Studies on sepiapterin reductase: Further characterization of the reaction product

doi:10.1016/0003-9861(68)90427-X

Archives of Biochemistry and Biophysics

Volume 126, Issue 2, August 1968, Pages 426-435

https://doi.org/10.1016/0003-9861(68)90427-X Get rights and content

Abstract

Sepiapterin reductase from rat liver catalyzes reduction of sepiapterin in the presence of NADPH. The reaction product was isolated from the reaction mixture via Sephadex column chromatography and its properties were compared with those of synthetic dihydrobiopterin. All of the chemical and enzymatic characteristics examined distinctly indicated that two compounds are identical. Stoichiometry of sepiapterin reductase-catalyzed reaction was established from balance studies. Evidence is presented which indicates that dihydrobiopterin will be reduced to tetrahydrobiopterin by dihydrofolate reductase in the presence of NADPH. Stoichiometric formulation was proposed for the enzymatic reduction of dihydrobiopterin. From spectral characteristics and behaviors toward enzymatic reduction, a possibility of 7,8-dihydro structure of dihydrobiopterin has been discussed.

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Cited by (35)

Chemical reduction of pterins to dihydropterins as substrates for enzymatic reactions
2006, Analytical Biochemistry
Citation Excerpt :
After hydrogenation at the lower pH of 7.0, three peaks were visible with maxima at 232, 278, and 335 nm (Fig. 2, trace b). These values are consistent with published values of 230, 280, and 330 nm observed for the dihydro form of different pterins [27,28]. The DHPR assay indicated that only 7% of the product was in the fully reduced tetrahydro form.
Dihydropterins are important intermediates in various metabolic pathways, including the biosynthesis of tetrahydrofolate and tetrahydromethanopterin, a key coenzyme in the one-carbon metabolism of methanogenic Archaea. Some procedures for the reduction of pterins to dihydropterins may produce undesirable tetrahydropterin contaminants. This work describes a procedure for the rapid reduction of pterins to dihydropterins while minimizing tetrahydropterin production that may be particularly useful in producing substrates for enzyme reactions when the dihydropterin substrate cannot be purchased commercially.
Tetrahydrobiopterin uptake in supplemental administration: Elevation of tissue tetrahydrobiopterin in mice following uptake of the exogenously oxidized product 7,8-dihydrobiopterin and subsequent reduction by an anti-folate- sensitive process
2004, Journal of Pharmacological Sciences
In order to increase the tissue level of tetrahydrobiopterin (BH₄), supplementation with 6R-tetrahydrobiopterin (6RBH₄) has been widely employed. In this work, the effectiveness of 6RBH₄ was compared with 7,8-dihydrobiopterin (7,8BH₂) and sepiapterin by administration to mice. Administration of 6RBH₄ was the least effective in elevating tissue BH₄ levels in mice while sepiapterin was the best. In all three cases, a dihydrobiopterin surge appeared in the blood. The appearance of the dihydrobiopterin surge after BH₄ treatment suggested that systemic oxidation of the administered BH₄ had occurred before accumulation of BH₄ in the tissues. This idea was supported by the following evidences: 1) An increase in tissue BH₄ was effectively inhibited by methotrexate, an inhibitor of dihydrofolate reductase which reduces 7,8BH₂ to BH₄. 2) When the unnatural diastereomer 6SBH₄ was administered to mice, a large proportion of the recovered BH₄ was in the form of the 6R-diastereomer, suggesting that this BH₄ was the product of a dihydrofolate reductase process by which 7,8BH₂ converts to 6RBH₄. These results indicated that the exogenous BH₄ was oxidized and the resultant 7,8BH₂ circulated through the tissues, and then it was incorporated by various other tissues and organs through a pathway shared by the exogenous sepiapterin and 7,8BH₂ in their uptake. It was demonstrated that maintaining endogenous tetrahydrobiopterin in tissues under ordinary conditions was also largely dependent on an methotrexate-sensitive process, suggesting that cellular tetrahydrobiopterin was maintained both by de novo synthesis and by salvage of extracellular dihydrobiopterin.
The hydroxymethyldihydropterin pyrophosphokinase domain of the multifunctional folic acid synthesis fas protein of pneumocystis carinii expressed as an independent enzyme in escherichia coli: Efolding and characterization of the recombinant enzyme
1994, Protein Expression and Purification
The folic acid synthesis (Fas) protein of Pneumocystis carinii is a multifunctional enzyme containing dihydroneopterin aldolase, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (PPPK), and dihydropteroate synthase activities. Isolation of the stretch of fas cDNA shown by amino acid similarity to the bacterial counterparts to code for PPPK activity (fasC domain) is described. FasC was expressed to high levels in Escherichia coli inclusion bodies using an inducible tac promoter expression system. Solubilization of the inclusion bodies in 6 M guanidine hydrochloride and refolding of the recombinant protein yielded enzymatically active PPPK which was purified to homogeneity by anion-exchange and gel-filtration chromatography. Sequence analysis showed that the first 13 amino acids of the purified protein were in agreement with those predicted from the DNA sequence and, furthermore, that the amino-terminal methionine had been removed. The enzyme is active in the monomeric form, exhibiting maximum activity at around pH 8.0. Isoelectric focusing gave a pI of 9.1. The K_m value for 6-hydroxymethyl-7,8-dihydropterin was 3.6 μM in 50 mM Tris buffer, pH 8.2. The production of independently folded, active P. carinii PPPK will allow detailed biochemical and structural studies, increasing our understanding of this enzyme domain.
On the role of sepiapterin reductase in the biosynthesis of tetrahydrobiopterin
1987, Archives of Biochemistry and Biophysics
Rat erythrocyte sepiapterin reductase can catalyze the NADPH-dependent reduction of tetrahydropterin substrates with relative velocities of sepiapterin > lactoyltetrahydropterin ⩾ pyruvoyltetrahydropterin > 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
1985, BBA - General Subjects
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.
Dyspropterin, an intermediate formed from dihydroneopterin triphosphate in the biosynthetic pathway of tetrahydrobiopterin
1985, BBA - General Subjects
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-catalysed 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.

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Archives of Biochemistry and Biophysics

Studies on sepiapterin reductase: Further characterization of the reaction product

Abstract

Biochim. Biophys. Acta

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

J. Chromatog

Biochim. Biophys. Acta

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

Biochem. Biophys. Res. Commun

Chemical reduction of pterins to dihydropterins as substrates for enzymatic reactions

Tetrahydrobiopterin uptake in supplemental administration: Elevation of tissue tetrahydrobiopterin in mice following uptake of the exogenously oxidized product 7,8-dihydrobiopterin and subsequent reduction by an anti-folate- sensitive process

The hydroxymethyldihydropterin pyrophosphokinase domain of the multifunctional folic acid synthesis fas protein of pneumocystis carinii expressed as an independent enzyme in escherichia coli: Efolding and characterization of the recombinant enzyme

On the role of sepiapterin reductase in the biosynthesis of tetrahydrobiopterin

Carbonyl reductase activity of sepiapterin reductase from rat erythrocytes

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