Masahiro Tazawa

(6R)-5,6,7,8-Tetrahydro-L-Monapterin from Escherichia coli, a Novel Natural Unconjugated Tetrahydropterin

Abstract The structure of the major tetrahydropterin in Escherichia coli was determined as (6R)-5,6,7,8-tetrahydroLmonapterin, i. e. (6R)-2-amino-5,6,7,8-tetrahydro 6-[(1S,2S)-1,2,3-trihydroxypropyl]pteridin-4(3H)one. Although the stereochemical structure of the trihydroxypropyl side chain has been determined previously by fluorescence detected circular dichroism analysis on its aromatic derivative, the most important configuration at C(6) has not been clarified. The major difficulties for the determination of the chirality were instability toward air oxidation and very low concentration of the tetrahydropterin derivative. In the present study, the C(6)configuration was determined as R by comparing its stable hexaacetyl derivative with authentic (6R) and (6S)hexaacetyl-5,6,7,8- tetrahydroLmonapterins by high performance liquid chromatography (HPLC) and HPLCmass spectrometry (LCMS). (6R)-5,6,7,8-TetrahydroLmonapterin is a new unconjugated tetrahydropterin from natural sources.

cAMP inhibits cytokine-induced biosynthesis of tetrahydrobiopterin in human umbilical vein endothelial cells

We studied the effects of cAMP on cytokine (interferon-gamma plus tumor necrosis factor-alpha)-induced stimulation of tetrahydrobiopterin (BH4) synthesis in human umbilical vein endothelial cells (HUVEC). The cytokine mixture caused a marked increase in the biosynthesis and release of BH4 by HUVEC. Dibutyryl-cAMP produced a dose-dependent inhibition of this cytokine-induced stimulation of synthesis and release of BH4 by these cells. 8-Bromo-cAMP also caused a significant inhibition, although the effects were less marked than those of dibutyryl-cAMP. Both forskolin and the stable analog of prostacyclin, iloprost, caused cAMP accumulation and a concomitant diminution of the cytokine-induced BH4 synthesis in HUVEC. Dibutyryl-cAMP and iloprost also significantly inhibited the cytokine-induced stimulation of GTP cyclohydrolase I (GCHI) activity and mRNA production. We concluded that the suppression by the cAMP messenger system of cytokine-induced stimulation of synthesis and release of BH4 by HUVEC can be attributed to the inhibition of the activity of GCHI, the rate-limiting enzyme in BH4 biosynthetic pathway, in HUVEC. The data also suggest that the cAMP-mediated reduction in the GCHI mRNA level may at least partially explain the decline in GCHI activity. It is reasoned that under inflammatory conditions, cAMP-elevating agents such as prostacyclin exert regulatory effects on circulation by inhibiting cytokine-induced synthesis and release of BH4 by HUVEC.

GTP cyclohydrolase I from Tetrahymena pyriformis: cloning of cDNA and expression.

A full-length cDNA clone for GTP cyclohydrolase I (EC 3.5.4.16) was isolated from a Tetrahymena pyriformis cDNA library by plaque hybridization. The nucleotide sequence determination revealed that the length of the cDNA insert was 1516 bp. The coding region encoded a protein of 223 amino acid residues with a calculated molecular mass of 25 416 Da. The deduced amino acid sequence of Tetrahrymena GTP cyclohydrolase I showed sequence identity with that of Escherichia coli (55%). The identity of T. pyriformis GTP cyclohydrolase I with sequences of Dictyostelium discoideum, Saccharomyces cerevisiae, Drosophila melanogaster, mouse, rat, and human enzymes was less marked and was 30, 30, 25, 28, 28, and 27%, respectively. RNA blot analysis showed a single mRNA species of 2.1 kb in this protozoan. The mRNA level of GTP cyclohydrolase I increased during synchronous cell division induced by intermittent heat treatment. The results suggest that the mRNA expression is associated with the cell cycle of T. pyriformis.

Determination of Tetrahydropterins as Native Pteridines in Two Microorganisms, Tetrahymena Pyriformis and E. Coli

Unique pterins were identified as a major pterin in several microorganisms. D-Monapterin was identified as major pterin in Tetrahymena pyriformis (1 ), which is a protozoa living in fresh water, and L-monapterin was identified in E. coli (2), respectively. However, their biological functions have not been revealed yet. We recently reported presence of reduced forms of those monapterins in each microorganism, and the reduced form was estimated 5,6,7,8-tetrahydro form (3, 4). In this study, we would describe structural determination of unstable natural 5,6,7,8-tetrahydromonapterins in the two microorganisms. First, the configuration of 1′,2′,3′-trihydroxypropyl side chain was determined by fluorescence detected circular dichroism (FDCD) spectra using stable aromatic derivatives, which has strong fluorescence nature, and then configurations of 6-position was determined by LC-MS analysis using stable completely acetyl derivatives.