Biosynthesis of "drosopterins" in Ḏṟo̲s̲o̲p̲ẖi̲ḻa̲ m̲e̲ḻa̲ṉo̲g̲a̲s̲ṯe̲ṟ

To test the hypothesis that sepiapterin is a "drosopterin" precursor, radioactively-labeled H₂-neopterin-(P)₃ was used as a substrate for sepiapterin and "drosopterin" synthesis by enzymes in Drosophila extracts to compare the requirements for biosynthesis. The reaction products were separated by two-dimensional cellulose thin-layer chromatography. Results were obtained to indicate that sepiapterin synthase actually consists of two enzymes, the first requiring Mg²⁺ and the second, NADPH. The product of the Mg²⁺-dependent enzyme [known hereafter as the sepiapterin synthase intermediate or (X)], and not sepiapterin, serves as a "drosopterin" precursor in the presence of either NADH or NADPH. The evidence was: (1) the pr mutant, low in accumulated sepiapterin and "drosopterins" is known to have only 20% wild-type sepiapterin synthase activity, (2) unlabeled sepiapterin did not cause isotope dilution of "drosopterin" biosynthesis from [U-¹⁴H₂-neopterin-(P)₃, (3) the 600g pellet prepared from a wild-type homogenate contains "drosopterin" synthesizing activity and no sepiapterin synthetic activity, yet a heat-labile factor in this fraction stimulates sepiapterin synthesis in the 100,000g supernatants of both wild-type and pr flies, (4) sepiapterin and "drosopterin" synthesis require Mg²⁺, and (5) sepiapterin synthesis is stimulated by NADPH while "drosopterin" synthesis responds to either NADPH or NADH. The sepiapterin synthase intermediate could not be substituted for by H₂-neopterin.

A high performance liquid chromatography (HPLC) assay for (X) was developed. The sepiapterin synthase intermediate was identified by its dependence on Mg²⁺ for enzymatic synthesis from H₂-neopterin-(P)₃, the decreased yield with a concomitant increase in the yield of sepiapterin in the presence of NADPH, the incorporation of ³H and ¹⁴C from [U-¹⁴C][3’-³H]H₂-neopterin-(P)₃ in the same ratio as sepiapterin, and the observation that the pr mutant had only 20% wild-type (X) synthetic activity.

It was found that (X) and two related compounds [(X1) and (X2)] could be synthesized nonenzymatically from H₂-neopterin-(P)₃ in a reaction catalyzed by Tris base, dependent on H₂-neopterin-(P)₃ concentration, significant at temperatures greater than 80°C, and maximal between pH 8.5 and 9.0 (25°C). These compounds were purified and it was found that both (X) and (X1) could serve as substrates for the enzymatic NADPH-dependent synthesis of sepiapterin. This observation, the kinetics of formation from H₂-neopterin-(P)₃, and the similarity of the ultraviolet absorption spectra led to the hypothesis that (X), (X1), and (X2) are isomers. None of the compounds could be reduced by NaBH₄ and only (X1) was sensitive to oxidation with NaIO₄. All three were oxidized by iodine to give rise to more fluorescent compounds which in turn could be reduced with NaBH₄, to give rise to the original compounds. The latter observations indicate that (X), (X1), and (X2) are dihydropterins.

Since it had been observed that the pr and se mutants both contained low amounts of quench spot (Q.S.), a pterin-like compound and no “drosopterins” while Henna-recessive-3 (Hn^r3) flies accumulate both Q.S. and sepiapterin but no "drosopterins" (Wilson & Jacobson, 1977), it was postulated that Q.S. is a "drosopterin" precursor. Evidence was obtained that Q.S. is an intermediate in the conversion of (X) to "drosopterins" and that there exists at least one other "drosopterin" precursor which apparently arises from H₂-neopterin-(P)₃ via a separate pathway. Q.S. biosynthetic activity was found in Drosophila extracts (using a two-dimensional cellulose thin-layer chromatography separation of Q.S. for the assay) and label from [U-¹⁴C] but not [3'-³H]H₂-neopterin-(P)₃ was incorporated into Q.S. Mg²⁺ was required for Q.S. synthesis but either NADH or NADPH diminished the incorporation of label. Extracts from pr^bwcn heads contained only 30% of the synthetic activity found in Oregon-R heads and purified Q.S. (from Oregon-R heads), when added to in vitro biosynthetic reactions utilizing either Oregon-R or pr^bwcn extracts, stimulated the incorporation of label from [U-¹⁴C]H₂-neopterin-(P)₃ into the "drosopterins".

The chemical properties of Q.S. were investigated. It demonstrated a molecular ion of m/z=221 with a composition of C₉H₁₁N₅O₂ in the mass spectrum. Q.S. has an ultraviolet absorption spectrum (maxima at 260, 360, and 384 nm at pH 6.0) and pKa values similar to pterins but it is nonfluorescent. Upon drying of Q.S. solutions, an orange compound was formed from which Q.S. could be regenerated by dissolving and diluting in H₂O. Q.S. was sensitive to oxidation by alkaline KMnO₄, but did not give rise to 6-carboxypterin as a product. It was oxidized by NaIO₄ and could be reduced with NaBH₄. Q.S. reacted with 2,4-dinitrophenylhydrazine to form a red precipitate. Iodine oxidized Q.S. but the products were nonfluorescent. These observations and the observation that Q.S. is an intermediate in the conversion of (X) to "drosopterins" led to two postulated structures for Q.S. in which it has retained the three carbon side chain from H₂-neopterin(P)₃ but two atoms (C1’ and C2’) of the side chain form a four-membered ring with two atoms of the pyrazine ring (N5 and C6) by the presence of a bond between N5 and C2'. The terminal carbon of the side chain is a methyl group in both structures. One of the proposed structures is an epoxide with an oxygen atom forming a three-membered ring with C1' and C2' and the other structure is a 2'-oxo compound. The latter is postulated to equilibrate with a hydrated form (1',2'-dihydroxy) in aqueous solution. For either of these structures to be converted to "drosopterins" would require a rearrangement of the four-membered ring.