Terumi Sueoka

Direct inhibition of brain sepiapterin reductase by a catecholamine and an indoleamine

Summary Rat brain sepiapterin reductase, which is involved in the biosynthesis of tetrahydrobiopterin, was directly inhibited by a catecholamine and an indoleamine which require tetrahydrobiopterin as a cofactor in the biosyntheses of themselves. A competitive inhibition appeared for sepiapterin reductase with respect to pterin substrate by 1-norepinephrine (catecholamine) and serotonin, N-acetyl-serotonin and melatonin (indoleamines): Ki values of them were estimated as 3.4 mM, 2.3 mM, 0.20 μM and 30 μM, respectively with partially purified sepiapterin reductase from whole rat brain. Some possible co-regulatory mechanisms to control the formation of catecholamines or indoleamines and of tetrahydrobiopterin are discussed.

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.

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.

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

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-catalyzed 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.

Phosphorylation by Ca2+/calmodulin-dependent protein kinase II and protein kinase C of sepiapterin reductase, the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin

Sepiapterin reductase, the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin, was stoichiometrically phosphorylated by Ca2+/calmodulin‐dependent protein kinase II and protein kinase C (Ca2+/phospholipid‐dependent protein kinase) in vitro. Maximal incorporation of phosphate into the enzyme subunit by these was 3.05 ± 0.05 (n = 4) and 0.74 ± 0.03 (n = 5) 32P mol per mol enzyme subunit, respectively. The enzyme was not phosphorylated by cyclic nucleotide‐dependent protein kinase of either the cAMP‐dependent or cGMP‐dependent type in this study. Dihydropteridine reductase, another enzyme working in direct supply of tetrahydrobiopterin, was also a good substrate for Ca2+/calmodulin‐dependent protein kinase II. Phosphorylation of sepiapterin reductase by these protein kinases modified the kinetic properties of the enzyme. It is likely that these multifunctional Ca2+‐activated protein kinases may play a role in the regulation of the physiological function of the BH4‐generating enzymes in vivo, as was previously found in the case of BH4‐requiring enzymes.

The complete amino acid sequence of the mature form of rat sepiapterin reductase

The partial amino acid sequence of rat sepiapterin reductase was determined using peptides generated by cleavage of the S-carboxyamidomethylated protein with Achromobacter protease I, cyanogen bromide, chymotrypsin or BNPS-skatole. The protein began with N-acetyl methionyl residue at the N-terminus and ended with isoleucyl residue at the C-terminus. The present results essentially coincided with the amino acid sequence predicted from the nucleotide sequence of the cDNA recently reported by Citron et al. (Proc. Natl. Acad. Sci. USA 87, 6436-6440 (1990)), clarified the processing event during the biosynthesis and provided the complete amino acid sequence of the mature form of the enzyme.

Biopterin and neopterin in human saliva

Presence of biopterin and neopterin in human saliva was investigated by HPLC after iodine oxidation in acidic medium. Concentrations of biopterin and neopterin (M +/- SEM) were 1.271 +/- 0.254 and 0.358 +/- 0.075 ng per ml, respectively, in saliva of apparently healthy young male adults, ages 20 to 22 years (n = 9). Nearly identical value of the neopterin/biopterin ratio (0.29 +/- 0.07) was obtained for each of these specimens. Monapterin, the L-threo-isomer of neopterin (0.084 +/- 0.022 ng per ml saliva), and other unconjugated pterins such as xanthopterin, 6-hydroxymethylpterin and pterin were also found in the saliva. These pterins were all detectable in saliva of young female adults with similar levels to those of male saliva. Another fluorescent compound which was identical with 7-iso biopterin in retention time on HPLC was observed in all specimens of normal saliva examined.

Neopterin in the sepia mutant of adult Drosophila melanogaster

Abstract Neopterin (2-amino-4-hydroxy-6-trihydroxypropyl pteridine) has been found in adult flies of the sepia mutant of Drosophila melanogaster . Procedures for the isolation and purification of this compound are described. Neopterin from sepia flies was identified as the d -erythro-form, of the four possible stereoisomers of neopterin by descending paper chromatography and by circular dichroism studies. The occurrence of d -erythro-neopterin in Drosophila melanogaster suggests that a neopterin-compound (possibly d -erythro-dihydroneopterin triphosphate) is an important intermediate in the biosynthesis of unconjugated pteridines from purine nucleotide in insects.

Cloning and sequencing of cDNA encoding human sepiapterin reductase

Sepiapterin reductase (7,8-dihydrobiopterin: N A D P + oxidoreductase, EC 1.1.1.153; SPR) catalyzes the NADPH-dependent reduction of various carbonyl substances including derivatives of pteridines, and belongs to a group of enzymes called aldo-keto reductases. Many reports supports that SPR plays an important role for the biosynthesis of BH4. It is uncertain, however, whether two carbonyl groups on the side chain of 6-pyruvoyl tetrahydropterin, an intermediate involved in BH4 formation, are reduced, in vivo, by the sequential action of SPR alone or by the two enzymatic reactions with SPR and aldose reductase (EC 1.1.1.21). Molecular probes are required to study the metabolism of BH4 in a new aspect.

Dihydropteridine reductase and tetrahydropterin in Crithidia fasciculata cells.

Dihydropteridine reductase was found in extracts of Crithidia fasciculata and was demonstrated by the fact that the enzyme required both quinonoid-dihydropterin and NADH as substrates. 7,8-Dihydropterin and dihydrofolate failed to serve as substrates; tetrahydropterin was formed as the reaction product. The molecular weight of the enzyme was estimated to be about 55 000 by Sephadex G-100 gel filtration. NADH was more effective than NADPH as substrate for the enzyme. Tetrahydropterin (1.35 nmol tetrahydrobiopterin equivalents/g cells) was also detected in C. fasciculata.