Formation of the Δ^18,19 Double Bond and Bis(spiroacetal) in Salinomycin Is Atypically Catalyzed by SlnM, a Methyltransferase‐like Enzyme

Abstract

Salinomycin is a widely used polyether coccidiostat and was recently found to have antitumor activities. However, the mechanism of its biosynthesis remained largely speculative until now. Reported herein is the identification of an unprecedented function of SlnM, homologous to O‐methyltransferases, by correlating its activity with the formation of the Δ18,19 double bond and bis(spiroacetal). Detailed in vivo and in vitro investigations revealed that SlnM, using positively charged S‐adenosylmethionine (SAM) or sinefungin as the cofactor, catalyzed the spirocyclization‐coupled dehydration of C19 in a highly atypical fashion to yield salinomycin.

The biosynthetic pathway to ossamycin, a macrocyclic polyketide bearing a spiroacetal moiety

“…An alternative precedent is offered by the biosynthesis of avermectins in Streptomyces avermitilis , where the enzyme AveC (with no structural similarity to RevJ) has been shown to be essential both for stereospecific spiroketal formation from a dihydroxy-ketone polyketide intermediate and for optional dehydration at C22-C23 [57]. Another bifunctional spirocyclase, from biosynthesis of the polyether salinomycin, is the methyltransferase-like SlnM [58] (also known as SalE [59]) whose disruption prevents both formation of a 6,6-spiroacetal and dehydration to give a cis double bond at C-18-C-19 [60,61]. Furthermore, formation of the spiroacetal in vitro has been directly shown to require SlnM [61].…”

“…Another bifunctional spirocyclase, from biosynthesis of the polyether salinomycin, is the methyltransferase-like SlnM [58] (also known as SalE [59]) whose disruption prevents both formation of a 6,6-spiroacetal and dehydration to give a cis double bond at C-18-C-19 [60,61]. Furthermore, formation of the spiroacetal in vitro has been directly shown to require SlnM [61].…”

Enzymology of Pyran Ring A Formation in Salinomycin Biosynthesis

“…The gene salBIII [ 9a ] encodes a third member of the MonB-family of epoxide hydrolases/cyclases (Supporting Information, Figure S10 ), and we wished to test the hypothesis that this enzyme catalyzes pyran formation. Previous analysis of a mutant deleted in the salE dehydratase gene has shown that the metabolites 5 , 6 , and 7 that accumulate (Supporting Information, Figure S15 ) all have pyran ring A present,[ 9c , d ] as does diene 3 (Figure 2 ), from a mutant lacking the epoxidase SalC. [ 9a ] These results imply that pyran formation is an early post-PKS event.…”

“…This in turn is ring-opened by tandem action of SalBI and SalBII, from the MonB-family of epoxide hydrolases/cyclases. The sal PKS shows several unusual features, including a cis -double bond at C18–C19 inserted by the novel dehydratase[ 9c , d ] (and putative spirocyclase[ 9d ]) SalE, and the lack of a conventional off-loading enzyme. [ 9 ] Notably, the final extension module of the PKS contains neither a DH domain nor a pederin-like PS domain, so the origin of the pyran ring at the western end of the molecule has been obscure.…”

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“…17 In vivo and in vitro studies have shown that SlnM utilises the positive charge of S-adenosylmethionine to stabilise the active conformation of SlnM and promote the reaction sequence. 17 In vivo and in vitro studies have shown that SlnM utilises the positive charge of S-adenosylmethionine to stabilise the active conformation of SlnM and promote the reaction sequence.…”