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

The biosynthesis of tetrahydrobiopterin from either dihydroneopterin triphosphate, sepiapterin, dihydrosepiapterin or dihydrobiopterin was investigated using extracts from human liver, dihydrofolate reductase and purified sepiapterin reductase from human liver and rat erythrocytes. The incorporation of hydrogen in tetrahydrobiopterin was studied in either 2H2O or in H2O using unlabeled NAD(P)H or (R)-(4-2H)NAD(P)H or (S)-(4-2H)NAD(P)H. Dihydrofolate reductase catalyzed the transfer of the pro-R hydrogen of NAD(P)H during the reduction of 7,8-dihydrobiopterin to tetrahydrobiopterin. Sepiapterin reductase catalyzed the transfer of the pro-S hydrogen of NADPH during the reduction of sepiapterin to 7,8-dihydrobiopterin. In the presence of partially purified human liver extracts one hydrogen from the solvent is introduced at position C(6) and the 4-pro-S hydrogen from NADPH is incorporated at each of the C(1') and C(2') position of BH4. Label from the solvent is also introduced into position C(3'). These results suggest that dihydrofolate reductase is not involved in the biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate. They are consistent with the assumption of the occurrence of a 6-pyruvoyl-tetrahydropterin intermediate, which is proposed to be formed upon triphosphate elimination from dihyroneopterin triphosphate, and via an intramolecular redox reaction. Our results suggest that the reduction of 6-pyruvoyl-tetrahydropterin might be catalyzed by sepiapterin reductase.     

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.     

Carbonyl reductase activity of sepiapterin reductase from rat erythrocytes

A homogeneous preparation of sepiapterin reductase, an enzyme involved in the biosynthesis of tetrahydrobiopterin, from rat erythrocytes was found to be responsible for the reduction with NADPH of various carbonyl compounds of non-pteridine derivatives including some vicinal dicarbonyl compounds which were reported in the previous paper (Katoh, S. and Sueoka, T. (1984) Biochem, Biophys. Res. Commun. 118, 859-866) in addition to the general substrate, sepiapterin (2-amino-4-hydroxy-6-lactoyl-7,8-dihydropteridine). The compounds sensitive as substrates of the enzyme were quinones, e.g., p-quinone and menadione; other vicinal dicarbonyls, e.g., methylglyoxal and phenylglyoxal; monoaldehydes, e.g., p-nitrobenzaldehyde; and monoketones, e.g., acetophenone, acetoin, propiophenone and benzylacetone. Rutin, dicoumarol, indomethacin, and ethacrynic acid inhibited the enzyme activity toward either a carbonyl compound of a non-pteridine derivative or sepiapterin as substrate. Sepiapterin reductase is quite similar to general aldo-keto reductases, especially to carbonyl reductase.     

Carbonyl reductase activity of sepiapterin reductase from rat erythrocytes

A homogeneous preparation of sepiapterin reductase, an enzyme involved in the biosynthesis of tetrahydrobiopterin, from rat erythrocytes was found to be responsible for the reduction with NADPH of various carbonyl compounds of non-pteridine derivatives including some vicinal dicarbonyl compounds which were reported in the previous paper (Katoh, S. and Sueoka, T. (1984) Biochem. Biophys. Res. Commun. 118, 859–866) in addition to the general substrate, sepiapterin (2-amino-4-hydroxy-6-lactoyl-7,8-dihydropteridine). The compounds sensitive as substrates of the enzyme were quinones, e.g., p-quinone and menadione; other vicinal dicarbonyls, e.g., methylglyoxal and phenylglyoxal; monoaldehydes, e.g., p-nitrobenzaldehyde; and monoketones, e.g., acetophenone, acetoin, propiophenone and benzylacetone. Rutin, dicoumarol, indomethacin, and ethacrynic acid inhibited the enzyme activity toward either a carbonyl compound of a non-pteridine derivative or sepiapterin as substrate. Sepiapterin reductase is quite similar to general aldo-keto reductases, especially to carbonyl reductase.     

Immunological studies on the participation of 6-pyruvoyl tetrahydropterin (2'-oxo) reductase, an aldose reductase, in tetrahydrobiopterin biosynthesis

The NADPH-dependent reduction of the two carbonyl groups in the side chain of the first tetrahydropterin intermediate on the tetrahydrobiopterin biosynthetic pathway, 6-pyruvoyl tetrahydropterin, proceeds in a sequential manner whose order has not yet been resolved. Sepiapterin reductase can catalyze the reduction of both carbonyl groups starting with the 1'-oxo. 6-Pyruvoyl tetrahydropterin (2'-oxo) reductase, which has now been shown to be a member of the aldose reductase family, catalyzes the formation of only the 2'-hydroxy-1'-oxo intermediate which still requires sepiapterin reductase for final conversion to tetrahydrobiopterin. Inhibiting antibodies to the 2'-oxo reductase have been prepared and utilized to explore the distribution of this reductase in rat brain. The antiserum also maximally inhibited in vitro tetrahydrobiopterin synthesis in crude rat brain extracts by 60%, indicating that the majority of tetrahydrobiopterin biosynthesis in vivo may proceed via the 2'-hydroxy-1'-oxo intermediate. However, analogous experiments with rat liver extracts demonstrate that inhibition of the 2'-oxo reductase activity does not inhibit the conversion of 6-pyruvoyl tetrahydropterin to tetrahydrobiopterin, suggesting that tetrahydrobiopterin biosynthesis may proceed via different pathways in rat brain and liver.     

Human carbonyl and aldose reductases: new catalytic functions in tetrahydrobiopterin biosynthesis

New catalytic functions of human carbonyl- and aldose reductase in tetrahydrobiopterin biosynthesis are proposed. 6-Pyruvoyl tetrahydropterin, an intermediate in the biosynthesis of tetrahydrobiopterin, was converted to 6-lactoyl tetrahydropterin and 1'-hydroxy-2'-oxopropyl tetrahydropterin by carbonyl reductase under anaerobic condition. 1'-Hydroxy-2'-oxopropyl tetrahydropterin was subsequently metabolized to tetrahydrobiopterin by aldose reductase. Based on these results alternative pathways for the synthesis of tetrahydrobiopterin in patients with genetic defects of sepiapterin reductase are suggested.     

Development of the pteridine pathway in the zebrafish, Danio rerio

In the zebrafish, the peripheral neurons and the pigment cells are derived from the neural crest and share the pteridine pathway, which leads either to the cofactor tetrahydrobiopterin or to xanthophore pigments. The components of the pteridine pattern were identified as tetrahydrobiopterin, sepiapterin, 7-oxobiopterin, isoxanthopterin, and 2,4,7-trioxopteridine. The expression of GTP cyclohydrolase I activity during the first 24-h postfertilization, followed by 6-pyruvoyl-5,6,7,8-tetrahydropterin synthase and sepiapterin reductase, suggest an early supply of tetrahydrobiopterin for neurotransmitter synthesis in the neurons and for tyrosine supply in the melanophores. At 48-h postfertilization, sepiapterin formation branches off the de novo pathway of tetrahydrobiopterin synthesis. Sepiapterin, via 7,8-dihydrobiopterin and biopterin, serves as a precursor for the formation of 7-oxobiopterin, which may be further catabolized to isoxanthopterin and 2,4,7-trioxopteridine. Neither 7, 8-dihydrobiopterin nor biopterin is a substrate for xanthine oxidoreductase. In contrast, both of these compounds are oxidized at C-7 by a xanthine oxidase variant form, which is inactivated by KCN, but is insensitive to allopurinol. The oxidase and the dehydrogenase form of xanthine oxidoreductase as well as the xanthine oxidase variant have specific developmental patterns. It follows that GTP cyclohydrolase I, the formation of sepiapterin, and the xanthine oxidoreductase family control the pteridine pathway in the zebrafish.     

Sepiapterin reductase: Characteristics and role in diseases

Sepiapterin reductase, a homodimer composed of two subunits, plays an important role in the biosynthesis of tetrahydrobiopterin. Furthermore, sepiapterin reductase exhibits a wide distribution in different tissues and is associated with many diseases, including brain dysfunction, chronic pain, cardiovascular disease and cancer. With regard to drugs targeting sepiapterin reductase, many compounds have been identified and provide potential methods to treat various diseases. However, the underlying mechanism of sepiapterin reductase in many biological processes is unclear. Therefore, this article summarized the structure, distribution and function of sepiapterin reductase, as well as the relationship between sepiapterin reductase and different diseases, with the aim of finding evidence to guide further studies on the molecular mechanisms and the potential clinical value of sepiapterin reductase. In particular, the different effects induced by the depletion of sepiapterin reductase or the inhibition of the enzyme suggest that the non‐enzymatic activity of sepiapterin reductase could function in certain biological processes, which also provides a possible direction for sepiapterin reductase research.     

Sepiapterin Reductase Mediates Chemical Redox Cycling in Lung Epithelial Cells

In the lung, chemical redox cycling generates highly toxic reactive oxygen species that can cause alveolar inflammation and damage to the epithelium, as well as fibrosis. In this study, we identified a cytosolic NADPH-dependent redox cycling activity in mouse lung epithelial cells as sepiapterin reductase (SPR), an enzyme important for the biosynthesis of tetrahydrobiopterin. Human SPR was cloned and characterized. In addition to reducing sepiapterin, SPR mediated chemical redox cycling of bipyridinium herbicides and various quinones; this activity was greatest for 1,2-naphthoquinone followed by 9,10-phenanthrenequinone, 1,4-naphthoquinone, menadione, and 2,3-dimethyl-1,4-naphthoquinone. Whereas redox cycling chemicals inhibited sepiapterin reduction, sepiapterin had no effect on redox cycling. Additionally, inhibitors such as dicoumarol, N-acetylserotonin, and indomethacin blocked sepiapterin reduction, with no effect on redox cycling. Non-redox cycling quinones, including benzoquinone and phenylquinone, were competitive inhibitors of sepiapterin reduction but noncompetitive redox cycling inhibitors. Site-directed mutagenesis of the SPR C-terminal substrate-binding site (D257H) completely inhibited sepiapterin reduction but had minimal effects on redox cycling. These data indicate that SPR-mediated reduction of sepiapterin and redox cycling occur by distinct mechanisms. The identification of SPR as a key enzyme mediating chemical redox cycling suggests that it may be important in generating cytotoxic reactive oxygen species in the lung. This activity, together with inhibition of sepiapterin reduction by redox-active chemicals and consequent deficiencies in tetrahydrobiopterin, may contribute to tissue injury.     

Immunological Evidence for the Requirement of Sepiapterin Reductase for Tetrahydrobiopterin Biosynthesis in Brain

Specific antibodies to sepiapterin reductase were used to investigate its involvement in de novo (6R)-5,6,7,8-tetrahydrobiopterin (BH4) biosynthesis in rat brain. Antisepiapterin reductase (anti-SR) serum totally inhibited NADPH-dependent sepiapterin reductase activity in supernatants from discrete rat brain areas and liver. The anti-SR serum also inhibited the conversion of 7,8-dihydroneopterin triphosphate to BH4 in rat brain extracts. The inhibition was accompanied by a concentration-dependent increase in the formation of 6-lactoyltetrahydropterin (6LPH4), a proposed intermediate in BH4 biosynthesis. In addition, anti-SR serum was used to characterize the distribution and molecular properties of sepiapterin reductase in rat tissues. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting indicated that there was a single polypeptide with the same molecular weight (28,000) as that of the subunit of pure sepiapterin reductase present in all tissues examined except for liver, where an immunoreactive protein of higher molecular weight (30,500) also was detected. Two-dimensional gel electrophoresis of rat striatum and liver demonstrated that the isoelectric point of sepiapterin reductase from both tissues was 6.16 and that the higher molecular weight immunoreactive material in liver had an isoelectric point of 7.06. Our studies with specific anti-SR serum confirmed the results of previous studies using chemical inhibitors of sepiapterin reductase, which suggested that sepiapterin reductase activity was essential for BH4 biosynthesis in the CNS and that 6LPH4 could be a precursor of BH4.     

Cloning and sequencing of cDNA encoding human sepiapterin reductase--an enzyme involved in tetrahydrobiopterin biosynthesis

A full-length cDNA clone for sepiapterin reductase, an enzyme involved in tetrahydrobiopterin biosynthesis, was isolated from a human liver cDNA library by plaque hybridization. The nucleotide sequence of hSPR 8-25, which contained an entire coding region of the enzyme, was determined. The clone encoded a protein of 261 amino acids with a calculated molecular mass of 28,047 daltons. The predicted amino acid sequence of human sepiapterin reductase showed a 74% identity with the rat enzyme. We further found a striking homology between human SPR and carbonyl reductase, estradiol 17 beta-dehydrogenase, and 3 beta-hydroxy-5-ene steroid dehydrogenase, especially in their N-terminal region.     

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.     

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.     

Tetrahydrobiopterin biosynthetic activities in human macrophages, fibroblasts, THP-1, and T 24 cells. GTP-cyclohydrolase I is stimulated by interferon-gamma, and 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase are constitutively present

Interferon-gamma induces tetrahydrobiopterin biosynthesis in human cells and cell lines. Macrophages are peculiar in the formation of large amounts of neopterin derivatives as compared to tetrahydrobiopterin (Werner, E. R., Werner-Felmayer, G., Fuchs, D., Hausen, A., Reibnegger, G., and Wachter, H. (1989) Biochem J. 262, 861-866). Here we compare the impact of interferon-gamma treatment on activities of GTP-cyclohydrolase I (EC 3.5.4.16), 6-pyruvoyl tetrahydropterin synthase, and sepiapterin reductase (EC 1.1.1.153) in human peripheral blood-derived macrophages, normal dermal fibroblasts, THP-1 myelomonocytic cells, and the T 24 bladder transitional-cell carcinoma line. Upon interferon-gamma treatment, GTP-cyclohydrolase I activity is increased 7- to 40-fold, whereas 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase activities, which are constitutively present in all four investigated cells, remain unchanged. In fibroblasts and T 24 cells GTP cyclohydrolase I activity is the rate-limiting step of tetrahydrobiopterin biosynthesis. In macrophages and in THP-1 cells, however, the induced GTP cyclohydrolase I activity is higher than the 6-pyruvoyl tetrahydropterin synthase activity, leading to the accumulation of neopterin and neopterin phosphates.     

6-Acetyl-7,7-dimethyl-5,6,7,8-tetrahydropterin is an activator of nitric oxide synthases

6-Acetyl-7,7-dimethyl-7,8-dihydropterin 3 has been shown to be able to substitute for the natural cofactor of nitric oxide synthases, tetrahydrobiopterin 1, in cells and tissues that contain active nitric oxide synthases (NOSs). In both macrophages, which produce iNOS, and endothelial cells, which produce eNOS, in which tetrahydrobiopterin biosynthesis has been blocked by inhibition of GTP cyclohydrolase 1, dihydropterin 3 restored production of nitric oxide by these cells. In tissues, 3 caused relaxation in preconstricted rat aortic rings, again in which tetrahydrobiopterin biosynthesis had been inhibited, an effect that was blocked by the NOS inhibitor, L-NAME. However, dihydropterin 3 was not itself an active cofactor in purified NOS (nNOS) preparations free of tetrahydrobiopterin suggesting that intracellular reduction to 6-acetyl-7,7-dimethyl-5,6,7,8-tetrahydropterin 4 is required for activity. Compound 4 was prepared by reduction of the corresponding 7,8-dihydropterin with sodium cyanoborohydride and has been shown to be a competent cofactor for nitric oxide production by nNOS. Together, the results show that the 7,7-dimethyl-7,8-dihydropterin is a novel structural framework for effective tetrahydrobiopterin analogues.     

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.     

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.     

Regulation of Guanosine Triphosphate Cyclohydrolase and Tetrahydrobiopterin Levels and the Role of the Cofactor in Tyrosine Hydroxylation in Primary Cultures of Adrenomedullary Chromaffin Cells

Selective modification of the tetrahydrobiopterin levels in cultured chromaffin cells were followed by changes in the rate of tyrosine hydroxylation. Addition of sepiapterin, an intermediate on the salvage pathway for tetrahydrobiopterin synthesis, rapidly increased intracellular levels of tetrahydrobiopterin and elevated the rate of tyrosine hydroxylation in the intact cell. Tyrosine hydroxylation was also enhanced when tetrahydrobiopterin was directly added to the incubation medium of intact cells. When the cultured chromaffin cells were treated for 72 h with N-acetylserotonin, an inhibitor of sepiapterin reductase, tetrahydrobiopterin content and the rate of tyrosine hydroxylation were decreased. Addition of sepiapterin or N-acetylserotonin had no consistent effect on total extractable tyrosine hydroxylase activity or on catecholamine content in the cultured chromaffin cells. Three-day treatment of chromaffin cell cultures with compounds that increase levels of cyclic AMP (forskolin, cholera toxin, theophylline, dibutyryl- and 8-bromo cyclic AMP) increased total extractable tyrosine hydroxylase activity and GTP-cyclohydrolase, the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. Tetrahydrobiopterin levels and intact cell tyrosine hydroxylation were markedly increased after 8-bromo cyclic AMP. The increase in GTP-cyclohydrolase and tetrahydrobiopterin induced by 8-bromo cyclic AMP was blocked by the protein synthesis inhibitor cycloheximide. Agents that deplete cellular catecholamines (reserpine, tetrabenazine, and brocresine) increased both total tyrosine hydroxylase and GTP-cyclohydrolase activities, although treating the cultures with reserpine or tetrabenazine resulted in no change in cellular levels of cyclic AMP. Brocresine and tetrabenazine increased tetrahydrobiopterin levels, but the addition of reserpine to the cultures decreased catecholamine and tetrahydrobiopterin content and resulted in a decreased rate of intact cell tyrosine hydroxylation in spite of the increased activity of the total extractable enzyme. These data indicate that in cultured chromaffin cells GTP-cyclohydrolase activity like tyrosine hydroxylase activity is regulated by both cyclic AMP-dependent and cyclic AMP-independent mechanisms and that the intracellular level of tetrahydrobiopterin is one of the many factors that control the rate of tyrosine hydroxylation.     

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.     

The 1.25 A crystal structure of sepiapterin reductase reveals its binding mode to pterins and brain neurotransmitters.

Sepiapterin reductase catalyses the last steps in the biosynthesis of tetrahydrobiopterin, the essential co-factor of aromatic amino acid hydroxylases and nitric oxide synthases. We have determined the crystal structure of mouse sepiapterin reductase by multiple isomorphous replacement at a resolution of 1.25 A in its ternary complex with oxaloacetate and NADP. The homodimeric structure reveals a single-domain alpha/beta-fold with a central four-helix bundle connecting two seven-stranded parallel beta-sheets, each sandwiched between two arrays of three helices. Ternary complexes with the substrate sepiapterin or the product tetrahydrobiopterin were studied. Each subunit contains a specific aspartate anchor (Asp258) for pterin-substrates, which positions the substrate side chain C1'-carbonyl group near Tyr171 OH and NADP C4'N. The catalytic mechanism of SR appears to consist of a NADPH-dependent proton transfer from Tyr171 to the substrate C1' and C2' carbonyl functions accompanied by stereospecific side chain isomerization. Complex structures with the inhibitor N-acetyl serotonin show the indoleamine bound such that both reductase and isomerase activity for pterins is inhibited, but reaction with a variety of carbonyl compounds is possible. The complex structure with N-acetyl serotonin suggests the possibility for a highly specific feedback regulatory mechanism between the formation of indoleamines and pteridines in vivo.