Sodorifen Biosynthesis in the Rhizobacterium Serratia plymuthica Involves Methylation and Cyclization of MEP-Derived Farnesyl Pyrophosphate by a SAM-Dependent C-Methyltransferase

Abstract

The rhizobacterium Serratia plymuthica 4Rx13 releases a unique polymethylated hydrocarbon (CH) with a bicyclo[3.2.1]octadiene skeleton called sodorifen. Sodorifen production depends on a gene cluster carrying a C-methyltransferase and a terpene cyclase along with two enzymes of the 2- C-methyl-d-erythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis. Comparative analysis of wild-type and mutant volatile organic compound profiles revealed a C-methyltransferase-dependent C alcohol called pre-sodorifen, the production of which is upregulated in the terpene cyclase mutant. The monocyclic structure of this putative intermediate in sodorifen biosynthesis was identified by NMR spectroscopy. In vitro assays with the heterologously expressed S. plymuthica C-methyltransferase and terpene cyclase demonstrated that these enzymes act sequentially to convert farnesyl pyrophosphate (FPP) into sodorifen via a pre-sodorifen pyrophosphate intermediate, indicating that the S-adenosyl methionine (SAM)-dependent C-methyltransferase from S. plymuthica exhibits unprecedented cyclase activity. In vivo incorporation experiments with C-labeled succinate, l-alanine, and l-methionine confirmed a MEP pathway to FPP via the canonical glyceraldehyde-3-phosphate and pyruvate, as well as its SAM-dependent methylation in pre-sodorifen and sodorifen biosynthesis.C{H} NMR spectroscopy facilitated the localization of C labels and provided detailed insights into the biosynthetic pathway from FPP via pre-sodorifen pyrophosphateto sodorifen.

Volatilomes of Bacterial Infections in Humans

“…The most prominent bacterial terpenes are geosmin and methyl isoborneol (summarized in Dickschat, 2016). Recently a sesquiterpene with a unique structure was isolated from Serratia plymuthica (von Reuss et al, 2010(von Reuss et al, , 2018. Terpene synthases are fascinating enzymes as they are quite limited regarding their substrate usage (GPP, FPP, GGPP, NPP, and/or respective enantiomers), however their reaction mechanisms (including several cyclization cascades) can release a multitude of products (multi product enzymes).…”

“…Terpene synthases are fascinating enzymes as they are quite limited regarding their substrate usage (GPP, FPP, GGPP, NPP, and/or respective enantiomers), however their reaction mechanisms (including several cyclization cascades) can release a multitude of products (multi product enzymes). It is interesting to note, that the elucidation of the sodorifen biosynthesis led to a change of paradigm, as the substrate for the sodorifen terpene synthase is a methylated and cyclic FPP, produced by an upstream acting SAM-dependent C-methyltransferase (von Reuss et al, 2018).…”

The hidden enzymology of bacterial natural product biosynthesis

“…Many of the TCs identified in these studies appear to be transcriptionally silent but their expression in an engineered Streptomyces heterologous host has enabled the identification of a number of unprecedented terpene NPs 108 , and novel TC families will surely continue to be discovered regularly. Indeed, it was recently shown that the organic volatile sodorifen (20) produced by S. plymuthica 4Rx13 is the product of a TC 109,110 , a remarkable finding given that every C atom in the bicyclic ring structure of the compound is substituted with either a methyl or methylene group, which previously obscured the biosynthetic origins of this molecule 111 . Unusual methylations of C5 building blocks have also been reported for the biosynthesis of a number of other structurally unique bacterial terpenes, with longestin 112 (21) and the teleocidins 113 (22) representing additional characterized examples.…”

An Unusual Skeletal Rearrangement in the Biosynthesis of the Sesquiterpene Trichobrasilenol from Trichoderma

“…A similar mechanism may also be relevant for the biosynthesis of sodorifen, [28] a polymethylated homosesquiterpene in which one additional Me group equivalent is introduced by an SAM-dependent methylation, but in this case, three more Me groups seem to originate from internal methylene groups of FPP. [29] Despite preliminary results from labelling experiments that have resulted in a biosynthetic hypothesis, [29] a detailed mechanistic investigation for the biosynthesis of this molecule is lacking. In the case of the homomonoterpene 2-methylisoborneol, one additional Me group is introduced by SAM-dependent methylation of GPP, but the subsequent terpene cyclisation of the resulting 2methyl-GPP is regular.…”