Sepiapterin reductase: Characteristics and role in diseases

Abstract 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.

Due to the important role of BH 4 in various biological processes, SPR is inferred to be required for many functions at the in vitro and in vivo levels. Therefore, tremendous efforts have been made in attempts to unfold the molecular basis of the function of SPR. Based on the analysis of crystals and nuclear magnetic resonance (NMR) studies, the structure of SPR has been solved for various species.
Moreover, gene cloning, recombinant expression and mutagenesis studies have enabled people to understand the biological functions and roles of SPR in different diseases. Meanwhile, many compounds have been identified in relation to SPR, which has provided potential therapeutics for brain dysfunction, cardiovascular disease and cancer.
However, there are still many questions unresolved. In particular, there F I G U R E 1 The biosynthetic pathway of tetrahydrobiopterin: de novo pathway from GTP and two other salvage pathways from sepiapterin and 1′-hydroxy-2′-oxopropyl-tetrahydropterin, respectively. DHFR: dihydrofolate reductase are different effects on biological processes induced by SPR inhibitors and SPR knockdown, for example effects on nitric oxide (NO) generation. Therefore, the main focus in this review will be on the structure, function, distribution and regulation of sepiapterin reductase. In addition, the relationship between SPR and different diseases will also be summarized to determine the possible clinical value of SPR.

| S TRUC TURE OF S EPIAP TERIN REDUC TA S E
Sepiapterin reductase has structural similarity to that of members of the NADP(H)-preferring short-chain reductase family, which contain a strictly conserved Tyr-Xaa-Xaa-Xaa-Lys sequence motif. Specifically, SPR exists in solution as a homodimer composed of two subunits with a molecular mass calculated to be approximately 28 kDa. [10][11][12][13] The crystal structures of SPR ( Figure 2) indicate that 261 amino acids of each monomer fold into a single domain with an α/β-structure. A seven-stranded anti-parallel oriented β-sheet in the centre of the molecule is sandwiched by two arrays of three α-helices. Six of these strands could form a classic nicotinamide-binding motif composed of βαβ units. The association of two monomers into the active homodimeric SPR leads to the formation of a four-helix bundle (helices αE and αF of each monomer). 14,15 In addition to human beings, this enzyme has been identified in rats, mice, monkeys, Chlorobium tepidum (C tepidum), and Drosophila.
In addition, the amino acid sequence comparison derived from cDNAs reveals high homology among these different species; 16,17 for example, the sequence of human SPR (hSPR) shows 74% identity with the rat sequence, 10 which suggests that the tertiary and quaternary structures of SPR may be conserved. Typically, the NADP(H) binding domain and the positions of active sites show high similarity. Remarkable differences among SPR from various sources are F I G U R E 2 The overall structure of CT-SPR. A, Stereoview of the ribbon representation of CT-SPR monomer which binds with NADP and sepiapterin (PDB code 2BD0). β-Strands and α-helices are labelled in alphabetical order from the N terminus; three amino acid residues that are essential for catalysis and substrate binding have been labelled by a different colour (F99 is in green; S158 is in red; W196 is in yellow) (B) Ribbon representation of a CT-SPR tetramer formed by two dimers in the asymmetric unit. NADP is in orange; sepiapterin is in white. C, The comparison of SPR monomer from C tepidum (orange), mouse (blue, PDB code 1SEP) and human (magenta, PDB code 4Z3K). Arrows show the largest differences among the three structures present in the substrate-binding regions around the active sites. For example, the SPR from C tepidum (CT-SPR) contains a shorter loop and longer C-terminal extension compared to mouse SPR and hSPR, resulting in diverse stereospecific catalysis reactions. 15 Furthermore, the active sites have been explored by constructing truncation mutants and through the use of site-directed mutagenesis. Unlike the N-terminal A-X-L-L-S sequence of other BH 4 -requiring aromatic amino acid hydroxylases, the region of SPR is speculated to preferably act as the coenzyme NADP(H) binding site. 18 Amino acid residues including Ser-158, Tyr-171 and Lys-175 play an important role in proton transfer and stabilization for the carbonyl group of substrates, according to the SPR crystal structure and kinetic properties of site-directed mutants. In addition, the catalytic activity could not be detected in the double-point mutant, SPRY171V + S158D, as opposed to the single-point mutant, suggesting that the remaining residue might function alone and show low activity if either of the important residues is mutated. 18,19 However, Trp-196 and Phe-99 are indispensable for substrate binding in CT-SPR because of the swivelled sepiapterin binding mode. 20 In brief, all of these revelations regarding the structure of SPR make it possible to explore its function and develop therapeutic strategies.

| B IOLOG IC AL FUN C TIONS
It is well known that sepiapterin reductase acts as a key enzyme in the biosynthetic pathway of tetrahydrobiopterin cofactor. As shown in Figure 1, sepiapterin reductase takes part not only in the salvage biosynthetic pathway of tetrahydrobiopterin, in which it catalyses the NADPH-mediated reduction of sepiapterin to dihydrobiopterin, 21,22 but also in the de novo synthetic pathway, in which it catalyses the conversion of 1′-oxo-2′-hydroxypropyl-BH 4 to BH 4 . [23][24][25][26] Moreover, another new activity of SPR has been identified, namely 'lactoyl-BH 4 isomerase' activity, which converts 1′-hydroxy-2′-oxopropyl-BH 4 into 1′-oxo-2′-hydroxypropyl-BH 4 independently of NADPH. [27][28][29] Additionally, many non-pteridine derivatives, including quinones, for example p-quinone and menadione; other vicinal dicarbonyls, for example methylglyoxal and phenylglyoxal; monoaldehydes, for example p-nitrobenzaldehyde; and monoketones, for example acetophenone, acetoin, propiophenone and benzylacetone, are sensitive as substrates of SPR. 30,31 Furthermore, it has been demonstrated that carbonyl reductases (CR) and aldose reductases (AR), which are primarily active in the liver, could take the place of SPR by an alternative pathway in the biosynthesis of BH 4 . Specifically, CR could also catalyse the conversion of sepiapterin, and AR serves a catalytic function in con- The important role of SPR in the biosynthesis of nitric oxide has also been studied based on the conclusion that tetrahydrobiopterin is a limiting factor of nitric oxide generation. According to these results, SPR inhibitors could abolish cytokine-induced NO production in various cell types, [35][36][37][38] such as murine macrophages and endothelial cells, but do not affect the constitutive level of NO. 37,38 Nevertheless, knockdown or overexpression of SPR could significantly affect the constitutive level of NO both in vitro and in vivo. 39 One hypothetical reason for this controversial conclusion is the function of the non-enzymatic activity of SPR in the regulation of NO generation. On the other hand, SPR is also involved in oxidative stress. It has been reported that SPR inhibitors could prevent the protective effect of sepiapterin against cell injury induced by H 2 O 2 in endothelial cells. 40 The knockout of SPR could impair mitochondrial function and increase the susceptibility of Dictyostelium discoideum Ax2 to oxidative stress. 41 Meanwhile, these results have been proven by the SPR enzymatic inhibitor -SPRi3 in CD 4+ T cells. 42 However, site-directed mutagenesis of SPR indicates that mutation of Asp-257 to histidine abolished sepiapterin reduction activity but had minimal effects on reactive oxygen species production, 43 suggesting the biological function of the non-enzymatic activity of SPR (Table 1). Moreover, our published study indicated that SPR could promote hepatocellular carcinoma progression via FoxO3a/Bim signalling in a non-enzymatic manner, while its enzymatic activity might have no effect on hepatocellular carcinoma development. 44 Overall, SPR plays a key role in different biological processes, and these effects might be related not only to its enzymatic activity but also to its non-enzymatic function. Increasing studies prove that many enzymes are involved in tumour progression independent of their catalytic activities. For example, phosphoglycerate mutase 1, aside from its glycolytic enzymatic activity, could directly interact with α-smooth muscle actin and modulates cancer cell migration. 45 In addition to its pyruvate kinase function, the M2 isoform of pyruvate kinase also promotes cyclin D1 expression through binding to β-catenin. 46 Moreover, the interaction between lysine-specific histone demethylase 1 (LSD1) and the transcription factor is necessary for acute myeloid leukaemia survival, instead of the demethylase activity of LSD1. 47,48 Thus, further studies are necessary to construct an integrated map of the molecular mechanisms of SPR.

| D IS TRIBUTI ON AND REG UL ATI ON OF S EPIAP TERIN REDUC TA S E
It is reported that sepiapterin reductase has a wide distribution. As mentioned above, SPR has been detected in various species such as C tepidum, Drosophila, chicken, rat, horse and humans. It has also been found in multiple tissues, including liver, kidney, thymus, brain, spleen, testis and blood. 43,[49][50][51][52][53] Specifically, in rats, SPR activities average 130 and 80 pmol/h/ mg in the liver and the erythrocyte fraction of blood, respectively, while no activity is detected in the intestine and muscle. 54 The enzyme mRNA presents in nearly all the peripheral tissues of goldfish, including intestine and muscle; meanwhile, expression in the brain could be affected by fasting. 55 Quantitative transcriptomics analysis and microarray-based immunohistochemistry have been used to analyse the tissue-specific expression of SPR in a representative set of major human organs and tissues. 56,57 According to the results, which are presented in a database (gtexportal.org), SPR in normal tissue exhibits relatively high expression levels in the liver, kidney and colon ( Figure 3). Furthermore, the distribution of SPR in the human brain has been demonstrated, and the data show that SPR is localized in the pyramidal neurons of the cerebral cortex, in a small number of striatal neurons, and neurons of the hypothalamic and brainstem monoaminergic region and olivary nucleus. 58 Furthermore, the expression of the SPR gene is also high in liver cancer and colorectal cancer (Figure 3), based on data from The Cancer Genome Atlas dataset (cancergenome.nih.gov). Therefore, SPR might become a clinical biomarker of cancer.
Although the distribution of SPR is wide, no SPR mRNA has been detected in the human NK-like cell line YT or the murine erythroleukaemic cell line B8/3. Furthermore, the activity and expression of SPR depend on the types of cell lines. For example, in contrast to the liver cell line HepG2, the T-cell line HuT102 shows lower SPR activity. 49 However, the enzymatic activity in human T lymphocytes could be continuously stimulated by lectin treatment and achieve a 4-fold increase. 59 Moreover, rapid enhancement of SPR activity could be induced in T cells by the synergism of IFN-γ and IL-2 rather than by IFN-γ or by IL-2 alone. 60 In the human neuroblastic cell line BE2-M17, SPR mRNA and protein levels could be down-regulated by overexpression of wild-type α-synuclein. 61 Lipopolysaccharide treatment has resulted in the up-regulation of SPR gene expression in the murine neuroblastoma (NB) cell line N1E-115 62 and the striatum, 63 while this effect of lipopolysaccharide has not been achieved in the murine locus coeruleus. 64 Moreover, the serine amino acid residues of rat SPR and hSPR could be phosphorylated by Ca 2+ /calmodulin-dependent protein kinase II and protein kinase C, but whether phosphorylation has an effect on the activity of SPR or not is controversial. 65,66 In summary, the study of SPR regulation is rare. To better understand the biological function of SPR and to find new targets for disease therapies, more studies are needed to elucidate the mechanism of SPR regulation.

| S EPIAP TERIN REDUC TA S E AND DISE A SE
As described above, SPR plays a key role in the de novo biosynthesis of BH 4 . BH 4 is a key cofactor for a set of metabolic enzymes and is associated with a large number of biological processes, such as monoamine neurotransmitter formation, immune response and pain sensitivity. Therefore, many studies have been conducted to examine the relationship between SPR abnormity and disease.

| Sepiapterin reductase and brain dysfunction
Tetrahydrobiopterin is a key cofactor for enzymatic control of the synthesis and secretion of monoamine neurotransmitters, including dopamine and serotonin. Deficiency in the BH 4  in the SPR gene has been generated. 78-81 SPR-knockout (SPR -/-) mice show lower levels of BH 4 , dopamine, serotonin and tyrosine hydroxylase in the brain and liver in contrast to the wild-type group.
In contrast to the data of human patients, the serum phenylalanine level in transgenic mice is significantly increased. One reason for this difference might be that the salvage biosynthetic pathway from 6-pyruvoyltetrahydropterin to BH 4 catalysed by 3α-hydroxysteroid dehydrogenase type 2 (HSDH2) and AR does not function in mice. 32 Moreover, SPR -/mice exhibit motor dysfunction and developed dwarfism. In addition, most SPR -/mice die within 1-2 months. The administration of BH 4 and neurotransmitter precursors could rescue the growth retardation and high phenylalanine levels, but the level of BH 4 and tyrosine hydroxylase in the brain depends on the method of administration. Furthermore, the tyrosine diet ameliorates the abnormal motor behaviours and enhances mTORC1 activity without affecting dopamine expression in SPR -/mice, suggesting that the mTORC1 signalling pathway in the brain is one of the possible targets in understanding the abnormal motor behaviours related to SPD. 82,83 Considering that the human SPR gene is located within the

| Sepiapterin reductase and chronic pain
The data of gene expression profiling in the dorsal root ganglion in three different models of neuropathic pain showed that three of the enzymes, including GTPCH, SPR and quinoid dihydropteridine reductase, which are critical to the control of intracellular levels of BH 4 , were highly regulated within injured sensory neurons. 87 Although GTPCH, an obligate rate-limiting enzyme in BH 4 synthesis, plays an important role in chronic pain, 88

| Sepiapterin reductase and cardiovascular disease
Nitric oxide, synthesized by three nitric oxide synthases, is an important regulator of vascular tone. All NOS isoforms require BH 4 as cofactor for catalytic activity, reiterating that SPR might be implicated in cardiovascular disease. Many studies have demonstrated that cytokine-induced NO production in murine macrophages and endothelial cells could be abolished by SPR inhibitors. [35][36][37][38] Gao and coworkers 39 proved that the overexpression or depletion of SPR could affect NO production in endothelial cells and NO-dependent vasorelaxation in vivo. Furthermore, the endothelium-specific SPR deficiency in deoxycorticosterone acetate-salt hypertensive mice suggests the importance of SPR in maintaining normal blood pressure. 95 Nevertheless, aortic endothelial SPR expression is unaffected in angiotensin II-induced hypertensive mice 95 and mice with pulmonary hypertension induced by bleomycin. 96 Moreover, the therapeutic effects of sepiapterin, a substrate of SPR, in hypertension depend on the level of SPR in the arteries.
Therefore, strategies specifically targeting SPR might be necessary for restoring NOS function in different types of hypertension.
Recently, the cardiovascular function of SPR gene-disrupted mice has been analysed. 97 After weaning, SPR -/but not SPR +/− adult mice suffered from hypertension with fluctuation and bradycardia due to the decrease in endothelium-dependent relaxation. At the same time, the imbalance of the sympathetic and parasympathetic nervous systems found in the SPR -/mice might contribute to cardiovascular instability. Besides, the SPR inhibitor NAS could completely inhibit the sepiapterin-stimulated tube formation of bovine aortic endothelial cells in vitro. 98 Although the key role of SPR in cardiovascular disease has been proposed, the underlying mechanism remains unclear. Further studies are needed to answer the remaining questions.

| Sepiapterin reductase and cancer
In recent years, the role of BH 4 in the development of cancer has at- Sulfasalazine (SSZ), which has been identified as an SPR inhibitor, could inhibit the growth of NB cells and produce synergistic antiproliferative effects in combination with alpha-difluoromethylornithine. 103 Furthermore, oral/intraperitoneal SSZ co-administration resulted in measurable inhibition of NB tumour growth in vivo. 104 Recently, it is reported that SPR is required for the proliferation of mature T cells in vitro and in vivo and that the inhibition of cancer induced by SPR inhibitors links to the immunosuppressive tumour environment. 42 All of these studies provide potential targets for cancer therapy, although further research illuminating the role of SPR in cancer progression and the mechanism underlying the regulation is needed.

| FUTURE D IREC TI ON S
The important biological function of SPR has been demonstrated by using molecular biological techniques, ranging from protein X-ray structure determinations to mutagenesis studies.
Furthermore, a set of drugs targeting SPR have been identified, which makes the studies of the SPR enzymatic activity easier.
In addition, the discovery of patients with SPD has provided new insight into the role of SPR in disease and provided potential therapeutic strategies and biomarkers for brain dysfunction, chronic pain, cardiovascular disease and cancer. However, for obtaining an integrated map of the molecular biology of SPR, many questions remain to be solved, such as the following. Otherwise, we would like to thank Pei Wang for the collection of clinical data from datasets.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analysed in this study.