Young Shik Park

A Murine Model for Human Sepiapterin-Reductase Deficiency

Tetrahydrobiopterin (BH(4)) is an essential cofactor for several enzymes, including all three forms of nitric oxide synthases, the three aromatic hydroxylases, and glyceryl-ether mono-oxygenase. A proper level of BH(4) is, therefore, necessary for the metabolism of phenylalanine and the production of nitric oxide, catecholamines, and serotonin. BH(4) deficiency has been shown to be closely associated with diverse neurological psychiatric disorders. Sepiapterin reductase (SPR) is an enzyme that catalyzes the final step of BH(4) biosynthesis. Whereas the number of cases of neuropsychological disorders resulting from deficiencies of other catalytic enzymes involved in BH(4) biosynthesis and metabolism has been increasing, only a handful of cases of SPR deficiency have been reported, and the role of SPR in BH(4) biosynthesis in vivo has been poorly understood. Here, we report that mice deficient in the Spr gene (Spr(-/-)) display disturbed pterin profiles and greatly diminished levels of dopamine, norepinephrine, and serotonin, indicating that SPR is essential for homeostasis of BH(4) and for the normal functions of BH(4)-dependent enzymes. The Spr(-/-) mice exhibit phenylketonuria, dwarfism, and impaired body movement. Oral supplementation of BH(4) and neurotransmitter precursors completely rescued dwarfism and phenylalanine metabolism. The biochemical and behavioral characteristics of Spr(-/-) mice share striking similarities with the symptoms observed in SPR-deficient patients. This Spr mutant strain of mice will be an invaluable resource to elucidate many important issues regarding SPR and BH(4) deficiencies.

Characterization of a novel unconjugated pteridine glycoside, cyanopterin, in Synechocystis sp. PCC 6803.

A new pteridine glycoside, called cyanopterin, was isolated from Synechocystis sp. PCC 6803 and its structure was elucidated as 6-[1-(4-O-methyl-(alpha-d-glucuronyl)-(1, 6)-(beta-d-galactosyloxy]methylpterin by chemical degradation and 1H- and 13C-NMR spectroscopic means. Cyanopterin is constitutively synthesized at a relatively high intracellular concentration that is comparable to that of chlorophyll a in a molar ratio of approximately 1 to 1.6. The in vivo oxidation state of cyanopterin is primarily the fully reduced 5,6,7,8-tetrahydro form. The cellular function is unknown at present. The findings have established a model system, using Synechocystis sp. PCC 6803, for studies of the physiological functions of unconjugated pteridine glycosides found mostly in cyanobacteria.

Diminished expression of dihydropteridine reductase is a potent biomarker for hypertensive vessels

To identify the new targets for hypertension, we analyzed the protein expression profiles of aortic smooth muscle in spontaneously hypertensive rats (SHR) of various ages during the development of hypertension, as well as in age‐matched normotensive Wistar–Kyoto (WKY) rats, using a proteomic analysis. The expressions of seven proteins were altered in SHR compared with WKY rats. Of these proteins, NADH dehydrogenase 1α, GSTω1, peroxi‐redoxin I and transgelin were upregulated in SHR compared with WKY rats. On the other hand, the expression of HSP27 and Ran protein decreased in SHR. The diminution of dihydrobiopterin reductase, an enzyme located in the regeneration pathways of tetrahydrobiopterin (BH4), was also prominent in SHR. The results from a PCR analysis revealed that the expression of BH4 biosynthesis enzymes – GTP cyclohydrolase‐1 and sepiapterin reductase – decreased and increased, respectively, in SHR compared with WKY rats. The level of BH4 was less in aortic strips from SHR than from WKY rats. Moreover, treatment with BH4 inhibited aortic smooth muscle contraction induced by serotonin. These results suggest that the deficiency in BH4 regeneration produced by diminished dihydrobiopterin reductase expression is involved in vascular disorders in hypertensive rats.

Purification and characterization of UDP-glucose:tetrahydrobiopterin glucosyltransferase from Synechococcus sp. PCC 7942

Tetrahydrobiopterin (BH4)-glucoside was identified from Synechococcus sp. PCC 7942 by HPLC analysis and the enzymatic activity of a glycosyltransferase producing the compound from UDP-glucose and BH4. The novel enzyme, named UDP-glucose:BH4 glucosyltransferase, has been purified 846-fold from the cytosolic fraction of Synechococcus sp. PCC 7942 to apparent homogeneity on SDS-PAGE. The native enzyme exists as a monomer having a molecular mass of 39.2 kDa on SDS-PAGE. The enzyme was active over a broad range of pH from 6.5 to 10.5 but most active at pH 10.0. The enzyme required Mn(2+) for maximal activity. Optimum temperature was 42 degrees C. Apparent K(m) values for BH4 and UDP-glucose were determined as 4.3 microM and 188 microM, respectively, and V(max) values were 16.1 and 15.1 pmol min(-1) mg(-1), respectively. The N-terminal amino acid sequence was Thr-Ala-His-Arg-Phe-Lys-Phe-Val-Ser-Thr-Pro-Val-Gly-, sharing high homology with the predicted N-terminal sequence of an unidentified open reading frame slr1166 determined in the genome of Synechocystis sp. PCC 6803, which is known to produce a pteridine glycoside cyanopterin.

Modulation by melatonin of the cardiotoxic and antitumor activities of adriamycin.

In this study, we investigated the effects of melatonin on adriamycin-induced cardiotoxicity both in vivo in rats and in vitro, and on the antitumor activities of adriamycin on MDA-231 and NCI breast cancer cells. Rats that received a single intraperitoneal injection of 25 mg/kg adriamycin showed a mortality rate of 86%, which was reduced to 20% by melatonin treatment (10 mg/kg, SC for 6 days). Melatonin attenuated adriamycin-induced body-weight loss, hemodynamic dysfunction, and the morphologic and biochemical alterations caused by adriamycin. Melatonin also reduced adriamycin-induced nuclear DNA fragmentation, as assessed by the comet assay. In addition, the antitumor activity of adriamycin could be maintained using lower doses of this drug in combination with melatonin. Melatonin treatment in the concentration range of 0.1-2.5 mM inhibited the growth of human breast cancer cells. In terms of oncolytic activity, the combination of adriamycin and melatonin improved the antitumor activity of adriamycin, as indicated by an increase in the number of long-term survivors as well as decreases in body-weight losses resulting from adriamycin treatment. These results indicate that melatonin not only protects against adriamycin-induced cardiotoxicity but also enhances its antitumor activity. This combination of melatonin and adriamycin represents a potentially useful regimen for the treatment of human neoplasms because it allows the use of lower doses of adriamycin, thereby avoiding the toxic side effects associated with this drug.

The Role of Cyanopterin in UV/Blue Light Signal Transduction of Cyanobacterium Synechocystis sp. PCC 6803 Phototaxis

We analyzed the effects of inactivating the pteridine glycosyltransferase gene (pgtA) on the photomovement of the cyanobacterium Synechocystis sp. PCC 6803 under different light conditions. The pgtA mutant displayed abnormal photomovement under UV-A/blue light. In particular, the pgtA mutant showed a negative phototactic response under UV-A (315-400 nm), whereas the wild-type did not show any photomovement. Inhibition of pterin biosynthesis by N-acetylserotonin (NAS), an inhibitor of sepiapterin reductase, also inhibited a positive phototactic response of the wild-type under white and blue light. In addition, negative phototaxis of the pgtA mutant was observed under UV-A/blue light in the presence of NAS. These results indicated that the product of the PgtA enzyme, cyanopterin, is involved in the inhibition of the negative phototaxis of the wild-type by sensing the UV-A. However, 2,4-diamino-6-hydroxypyrimidine-mediated inhibition of GTP cyclohydrolase I, the rate-limiting enzyme for pterin biosynthesis, significantly increased the positive phototaxis toward UV-A in the wild-type and the pgtA mutant. Furthermore, we measured the action spectrum of phototaxis in vivo for the wild-type and pgtA mutant. Maximal activity of the wild-type was at 300, 380 and 440 nm, indicating absorption by pterins and flavin. In particular, the UV-A/ blue peak at 380 and 440 nm obtained from the action spectrum of phototaxis was found to be closely correlated with the in vitro absorption spectrum previously reported for the cyanobacterial cryptochrome DASH. By investigating the photomovement of the wild-type and pgtA mutant to UV and blue light, we suggest that pterin can function as the chromophore of putative UV/blue photoreceptor(s) in cyanobacterial phototaxis.

Identification of the genes involved in 1-deoxynojirimycin synthesis in Bacillus subtilis MORI 3K-85

Abstract1-Deoxynojirimycin (DNJ), a D-glucose analogue with a nitrogen atom substituting for the ring oxygen, is a strong inhibitor of intestinal α-glucosidase. DNJ has several promising biological activities, including its antidiabetic, antitumor, and antiviral activities. Nevertheless, only limited amounts of DNJ are available because it can only be extracted from some higher plants, including the mulberry tree, or purified from the culture broth of several types of soil bacteria, such as Streptomyces sp. and Bacillus sp. In our previous study, a DNJ-producing bacterium, Bacillus subtilis MORI, was isolated from the traditional Korean fermented food Chungkookjang. In the present study, we report the identification of the DNJ biosynthetic genes in B. subtilis MORI 3K-85 strain, a DNJ-overproducing derivate of the B. subtilis MORI strain generated by γ-irradiation, xhe genomic DNA library of B. subtilis MORI 3K-85 was constructed in Escherichia coli, and clones showing α-glucosidase inhibition activity were selected. After DNA sequencing and a series of subcloning, we were able to identify a putative Operon which consists of gabT1, yktc1, and gutB1 genes predicted to encode putative transaminase, phosphatase, and oxidoreductase, respectively. When a recombinant plasmid containing this Operon sequence was transformed into an E. coli strain, the resulting transformant was able to produce DNJ into the culture medium. Our results indicate that the gabT1, yktc1, and gutB1 genes are involved in the DNJ biosynthetic pathway in B. subtilis MORI, suggesting the possibility of employing these genes to establish a large-scale microbial DNJ overproduction system through genetic engineering and process optimization.

Maintenance of cellular tetrahydrobiopterin homeostasis

Tetrahydrobiopterin (BH4) is a multifunctional cofactor of aromatic amino acid hydroxylases and nitric oxide synthase (NOS) as well as an intracellular antioxidant in animals. Through regulation of NOS activity BH4 plays a pivotal role not only in a variety of normal cellular functions but also in the pathogenesis of cardiovascular and neurodegenerative diseases, which develop under oxidative stress conditions. It appears that a balanced interplay between BH4 and NOS is crucial for cellular fate. If cellular BH4 homeostasis maintained by BH4 synthesis and regeneration fails to cope with increased oxidative stress, NOS is uncoupled to generate superoxide rather than NO and, in turn, exacerbates impaired BH4 homeostasis, thereby leading to cell death. The fundamental biochemical events involved in the BH4-NOS interplay are essentially the same, as revealed in mammalian endothelial, cardiac, and neuronal cells. This review summarizes information on the cellular BH4 homeostasis in mammals, focusing on its regulation under normal and oxidative stress conditions.

Escherichia coli 6‐pyruvoyltetrahydropterin synthase ortholog encoded by ygcM has a new catalytic activity for conversion of sepiapterin to 7,8‐dihydropterin

The putative gene (ygcM) of Escherichia coli was verified in vitro to encode the ortholog of 6‐pyruvoyltetrahydropterin synthase (PTPS). Unexpectedly, the enzyme was found to convert sepiapterin to 7,8‐dihydropterin without any cofactors. The enzymatic product 7,8‐dihydropterin was identified by HPLC and mass spectrometry analyses, suggesting a novel activity of the enzyme to cleave the C6 side chain of sepiapterin. The optimal activity occurred at pH 6.5–7.0. The reaction rate increased up to 3.2‐fold at 60–80°C, reflecting the thermal stability of the enzyme. The reaction required no metal ion and was activated slightly by low concentrations (1–5 mM) of EDTA. The apparent K m value for sepiapterin was determined as 0.92 mM and the V max value was 151.3 nmol/min/mg. The new catalytic function of E. coli PTPS does not imply any physiological role, because sepiapterin is not an endogenous substrate of the organism. The same activity, however, was also detected in a PTPS ortholog of Synechocystis sp. PCC 6803 but not significant in Drosophila and human enzymes, suggesting that the activity may be prevalent in bacterial PTPS orthologs.

Molecular cloning and disruption of a novel gene encoding UDP‐glucose:tetrahydrobiopterin α‐glucosyltransferase in the cyanobacterium Synechococcus sp. PCC 7942

The gene encoding UDP‐glucose:tetrahydrobiopterin α‐glucosyltransferase (BGluT) was cloned from the genomic DNA of Synechococcus sp. PCC 7942. The encoded protein consisting of 359 amino acid residues was verified in vitro and in vivo to be responsible for the synthesis of tetrahydrobiopterin (BH4)‐glucoside produced in the organism. The BGluT gene is the first cloned in pteridine glycosyltransferases and also a novel one cloned so far in UDP‐glycosyltransferases. The mutant cells disrupted in the BGluT gene produced only aglycosidic BH4 at a level of 8.3% of the BH4‐glucoside in wild type cells and exhibited half of the wild type growth in normal photoautotrophic conditions. These results suggest that the glucosylation of BH4 is required for the maintenance of the high cellular concentration of the compound, thereby supporting the normal growth of Synechococcus sp. PCC 7942.