Sepiapterin reductase from horse liver: Purification and properties of the Enzyme

doi:10.1016/S0003-9861(71)80057-7

Archives of Biochemistry and Biophysics

Volume 146, Issue 1, September 1971, Pages 202-214

https://doi.org/10.1016/S0003-9861(71)80057-7 Get rights and content

Sepiapterin reductase, which catalyzes the NADPH-mediated reversible reduction of sepiapterin to dihydrobiopterin, has been purified approximately 5000-fold from an extract of horse liver. The coenzyme requirement, optimum pH, substrate specificity, and susceptibility to inhibitors have been described.

In the reverse reaction, l-erythro- and l-threo-dihydroneopterin, in addition to dihydrobiopterin, were oxidized in the presence of NADP at a very slow rate, while the corresponding d-isomers were inactive as substrates.

Determination of the equilibrium constant and the free-energy change of the reaction revealed that the equilibrium lies much in favor of reduction.

Intracellular localization in horse liver and distribution in rat tissues of the sepiapterin reductase activity have also been studied.

References (37)

MatsubaraM. et al.
Biochim. Biophys. Acta
(1966)
Nagai (Matsubara)M.
Arch. Biochem. Biophys.
(1968)
KaufmanS.
J. Biol. Chem.
(1967)
FukushimaT. et al.
Arch. Biochem. Biophys.
(1968)
GelotteB.
J. Chromatogr.
(1960)
ZakrzewskiS.F.
J. Biol. Chem.
(1960)
KornbergA. et al.
SeegmillerJ.E. et al.
J. Biol. Chem.
(1951)
LowryO.H. et al.
J. Biol. Chem.
(1951)
KalckarH.M. et al.
Biochim. Biophys. Acta
(1950)

DealW.C. et al.

Biochem. Biophys. Res. Commun.

(1963)

GuroffG. et al.

J. Biol. Chem.

(1969)

KidderG.W. et al.

J. Biol. Chem.

(1968)

JonesT.H. et al.

J. Biol. Chem.

(1967)

FukushimaT.

Arch. Biochem. Biophys.

(1970)

KaufmanS.

MatsubaraM. et al.

Experientia

(1964)

TsusueM. et al.

Zool. Mag.

(1965)

Cited by (59)

Uterine secretome: What do the proteins say about maternal-fetal communication in buffaloes?
2024, Journal of Proteomics
The aim was to compare the UF proteomics of pregnant and non-pregnant buffalo during early pregnancy. Forty-four females were submitted to hormonal estrus synchronization and randomly divided into two groups: pregnant (n = 30) and non-pregnant (n = 14). The pregnant group was artificially inseminated and divided into a further two groups: P12 (n = 15) and P18 (n = 15). Conceptus and uterine fluid samples were collected during slaughter at, respectively, 12 and 18 days after insemination. Of all the inseminated females, only eight animals in each group were pregnant, which reduced the sample of the groups to P12 (n = 8) and P18 (n = 8). The non-pregnant group was also re-divided into two groups at the end of synchronization: NP12 (n = 7) and NP18 (n = 7). The UF samples were processed for proteomic analysis. The results were submitted to multivariate and univariate analysis. A total of 1068 proteins were found in the uterine fluid in both groups. Our results describe proteins involved in the conceptus elongation and maternal recognition of pregnancy, and their action was associated with cell growth, endometrial remodeling, and modulation of immune and antioxidant protection, mechanisms necessary for embryonic maintenance in the uterine environment.
Uterine fluid is a substance synthesized and secreted by the endometrium that plays essential roles during pregnancy in ruminants, contributing significantly to embryonic development. Understanding the functions that the proteins present in the UF perform during early pregnancy, a period marked by embryonic implantation, and maternal recognition of pregnancy is of fundamental importance to understanding the mechanisms necessary for the maintenance of pregnancy. The present study characterized and compared the UF proteome at the beginning of pregnancy in pregnant and non-pregnant buffaloes to correlate the functions of the proteins and the stage of development of the conceptus and unravel their processes in maternal recognition of pregnancy. The proteins found were involved in cell growth and endometrial remodeling, in addition to acting in the immunological protection of the conceptus and performing antioxidant actions necessary for establishing a pregnancy.
Sepiapterin reductase mediates chemical redox cycling in lung epithelial cells
2013, Journal of Biological Chemistry
Citation Excerpt :
We found that these point mutations caused major losses (90–98%) in the ability of sepiapterin reductase to reduce sepiapterin confirming that in the wild type enzyme these amino acids bind tightly to NADPH, generating a unique structural conformation that is essential for substrate reduction. These data are also consistent with the requirement for the adenine nucleotide for optimal enzyme activity (25). Interestingly, NADPH-binding site mutant sepiapterin reductase enzymes still retained 25–55% of their redox cycling activity.
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.
Background: Enzymes mediating chemical redox cycling in the lung are poorly defined.
Results: Sepiapterin reductase was identified as a key mediator of redox cycling and was analyzed using inhibitors and site-directed mutagenesis.
Conclusion: Sepiapterin reductase generates reactive oxygen species during redox cycling in a mechanism distinct from sepiapterin reduction.
Significance: This is the first report demonstrating that sepiapterin reductase mediates chemical redox cycling.
Membrane transport of sepiapterin and dihydrobiopterin by equilibrative nucleoside transporters: A plausible gateway for the salvage pathway of Tetrahydrobiopterin biosynthesis
2011, Molecular Genetics and Metabolism
Citation Excerpt :
The coenzyme is converted to inactive 7,8-dihydrobiopterin (7,8BH2 or simply BH2 ) through spontaneous isomerization of qBH2. It has long been known that BH4 is produced from sepiapterin (SP; 6-lactyl-7,8-dihydropterin) by two distinct enzymes, sepiapterin reductase (EC 1.1.1.153, Km to SP, 21 μM [10]) and dihydrofolate reductase (DHFR, EC 1.5.1.3, Km to 7,8BH2, 5–15 μM [11,12]), the latter of which converts 7,8BH2 to active BH4 [11,13–15]. The route of BH4 retrieval from SP or BH2 was termed the “salvage pathway” as opposed to the de novo pathway of BH4 biosynthesis [16].
Tetrahydrobiopterin (BH₄) is synthesized de novo in particular cells, but in the case of a systemic or local BH₄ deficiency, BH₄ supplementation therapy is applied. BH₄-responsive PKU has also been effectively treated with BH₄ supplementation. However, the rapid clearance of the supplemented BH₄ has prevented the therapy from being widely accepted. Deposition of BH₄ after supplementation involves oxidation of BH₄ to dihydrobiopterin (BH₂) and subsequent conversion to BH₄ by the salvage pathway. This pathway is known to be almost ubiquitous in the body. However, the mechanism for the redistribution and exclusion of BH₄ across the plasma membrane remains unclear. The aim of this work was to search for the key transporter of the uptake precursor of the salvage pathway. Based on the observed sensitivity of pterin transport to nitrobenzylthioinosine (NBMPR), we examined the ability of ENT1 and ENT2, representative equilibrative nucleoside transporters, to transport sepiapterin (SP), BH₂ or BH₄ using HeLa cell and Xenopus oocyte expression systems. hENT2 was capable of transporting the pterins with an efficiency of SP > BH₂ > BH₄. hENT1 could also transport the pterins but less efficiently. Non-transfected HeLa cells and rat aortic endothelial cells were able to incorporate the pterins and accumulate BH₄ via uptake that is likely mediated by ENT2 (SP > BH₂ > BH₄ ). When exogenous BH₂ was given to mice, it was efficiently converted to BH₄ and its tissue deposition was similar to that of sepiapterin as reported (Sawabe et al., 2004). BH₄ deposition after BH₂ administration was influenced by prior treatment with NBMPR, suggesting that the distribution of the administered BH₂ was largely mediated by ENT2, although urinary excretion appeared to be managed by other mechanisms. The molecular basis of the transport of SP, BH₂, and BH₄ across the plasma membrane has now been described for the first time: ENT2 is a transporter of these pterins and is a plausible gateway to the salvage pathway of BH₄ biosynthesis, at least under conditions of exogenous pterin supplementation. The significance of the gateway was discussed in terms of BH₂ uptake for BH₄ accumulation and the release for modifying the intracellular BH₂/BH₄ ratio.
The silkworm mutant lemon (lemon lethal) is a potential insect model for human sepiapterin reductase deficiency
2009, Journal of Biological Chemistry
Citation Excerpt :
The blot was visualized using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences) and a LAS 1000 imaging system (Fuji Film). Enzyme Assay—SPR activity was assayed according to the method reported by Katoh (21) with slight modifications. Ten micrograms of protein from whole body or 0.1-0.5 μg of recombinant BmSPR was used in the assessment.
Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic acid hydroxylases, which control the levels of monoamine neurotransmitters. BH4 deficiency has been associated with many neuropsychological disorders. An inherited defect in BH4 biosynthesis is caused by the deficiency of sepiapterin reductase (SPR), which catalyzes the biosynthesis of BH4 from guanosine triphosphate at the terminal step. The human SPR gene has been mapped at the PARK3 locus, which is related to the onset of Parkinson disease. In this study, we report that mutant strains, lemon (lem) and its lethal allele lemon lethal (lem¹) with yellow body coloration, of the silkworm Bombyx mori could be used as the first insect model for human SPR deficiency diseases. We demonstrated that mutations in the SPR gene (BmSpr) were responsible for the irregular body coloration of lem and lem^l. Moreover, biochemical analysis revealed that SPR activity in lem^l larvae was almost completely diminished, resulting in a lethal phenotype that the larvae cannot feed and that die immediately after the first ecdysis. Oral administration of BH4 and dopamine to lem^l larvae effectively increased their survival rates and feeding abilities. Our data demonstrate that BmSPR plays a crucial role in the generation of BH4, and monoamine neurotransmitters in silkworms and the lem (lem^l) mutant strains will be an invaluable resource to address many questions regarding SPR and BH4 deficiencies.
Cellular uptake of sepiapterin and push-pull accumulation of tetrahydrobiopterin
2008, Molecular Genetics and Metabolism
Cellular uptake of sepiapterin resulted in an efficient accumulation of tetrahydrobiopterin. Tetrahydrobiopterin is much less permeable across the cell membrane than sepiapterin or dihydrobiopterin, the precursors of the tetrahydrobiopterin-salvage pathway. The uptake of sepiapterin by the cell was examined under metabolic arrest with N-acetylserotonin, an inhibitor of sepiapterin reductase. The release profile of previously accumulated sepiapterin was also analyzed. Two routes were clearly distinguishable, namely rapid and slow. Both were apparently bi-directional and equilibrating in type. Each route was connected to non-mixable pools somehow separated in the cell. The rapid process was too fast to analyze by the current methods of cell handling. The slower process was associated with conversion of sepiapterin to tetrahydrobiopterin in the absence of N-acetylserotonin, suggesting that this route opens into the cytosolic compartment where use of the salvage pathway was strongly driven by sepiapterin reductase and dihydrofolate reductase with a supply of NADPH which favors tetrahydrobiopterin accumulation. Consequently, sepiapterin was enforcedly taken up by the cell where it accumulated tetrahydrobiopterin in the cytosol in continuous manner.
A brain-specific decrease of the tyrosine hydroxylase protein in sepiapterin reductase-null mice-as a mouse model for Parkinson's disease
2008, Biochemical and Biophysical Research Communications
Sepiapterin reductase (SPR) is an enzyme that acts in the third and final step of tetrahydrobiopterin (BH4) biosynthesis. The human Spr gene locates within the region of 2.5 MB mapped to PARK3, an autosomal dominant form of familial Parkinson’s diseases. In order to explore the role of SPR in the metabolism of BH4, we produced and analyzed Spr-deficient mice. Most of Spr-null mice survived beyond two weeks. Whereas the BH4 contents in the homozygous mutant mice were greatly decreased than those in wild-type and heterozygous mice, the substantial amounts of BH4 were remained even 17 days after delivery. Spr-null mice exhibited severe monoamine deficiencies and a tremor-like phenotype after weaning. The amount of TH protein in the brain of Spr-null mice was less than 10% of wild-type, while TH protein in the adrenal, phenylalanine hydroxylase protein in the liver, and nNOS in the brain were not altered. These data suggest an essential role of SPR in the biosynthesis of BH4, and that the SPR gene could be a candidate gene for PARK3.

View all citing articles on Scopus

¹: A part of this work was presented at the 4th International Symposium on Pteridines, Toba, July 21–25, 1969.

View full text

Sepiapterin reductase from horse liver: Purification and properties of the Enzyme1

Biochim. Biophys. Acta

Arch. Biochem. Biophys.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Chromatogr.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

Experientia

Zool. Mag.