Sulfonium Acids Loaded onto an Unusual Thiotemplate Assembly Line Construct the Cyclopropanol Warhead of a Burkholderia Virulence Factor

Abstract

Pathogenic bacteria of the Burkholderia pseudomallei group cause severe infectious diseases such as glanders and melioidosis. Malleicyprols were identified as important bacterial virulence factors, yet the biosynthetic origin of their cyclopropanol warhead has remained enigmatic. By a combination of mutational analysis and metabolomics we found that sulfonium acids, dimethylsulfoniumpropionate (DMSP) and gonyol, known as osmolytes and as crucial components in the global organosulfur cycle, are key intermediates en route to the cyclopropanol unit. Functional genetics and in vitro analyses uncover a specialized pathway to DMSP involving a rare prokaryotic SET‐domain methyltransferase for a cryptic methylation, and show that DMSP is loaded onto the NRPS‐PKS hybrid assembly line by an adenylation domain dedicated to zwitterionic starter units. Then, the megasynthase transforms DMSP into gonyol, as demonstrated by heterologous pathway reconstitution in E. coli.

Mechanistic insight into DsyB/DSYB, key enzymes in marine dimethylsulfoniopropionate synthesis

“…Finally, BurB is con ned to very closely related Burkholderia spp. that likely use DMSP as an intermediate in toxin production (32). This hierarchy of DMSP synthesis gene abundance in bacteria (DsyB > MmtN > BurB) was mirrored in marine environmental data.…”

“…Of the known DMSP synthesis enzymes DsyB is by far the most abundant in sequenced and/or isolated bacteria (65.8 % of cultured isolates) (Table S2) (7,10,11,32,33). DsyB is mostly found in alphaproteobacterial Rhodobacterales, Rhizobiales and Rhodospirillales, but is also sporadically found in e.g.…”

“…The presence or absence of DMSP synthesis genes in 111 cultured DMSP-producing bacteria (published since the discovery of bacterial DMSP synthesis (7,10,11,32,33) was analysed (Table S2). This is based previously published work that analysed their sequenced genomes and/or used degenerate primers to detect the presence of burB, dsyB and/or mmtN.…”

Recent insights into oceanic dimethylsulfoniopropionate biosynthesis and catabolism

“…Bacteria harbouring functional MmtN proteins are not monophyletic, suggesting that mmtN may have been horizontally transferred between these microorganisms (Williams et al, 2019). Recently, DMSP biosynthesis in Burkholderia thailandensis has been reported (Trottmann et al, 2020). By analogy to one of the established bacterial DMSP biosynthesis pathways, methionine undergoes S-methylation to form Smethylmethionine, decarboxylation, transamination and No added methylated sulfur compounds unless stated otherwise; 10 mM succinate was carbon source for all minimal media; 10 mM NH4Cl was nitrogen source for all media unless stated otherwise.…”

“…An in silico analysis of the bur gene locus revealed candidate genes for a methyltransferase (BurB), a decarboxylase (BurI), a transaminase (BurD), and a dehydrogenase (BurE) (Trottmann et al, 2020) (Fig. S2).…”