterin may result from the breakdown of an as yet unidentified intermediate that is actually on the pathway. It is speculated that NH 2 P 3 may be converted to a diketo-tetrahydropterin intermediate (or an equivalent tautomeric structure) by a mechanism involving an intramolecular oxidoreduction reaction. A diketo-tetrahydropterin intermediate could be converted to 5,6-dihydrosepiapterin, which also has a tetrahydropterin ring system and can be converted directly to BH 4 by sepiapterin reductase. This proposed pathway can explain how the tetrahydropterin ring system can be formed without sepiapterin, dihydrobiopterin, or dihydrofolate reductase being involved in BH 4 biosynthesis~vivo.

Tetrahydrobiopterin biosynthesis. Studies with specifically labeled (2H)NAD(P)H and 2H2O and of the enzymes involved

“…NHzP3 was prepared as described by Heintel et al [19]. Bis(trimethylsily1)trifluoroacetamide (BSTFA) was from Regis Chemical Co.…”

“…Bovine liver dihydrofolate reductase was from Sigma Chemical Co. Sepiapterin reductase from rat erythrocytes was partially purified by the method of Sueoka and Katoh [22], except that only the first two column steps were performed, Partially purified human liver extracts were prepared by ammonium sulfate fractionation and Sephadex G-25 chromatography as described earlier [19]. A partially purified sepiapterin reductase from human liver ( = hydroxyapatite fraction) was prepared by an adaptation of the method of Katoh and Sueoka [23].…”

Purification and Characterization of 6-Pyruvoyl Tetrahydropterin Synthase from Human Pituitary Gland

“…1) [4][5][6], PTPS catalyses the 6-Pyruvoyl tetrahydropterin synthase (PTPS) is one of the key enzymes in the bio synthesis of tetrahydrobiopterin (BH4), the natural cofactor of phenylalanine-4-hydroxylase, tyrosine-3-hydroxylase, and tryptophan-5-hydroxylase [1], Furthermore, BH4 serves as a cofactor for glycerol-ether mono from rat liver and a full-length cDNA encod ing this enzyme was cloned [18].…”

1H‐NMR and mass spectrometric studies of tetrahydropterins

“…The sepiapterin tautomer (B) can also be excluded because of its discrepant chromophore [43]. The quinonoid structure C might be compatible with the absorption spectra in the ultraviolet; this is very unlikely, however, since its FAB/MS behaviour contrasts sharply with that of quinonoid BH,, which rapidly takes up two hydrogen atoms prior or during FAB analysis, whereas in the case of PPH, such hydrogen uptake was not observed (Fiy 7 ) In conclusion. the 'H-NMR and the FAB/MS data can be safely interpreted as supporting the proposed structure for PPH4.…”