Biosynthesis of the Sphingolipid Inhibitors Sphingofungins in Filamentous Fungi Requires Aminomalonate as a Metabolic Precursor

Abstract

Sphingofungins belong to a group of structurally related sphingolipid inhibitors produced by fungi, which specifically inhibit serine palmitoyl transferases, enzymes catalyzing the initial step during sphingolipid biosynthesis. Sphingolipids are integral parts of the eukaryotic cell membrane, and disturbances in their homeostasis have been linked to various human diseases. It has been suggested that external interventions, via sphingolipid inhibitors, may represent a promising approach for alternative therapies. Here, we identified and elucidated the biosynthetic gene cluster responsible for the biosynthesis of sphingofungins B, C, and D in Aspergillus fumigatus. Moreover, in vitro analyses have shown that sphingofungin biosynthesis starts with the condensation of a C18 polyketide with the uncommon substrate aminomalonate. Furthermore, the investigations on sphingofungin E and F produced by Paecilomyces variotii pointed out that different aminomalonate derivatives are used as substrates for those chemical variants. This research boosts knowledge on the general biosynthesis of sphingolipid inhibitors in fungi.

Desaturation of the Sphingofungin Polyketide Tail Results in Increased Serine Palmitoyltransferase Inhibition

“…We then proceeded to compare the inhibitory activity of sphingofungin B (1) with sphingofungin C (2) and the novel sphingofungin derivatives B 2 (3) and C 2 (4) ( 17 ) ( Fig. 3A ).…”

“…Sphingofungins C 3 (5) and C 4 (6) were detected in extracts obtained from the previously created strain A. fumigatus xyl-G sphAPv ( 17 ) via HPLC-HRMS and tandem high-resolution mass spectrometry (HRMS 2 ) analyses (Fig. S5).…”

“…We successfully established and validated a new approach for determining SPT activity by coupling the synthesis of 3-ketodihydrosphingosine and the accompanying release of CoA to the quantifiable release of PPi by FadD through the ATP dependent esterification of palmitic acid with aforementioned CoA. We applied this approach to determine and compare the SPT inhibitory activity of novel sphingofungin derivatives ( 17 , 18 ) on the bacterial SPT of S. paucimobilis ( 19 ), which was used as a model to investigate the enzymatic mechanisms of SPT inhibition ( 10 , 28 ). The comparison of our in vitro data, gained using the bacterial SPT with a simple in vivo assay, the antifungal activity against S. cerevisiae , revealed a striking difference in the inhibitory activity of the investigated sphingofungins, likely due to the structural differences between the SPTs of these organisms.…”

“…Recently, we elucidated the biosynthesis of the known SPT inhibitors sphingofungins B-D in Aspergillus fumigatus ( 17 ). During this process we isolated the novel sphingofungin derivatives B 2 and C 2 .…”

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“…The biosynthetic gene cluster for the production of sphingofungins, such as sphingofungin B 29 , have been elucidated in Aspergillus fumigatus . 22 In vitro analysis showed that the first key step involves the condensation of a C18 polyketide with an aminomalonate substrate, using the aminotransferase SphA.…”