Mechanism and Biosynthesis of Enfumafungin

Enfumafungin was a fernane-type triterpene glycoside that was originally isolated from the fermentation of Hormonema sp. by Merck in 2000. Then it was found out that the natural product inhibited the synthesis of 1,3-β-D-glucan, a necessary component of the fungal cell wall, by inhibiting the enzyme glucan synthase.

Figure 1. Biosynthesis gene cluster of enfumafungin

Discovery

In 2000, Onishi group reported the identification of four triterpenoids as anti-fungal agents. One of the four compounds, enfumafungin, showed significant bioactivity, which could be comparable to that of the control L-733560. Moreover, they also found that enfumafungin specifically inhibited glucan synthesis in whole cells and in (1,3)-β-D-glucan synthase assays, altered the morphologies of yeasts and molds. To further explore the effects of enfumafungin on glucan synthase, enfumafungin was tested against Saccharomyces cerevisiae strains with point mutations in FKS1, the gene encoding the vegetatively expressed large subunit of glucan synthase. The result indicated that enfumafungin produced a unique response in S. cerevisiae strains with point mutations in FKS1, which support the conclusion that enfumafungin was specific inhibitors of glucan synthase. Thus, enfumafungin represented a new group of (1,3)-β-D-glucan synthase inhibitors.

Biosynthesis

Unfortunately, it showed weak activity in a murine model of disseminated candidiasis due to limited stability in vivo. Therefore, since its isolation, Merck undertook the challenge of improving oral efficacy and pharmacokinetic properties of enfumafungin through semi-synthetic modification of the natural product.

The biosynthesis of enfumafungin was still unclear until Kuhnert et al. reported on the preliminary identification of the enfumafungin biosynthetic gene cluster (BGC) based on genome sequencing, phylogenetic reconstruction, gene disruption, and cDNA sequencing studies. It was interesting to find out that enfumafungin synthase (efuA) consisted of a terpene cyclase domain (TC) fused to a glycosyltransferase (GT) domain and thus represented a novel multifunctional enzyme. Moreover, the TC domain bore a phylogenetic relationship to bacterial squalene-hopene cyclases (SHC) and included a typical DXDD motif within the active center suggesting that efuA evolved from SHCs. Phylogenetic reconstruction of the GT domain indicated that this portion of the fusion gene originated from fungal sterol GTs.

Then, in 2021, Li et al. reported the biosynthesis of group II fernane type triterpenoid, polytolypin. The authors have identified the biosynthetic gene cluster of polytolypin from its producer Polytolypa hystricis UAMH7299 and characterized its biosynthetic pathway. A new fernane-type triterpene cyclase was responsible for the biosynthesis of motiol, which was subsequently oxidized by three cytochrome P450s to give polytolypin. Moreover, the three P450 enzymes were employed in combination with two other new fungal fernane-type cyclases for isomotiol and fernenol biosynthesis to generate six polytolypin analogues.

Reference

[1] Liu C-Y, Zhang L, Liu S-X, Lu Y-F, Li C and Pei Y-H (2024) A review of the fernane-type triterpenoids as anti-fungal drugs. Front. Pharmacol. 15:1447450.

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