Identification of Biosynthetic and Metabolic Genes of 2-Azahypoxanthine in Lepista sordida Based on Transcriptomic Analysis

2-Azahypoxanthine was isolated from the fairy ring forming fungus Lepista sordida as a fairy ring-inducing compound. 2-Azahypoxanthine has an unprecedented 1,2,3-triazine moiety, and its biosynthetic pathway is unknown. The biosynthetic genes for 2-azahypoxanthine formation in L. sordida were predicted by a differential gene expression analysis using MiSeq. The results revealed that several genes in the purine and histidine metabolic pathways and the arginine biosynthetic pathway are involved in the biosynthesis of 2-azahypoxanthine. Furthermore, nitric oxide (NO) was produced by recombinant NO synthase 5 (rNOS5), suggesting that NOS5 can be the enzyme involved in the formation of 1,2,3-triazine. The gene encoding hypoxanthine-guanine phosphoribosyltransferase (HGPRT), one of the major phosphoribosyltransferases of purine metabolism, increased when 2-azahypoxanthine content was the highest. Therefore, we hypothesized that HGPRT might catalyze a reversible reaction between 2-azahypoxanthine and 2-azahypoxanthine-ribonucleotide. We proved the endogenous existence of 2-azahypoxanthine-ribonucleotide in L. sordida mycelia by LC-MS/MS for the first time. Furthermore, it was shown that recombinant HGPRT catalyzed reversible interconversion between 2-azahypoxanthine and 2-azahypoxanthineribonucleotide. These findings demonstrate that HGPRT can be involved in the biosynthesis of 2-azahypoxanthine via 2azahypoxanthine-ribonucleotide generated by NOS5.

Automated summary

2-Azahypoxanthine was isolated from Lepista sordida and found to induce fairy rings. The biosynthetic pathway of 2-Azahypoxanthine is unknown, but differential gene expression analysis predicted genes involved in its formation. Nitric oxide generated by NOS5 and reversible catalysis by HGPRT were found to be involved in its biosynthesis. These findings provide insights into the biosynthetic mechanism of 2-Azahypoxanthine.