Control of 6-(d-threo-1′,2′-dihydroxypropyl) pterin (dictyopterin) synthesis during aggregation of Dictyostelium discoideum. Involvement of the G-protein-linked signalling pathway in the regulation of GTP cyclohydrolase I activity

Abstract

6-(D-threo-1´,2´-Dihydroxypropylpterin (dictyopterin) has been identified in extracts of growing Dictyostelium discoideum cells [Klein, Thiery and Tatischeff (1990) Eur. J. Biochem. 187, 665–669]. We demonstrate that it originates from GTP by de novo biosynthesis and that the first committed step is catalysed by GTP cyclohydrolase I, yielding dihydroneopterin triphosphate [neopterin is 6-(D-erythro-1´,2´,3´-trihydroxypropyl) pterin]. The GTP cyclohydrolase I activity is found in the cytosolic fraction and in a membrane-associated form. The level of a 0.9 kb mRNA coding for GTP cyclohydrolase I decreases to about 10% of its initial value within 2 h after Dictyostelium cells start development induced by starvation. In the cytosolic fraction, the specific activities of GTP cyclohydrolase I, as well as the concentrations of (6R/S)-5,6,7,8-tetrahydrodictyopterin (H4dictyopterin), follow this decline of the mRNA level. In the particulate fraction, however, the specific activities of GTP cyclohydrolase I and, in consequence, H4dictyopterin synthesis, transiently increase and reach a maximum after 4–5 h of development. The time-course of H4dictyopterin concentrations in the starvation medium closely correlates with its production in the membrane fraction. The activity of membrane-associated GTP cyclohydrolase I can be increased by pre-incubation of the cell lysate with guanosine 5´-[γ-thio]triphosphate and Mg2+. This GTP analogue does not serve as a substrate and has no direct effect on the enzyme activity, indicating that a G-protein-linked signalling pathway is involved in the regulation of GTP cyclohydrolase I activity and thus in H4dictyopterin production during early development of D. discoideum.

Development of the Pteridine Pathway in the Zebrafish,Danio rerio

“…To determine the activity of the synthase, the extract containing it (plus Mg 2ϩ , to achieve a final concentration of 10 mM) was added, and the unstable intermediate 6-pyruvoyl-H 4 pterin was converted to H 4 biopterin by the addition of recombinant murine sepiapterin reductase and NADPH (200 M). After acidic iodine oxidation, biopterin was separated by HPLC (36). The activity of sepiapterin reductase was determined by reduction of sepiapterin to dihydrobiopterin with NADPH, acidic iodine oxidation, and HPLC separation of the resultant biopterin.…”

Phosphorylation of GTP Cyclohydrolase I and Modulation of Its Activity in Rodent Mast Cells

“…After acidic iodine oxidation, biopterin was separated by HPLC. Detailed assay conditions have been described in Gütlich et al (35). The activity of sepiapterin reductase was determined by reduction of sepiapterin to dihydrobiopterin with NADPH, acidic iodine oxidation, and HPLC separation of the product.…”

d‐threo‐tetrahydrobiopterin is synthesized via 1′‐oxo‐2′‐d‐hydroxypropyl‐tetrahydropterin inDictyostelium discoideumAx2

“…2). As known previously [11], wild type cells exhibited a small peak of L L-erythro-biopterin followed by a major peak of D D-threo-biopterin (dictyopterin), the fully oxidized form of DH4. Expectedly, the mutant cells manifested much decreased pteridine production, but not completely (Fig.…”

“…However, the DH4 synthesis remained unknown. The synthesis of DH4 was suspected to originate either from PPH4 or epimerization of L L-erythro-BH4, but no relevant activity was identified from the organism [11]. Recombinant Dictyostelium SR (Dic-SR) alone converted PPH4 to BH4 but never to DH4 [12], while Chlorobium SRs produced L L-threo-BH4 [13].…”

“…Hitherto, three isomers have been known in nature: L L-erythro isomer is a common form, L L-threo isomer from Chlorobium tepidum [9], and D D-threo isomer from Dictyostelium discoideum [10]. A multicellular eukaryote D. discoideum is an useful model organism for cellular and developmental research and is notorious for its synthesis of a D D-threo-BH4, named tetrahydrodictyopterin (DH4), together with a much lower amount of L L-erythro form [10,11]. However, the DH4 synthesis remained unknown.…”