Biosynthesis of tetrahydrobiopterin. Purification and characterization of 6-pyruvoyl-tetrahydropterin synthase from human liver

6-Pyruvoyl-tetrahydropterin synthase, which catalyzes the first step in the conversion of 7,8-dihydroneopterin triphosphate to tetrahydrobiopterin, was purified approximately 140,000-fold to apparent homogeneity from human liver. The molecular mass of the enzyme is estimated to be 83 kDa. 7,8-Dihydroneopterin triphosphate was a substrate of the enzyme in the presence of Mg2+, and the pH optimum of the reaction was 7.5 in Tris HCl buffer. The Km value for 7,8-dihydroneopterin triphosphate was 10 microM. The product of this enzymatic reaction was the presumed intermediate 6-pyruvoyl-tetrahydropterin. This latter compound was converted to tetrahydrobiopterin in the presence of NADPH and partially purified sepiapterin reductase from human liver. The conditions and the effect of N-acetylserotonin on this reaction, and on the formation of the intermediates 6-(1'-hydroxy-2'-oxopropyl)-tetrahydropterin and 6-(1' oxo-2'-hydroxypropyl)-tetrahydropterin have been studied.     

Biosynthesis of tetrahydrobiopterin: conversion of dihydroneopterin triphosphate to tetrahydropterin intermediates

It is known that the first step in the de novo synthesis of tetrahydrobiopterin from GTP is the conversion of GTP to dihydroneopterin triphosphate. Recent evidence supports the conclusion that beyond this first step, the pterin intermediates in the pathway are all at the tetrahydro level of reduction. We have now shown that partially purified fractions from rat liver, rat brain and bovine adrenal medulla catalyze the conversion of dihydroneopterin triphosphate to tetrahydrobiopterin, as well as to the putative intermediates in the pathway, 6-pyruvoyl-tetrahydropterin and 6-lactoyl-tetrahydropterin. Results of both enzymatic and chemical studies support the assigned structures for the latter two tetrahydropterins. We have also purified extensively from brain an enzyme, distinct from sepiapterin reductase, that catalyzes the TPNH-dependent reduction of 6-pyruvoyl-tetrahydropterin to 6-lactoyl-tetrahydropterin. The role of this reductase in tetrahydrobiopterin synthesis has not yet been established.     

Biosynthesis of tetrahydrobiopterin: possible involvement of tetrahydropterin intermediates

The biosynthetic pathway of tetrahydrobiopterin (BH₄) from dihydroneopterin triphosphate (NH₂P₃) 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. NH₂P₃ 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 BH₄ 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 BH₄ biosynthesis under normal $\text{in vivo}$ conditions. Rather, sepiapterin may result from the breakdown of an as yet unidentified intermediate that is actually on the pathway. It is speculated that NH₂P₃ 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 BH₄ by sepiapterin reductase. This proposed pathway can explain ho the tetrahydropterin ring system can be formed without sepiapterin, dihydrobiopterin, or dihydrofolate reductase being involved in BH₄ biosynthesis $\text{in vivo}$.     

Intermediates in the enzymic synthesis of tetrahydrobiopterin in Drosophila melanogaster

9 partially purified enzyme (Enzyme A) from Drosophila melanogaster Aatalyzes the conversion of 7,8- dihydroneopterin triphosphate to a compound that, from its ultraviolet absorption spectrum and other characteristics, appears to be 6- pyruvoyl -tetrahydropterin. This product can be converted to 6-lactoyl-tetrahydropterin in the presence of another partially purified enzyme (Enzyme B) and NADPH, and to 5,6,7,8-tetrahydrobiopterin in the presence of a third enzyme preparation (biopterin synthase) and NADPH. The enzymically-produced 6-lactoyl-tetrahydropterin, when exposed to air, is oxidized nonenzymically to sepiapterin (6-lactoyl-7,8- dihydropterin ). The results indicate that although 6-lactoyl-tetrahydropterin can be converted enzymically to tetrahydrobiopterin, neither it nor sepiapterin is an obligate intermediate in the conversion of 7,8- dihydroneopterin triphosphate to tetrahydrobiopterin.     

Synthesis, utilization, and structure of the tetrahydropterin intermediates in the bovine adrenal medullary de novo biosynthesis of tetrahydrobiopterin

The biosynthesis of two tetrahydropterin intermediates (H4pterin-1 and H4pterin-2), their conversion to tetrahydrobiopterin, and their overall chemical structures are described. A new high performance liquid chromatographic separation of these and other tetrahydropterins is also described. The biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate proceeds in the presence of the bovine adrenal medullary biosynthetic enzymes, Mg2+, NADPH. The biosynthesis of H4pterin-2 occurs under identical conditions, and the compound accumulates in the presence of 1 to 10 microM of N-acetylserotonin, an inhibitor of sepiapterin reductase. At higher concentrations of the inhibitor, the synthesis of H4pterin-2 is also inhibited, and H4pterin-1 accumulates. H4pterin-1 also accumulates in the absence of NADPH. In the presence of NADPH the biosynthetic enzymes convert both intermediates to tetrahydrobiopterin at rates which are greater than the rate of conversion of dihydroneopterin triphosphate to tetrahydrobiopterin. Electrochemical, UV/VIS, oxidation, and ionization properties identify the compounds as tetrahydropterins. The side chain structures of the compounds were determined by a combination of chemical means. The structures of the compounds are 6R-(1',2'-dioxopropyl)-tetrahydropterin (H4pterin-1) and 6R-(L-1'-hydroxy-2'-oxopropyl)-tetrahydropterin (H4pterin-2). The data indicate that the biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate proceeds in three steps: 1) formation of H4pterin-1 in the presence of Mg2+, 2) NADPH-dependent conversion of H4pterin-1 to H4pterin-2, and 3) NADPH-dependent conversion of H4pterin-2 to tetrahydrobiopterin.     

Purification of 6-pyruvoyl-tetrahydropterin synthase from human liver

The enzyme which catalyzes the first step in the conversion of dihydroneopterin triphosphate to tetrahydrobiopterin has been purified approx. 40,000-fold from human liver to apparent homogeneity. The enzyme has a native molecular weight of approximately 83,000 and consists of four identical subunits, each of which has a molecular weight of approximately 19,000. It contains carbohydrates and is remarkably stable to heat treatment. In the presence of purified sepiapterin reductase, Mg2+, and NADPH, this enzyme catalyzes efficiently the formation of tetrahydrobiopterin from dihydroneopterin triphosphate. This indicates that these two proteins are sufficient for the overall conversion.     

The enzyme that synthesizes tetrahydrobiopterin from 6-pyruvoyl-tetrahydropterin in the lemon mutant silkworm consists of two carbonyl reductases

Tetrahydrobiopterin plays an important role in the biosynthesis of certain neurotransmitters. Using DEAE-Sepharose FF column chromatography, we separated the enzyme that synthesizes tetrahydrobiopterin from 6-pyruvoyl-tetrahydropterin [which is different from sepiapterin reductase (EC 1.1.1.153)] in the lemon mutant of the silkworm Bombyx mori into two fractions, which were named carbonyl reductase I (CR I) and carbonyl reductase II (CR II). The CR I enzyme converted 6-pyruvoyl-tetrahydropterin to 6-lactoyl-tetrahydropterin, while CR II converted 6-pyruvoyl-tetrahydropterin to 1'-hydroxy-2'-oxopropyl-tetrahydropterin, both reactions occurring only in the presence of NADPH. Neither of the two carbonyl reductases alone was able to catalyze the conversion of 6-pyruvoyl-tetrahydropterin to tetrahydrobiopterin in the presence of NADPH. However, when CR I was mixed with CR II in the reaction mixture, 6-pyruvoyl-tetrahydropterin was reduced to tetrahydrobiopterin in the presence of NADPH. Moreover, CR I catalyzed the formation of tetrahydrobiopterin from 1'-hydroxy-2'-oxopropyl-tetrahydropterin, while CR II converted 6-lactoyl-tetrahydropterin to tetrahydrobiopterin, both reactions occurring only in the presence of NADPH. Our results suggest that there are two potential routes for formation of tetrahydrobiopterin from 6-pyruvoyl-tetrahydropterin in the lemon mutant silkworm. In the first route, 1'-hydroxy-2'-oxopropyl-tetrahydropterin is formed from 6-pyruvoyl-tetrahydropterin by CR II and then reduced to tetrahydrobiopterin by CR I, both reactions occurring only in the presence of NADPH. In the other route, 6-pyruvoyl-tetrahydropterin is reduced to 6-lactoyl-tetrahydropterin by CR I and then converted to tetrahydrobiopterin by CR II, both reactions occurring only in the presence of NADPH.     

Vanadate-dependent NAD(P)H oxidation by microsomal enzymes

Vanadate-dependent NAD(P)H oxidation, catalyzed by rat liver microsomes and microsomal NADPH-cytochrome P450 reductase (P450 reductase) and NADH-cytochrome b5 reductase (b5 reductase), was investigated. These enzymes and intact microsomes catalyzed NAD(P)H oxidation in the presence of either ortho- or polyvanadate. Antibody to P450 reductase inhibited orthovanadate-dependent NADPH oxidation catalyzed by either purified P450 reductase or rat liver microsomes and had no effect on the rates of NADH oxidation catalyzed by b5 reductase. NADPH-cytochrome P450 reductase catalyzed orthovanadate-dependent NADPH oxidation five times faster than NADH-cytochrome b5 reductase catalyzed NADH oxidation. Orthovanadate-dependent oxidation of either NADPH or NADH, catalyzed by purified reductases or rat liver microsomes, occurred in an anaerobic system, which indicated that superoxide is not an obligate intermediate in this process. Superoxide dismutase (SOD) inhibited orthovanadate, but not polyvanadate-mediated, enzyme-dependent NAD(P)H oxidation. SOD also inhibited when pyridine nucleotide oxidation was conducted anaerobically, suggesting that SOD inhibits vanadate-dependent NAD(P)H oxidation by a mechanism independent of scavenging of O2-.     

Enzymatic synthesis of biopterin from D-erythrodihydroneopterin triphosphate by extracts of kidneys from Syrian golden hamsters.

An enzyme system was found in either crude homogenates of dialyzed extracts of liver, kidney, lung, and brain from Syrian golden hamsters that catalyzed the synthesis of radioactive 6(L-erythro-1',2'-dihydroxypropyl)pterin (biopterin) from [U-14C]6(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropterin triphosphate (D-erythrolH2neopterin-PPP) preparation. The specific radioactivity of biopterin was found to be comparable to that of D-erythroH2neopterin-PPP. The enzyme system from hamster kidney was purified severalfold by fractionation with ammonium sulfate and with an Ultrogel AcA-34 column. It was demonstrated that (a) NADPH or NADAH was essential and that (b) Mg2+ was stimulatory for the enzymatic synthesis of biopterin from D-erythroH2-NEOPTERIN-PPP. Also GTP and nonphosphorylated neopterins were not converted to biopterin. Although 6-lactyl-7,8-dihydropterin (sepiapterin) was converted to biopterin in the presence of NADPH, sepiapterin was not detected from D-erythroH2neopterin-PPP in the absence of NADPH. A preliminary experiment was performed to identify dihydrobiopterin.     

Comparative activity of rat liver dihydrofolate reductase with 7,8-dihydrofolate and other 7,8-dihydropteridines

The various interactions of rat liver dihydrofolate reductase with two unconjugated 7,8-dihydropteridines, 7,8-dihydrobiopterin and 6-methyl-7,8-dihydropteridine, have been compared with those of 7,8-dihydrofolate and folate. Of particular interest was the reactivity demonstrated by 7,8-dihydrobiopterin because of the potential physiological significance of this reaction both in the regeneration of tetrahydrobiopterin, a cofactor for various biological hydroxylations, and as a step in the biosynthesis of this compound from GTP. Kinetic experiments gave Km values of 0.17, 6.42, and 10.2 microM for 7,8-dihydrofolate, 7,8-dihydrobiopterin, and 6-methyl-7,8-dihydropteridine, respectively, with Vmax = 6.22, 2.39, and 1.54 mumol min-1 mg-1. With folate the enzyme showed high affinity (Km = 0.88 microM) but low Vmax (0.20 mumol min-1 mg-1). The natural cofactor was NADPH and a Km of approximately 0.7 microM was measured with each substrate. The enzyme was activated by both p-hydroxymercuribenzoate and urea when assayed with 7,8-dihydrofolate but was inhibited when 7,8-dihydrobiopterin was the substrate. The pH optimum for dihydrofolate reduction was 4 with enhancement at pH greater than or equal to 5.5 in the presence of 1 M NaCl. Peak activity with 7,8-dihydrobiopterin occurred at pH 4.8; this was shifted to pH 5.3 but was not enhanced by 1 M NaCl. Inhibition with methotrexate was similar whether the enzyme was assayed with either the conjugated or unconjugated 7,8-dihydro derivatives. The rat liver enzyme, highly unstable after purification, was stabilized in the presence of the nonionic detergent, Tween-20 (0.1%); however, the comparative properties toward the conjugated and unconjugated substrates were not altered by this treatment.     

A sensitive high-performance liquid chromatographic-fluorometric assay for dihydrofolate reductase in adult rat brain, using 7,8-dihydrobiopterin as substrate

A liquid chromatographic-fluorometric assay has been developed to study the role of dihydrofolate reductase in adult rat brain since low levels of the enzyme preclude measurement by current spectrophotometric procedures. This method involves in vitro incubation of desalted, cell-free brain extracts with 7,8-dihydrobiopterin, NADPH, and an NADPH-regenerating system. The tetrahydrobiopterin formed is quantitatively converted to pterin using alkaline iodine oxidation, and the pterin formed is separated by liquid chromatography and detected fluorometrically. The method is linear from 100 fmol to greater than or equal to 1 nmol of product, and the sensitivity is at least 100 times greater than that of existing spectrophotometric assays. Enzyme activity of desalted brain extracts is linear with both time (to 100 min) and protein (from 50 to 620 micrograms). The enzyme shows an absolute requirement for NADPH, does not use NADH, and is completely inhibited by 10 nM methotrexate. The Km of the enzyme for NADPH was found to be 7.5 microM, while the Km for 7,8-dihydrobiopterin was 88 microM. Since brain dihydrobiopterin reductase has the same properties as dihydrofolate reductase, this fluorometric procedure can serve as a sensitive assay for dihydrofolate reductase.     

The biosynthesis of tetrahydrobiopterin in rat brain. Purification and characterization of 6-pyruvoyl tetrahydropterin (2'-oxo)reductase

An enzyme with 6-pyruvoyl tetrahydropterin (6PPH4) (2'-oxo)reductase activity was purified to near homogeneity from whole rat brains by a rapid method involving affinity chromatography on Cibacron blue F3Ga-agarose followed by high performance ion exchange chromatography and high performance gel filtration. The enzyme has a single subunit of Mr 37,000 and has a similar amino acid composition to previously described aldoketo reductases. The reductase activity is absolutely dependent on NADPH, will only catalyze the reduction of the C-2'-oxo group of 6PPH4, and is inactive towards the C-1'-oxo group. However, the enzyme also shows high activity towards nonspecific substrates, such as 4-nitrobenzaldehyde, phenanthrenequinone, and menadione. The role of this 6PPH4 reductase in the formation of tetrahydrobiopterin (BH4) was investigated. Measurements were made of the rate of conversion of 6PPH4, generated from dihydroneopterin triphosphate with purified 6PPH4 synthase, to BH4 in the presence of mixtures of pure sepiapterin reductase and the 6PPH4 (2'-oxo)reductase purified from rat brains. The results suggest that when sepiapterin reductase activity is limiting, a large proportion of BH4 synthesis proceeds through the 6-lactoyl intermediate. However, when sepiapterin reductase is not limiting, most of the BH4 is probably formed via reduction of the other mono-reduced intermediate which is produced from 6PPH4 by sepiapterin reductase alone.     

Dyspropterin, an intermediate formed from dihydroneopterin triphosphate in the biosynthetic pathway of tetrahydrobiopterin

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.     

On the role of sepiapterin reductase in the biosynthesis of tetrahydrobiopterin

Rat erythrocyte sepiapterin reductase can catalyze the NADPH-dependent reduction of tetrahydropterin substrates with relative velocities of sepiapterin greater than lactoyltetrahydropterin greater than or equal to pyruvoyltetrahydropterin greater than 1'-hydroxy-2'-oxopropyltetrahydropterin; L-erythrotetrahydrobiopterin is the product of the reduction of all three tetrahydropterins. The 1' position of the 1',2'-diketone, pyruvoyltetrahydropterin, is reduced first; the product of this first reduction is 1'-hydroxy-2'-oxopropyltetrahydropterin. Both steps are inhibited by N-acetylserotonin. An antibody to sepiapterin reductase purified from rat erythrocytes was produced in rabbits, and the purified antibody is highly specific for sepiapterin reductase. This antibody is an inhibitor of both sepiapterin reductase activity and tetrahydrobiopterin biosynthesis in crude extracts of rat adrenal and brain. The antibody inhibits the production of both the biosynthetic intermediate, 1'-hydroxy-2'-oxopropyltetrahydropterin, and tetrahydrobiopterin. The results indicate that sepiapterin reductase is on the biosynthetic pathway to tetrahydrobiopterin, and catalyzes the complete reduction of pyruvoyltetrahydropterin to tetrahydrobiopterin. In contrast, homogenates of whole rat adrenal also produce large quantities of lactoyltetrahydropterin which suggests that in some tissues this compound may also be an intermediate in tetrahydrobiopterin biosynthesis. The synthesis of lactoyltetrahydropterin is not inhibited by the antibody to sepiapterin reductase and therefore does not appear to be catalyzed by sepiapterin reductase. However, sepiapterin reductase is responsible for the conversion of lactoyltetrahydropterin to tetrahydrobiopterin. The source of sepiapterin in biosynthetic reactions was found to be oxidative decomposition of lactoyltetrahydropterin.     

A sensitive high-performance liquid chromatographic-fluoromotric assay for dihydrofolate reductase in adult rat brain, using 7,8-dihydrobiopterin as substrate

A liquid chromatographic-fluorometric assay has been developed to study the role of dihydrofolate reductase in adult rat brain since low levels of the enzyme preclude measurement by current spectrophotometric procedures. This method involves in vitro incubation of desalted, cell-free brain extracts with 7,8-dihydrobiopterin, NADPH, and an NADPH-regenerating system. The tetrahydrobiopterin formed is quantitatively converted to pterin using alkaline iodine oxidation, and the pterin formed is separated by liquid chromatography and detected fluorometrically. The method is linear from 100 fmol to ≥ 1 nmol of product, and the sensitivity is at least 100 times greater than that of existing spectrophotometric assays. Enzyme activity of desalted brain extracts is linear with both time (to 100 min) and protein (from 50 to 620 μg). The enzyme shows an absolute requirement for NADPH, does not use NADH, and is completely inhibited by 10 n methotrexate. The K_m of the enzyme for NADPH was found to be 7.5 μ , while the K_m for 7,8-dihydrobiopterin was 88 μ . Since brain dihydrobiopterin reductase has the same properties as dihydrofolate reductase, this fluorometric procedure can serve as a sensitive assay for dihydrofolate reductase.     

Control of pteridine biosynthesis in the natural killer-like cell line YT

The natural killer-like cell line YT constitutively expresses GTP-cyclohydrolase activity whereas 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase are absent. The product, dihydroneopterin triphosphate, is dephosphorylated and oxidized causing neopterin to accumulate in the cells. The activities of the H4biopterin synthesizing enzymes are not controlled by IFN-gamma or the synergistic action of both IFN-gamma and IL-2 as has been shown for monocytes/macrophages (Huber C. et al. (1984) J. Exp. Med. 160, 310) and CD4+ T cells, respectively (Ziegler I. et al. (1990) J. Biol. Chem. 265, 17026). Sepiapterin reductase specifically is induced by incubation of the cells with sepiapterin, leaving GTP-cyclohydrolase, 6-pyruvoyltetrahydropterin synthase and other enzymes related to pteridine metabolism (dihydropteridine reductase, dihydrofolate reductase) unaffected. The data indicate that H4biopterin synthesis is individually regulated in the diverse cellular components of the immune system.     

Computer studies on the stereostructure and quantum chemical properties of 6-pyruvoyl tetrahydropterin, the key intermediate of tetrahydrobiopterin biosynthesis

The optimized geometry of the conformation of atoms constituting the 6-pyruvoyl tetrahydropterin molecule, the labile key intermediate of tetrahydrobiopterin biosynthesis, was obtained by molecular orbital calculations within the MINDO/3 framework. The stereostructure of the molecule showing the preferred mode for binding to sepiapterin reductase or pyruvoyl tetrahydropterin reductase was drawn in perspective. The resulting structure with the equatorial staggered configuration of the 6-1',2'-dioxopropyl (pyruvoyl) side chain indicated that O(1') and H(6) were located in the trans position around the C(6)-C(1') bond and that the two vicinal carbonyls in the side chain were fixed in the incomplete trans form. The calculation of atomic charges and LUMO coefficients of these carbonyls suggests that the C2'-carbonyl may be more reactive toward NADPH than the C1'-carbonyl in the enzymatic reaction.     

Physarum polycephalum expresses a dihydropteridine reductase with selectivity for pterin substrates with a 6-(1', 2'-dihydroxypropyl) substitution

Physarum polycephalum is one of few non-animal organisms capable of synthesizing tetrahydrobiopterin from GTP. Here we demonstrate developmentally regulated expression of quinoid dihydropteridine reductase (EC 1.6.99.7), an enzyme required for recycling 6,7-[8H]-dihydrobiopterin. Physarum also expresses phenylalanine-4-hydroxylase activity, an enzyme that depends on dihydropteridine reductase. The 24.4 kDa Physarum dihydropteridine reductase shares 43% amino acid identity with the human protein. A number of residues important for function of the mammalian enzyme are also conserved in the Physarum sequence. In comparison to sheep liver dihydropteridine reductase, purified recombinant Physarum dihydropteridine reductase prefers pterin substrates with a 6-(1', 2'-dihydroxypropyl) group. Our results demonstrate that Physarum synthesizes, utilizes and metabolizes tetrahydrobiopterin in a way hitherto thought to be restricted to the animal kingdom.     

Lithosperm inhibition of anterior pituitary hormones.

The enzyme system for the synthesis of the pteridine pigment, sepiapterin, from $\text{2-amino-4-hydroxy-6-(D-}\text{erythro}\text{-1',2',3'-trihydroxyprophyl)}$ triphosphate (dihydroneopterin triphosphate) has been found in extracts of $\text{Drosophila melanogaster}$. NADP⁺ or NADPH and Mg²⁺ are required for this enzymatic transformation. No sepiapterin is produced when dihydroneopterin is supplied as substrate in place of dihydroneopterin triphosphate.     

The C-6 proton of tetrahydrobiopterin is acquired from water, not NADPH, during de novo biosynthesis

Tetrahydrobiopterin, the cofactor for the aromatic amino acid hydroxylases, is synthesized in mammals from GTP via a pathway involving both dihydropterin and tetrahydropterin intermediates. In this work, we have investigated the mechanism of conversion of the product formed from GTP, 7,8-dihydroneopterin triphosphate, into the tetrahydropterin intermediates. Tetrahydrobiopterin can be oxidized under conditions which yield pterin or pterin 6-carboxylate without exchange of the C-6 and C-7 protons. Using these techniques, a gas chromatography/mass spectrometry method was developed to determine that in the biosynthesis of tetrahydrobiopterin de novo, in preparations of bovine adrenal medulla, the C-6 proton of tetrahydrobiopterin is derived from water and not from NADPH. In contrast, the C-6 proton of tetrahydrobiopterin produced from sepiapterin (6-lactoyl-7,8-dihydropterin) comes from NADPH. The results are consistent with evidence for the formation of the first tetrahydropterin intermediate by a tautomerization without any requirement for NADPH.