The Interplay between a Multifunctional Dehydratase Domain and a C‐Methyltransferase Effects Olefin Shift in Ambruticin Biosynthesis

Abstract

The olefin shift is an important modification during polyketide biosynthesis. Particularly for type I cis-AT PKS, little information has been gained on the enzymatic mechanisms involved. We present our in vitro investigations on the olefin shift occurring during ambruticin biosynthesis. The unique, multifunctional domain AmbDH4 catalyzes consecutive dehydration, epimerization, and enoyl isomerization. The resulting 3-enethioate is removed from the equilibrium by α-methylation catalyzed by the highly specific C-methyltransferase AmbM. This thermodynamically unfavorable overall process is enabled by the high, concerted substrate specificity of the involved enzymes. AmbDH4 shows close relationship to DH domains and initial mechanistic studies suggest that the olefin shift occurs via a similar proton-shuttling mechanism as previously described for EI domains from trans-AT-PKS.

Molecular Basis for Olefin Rearrangement in the Gephyronic Acid Polyketide Synthase

“…Of the cis -AT DHs predicted to produce odd-to-even unsaturated final products, DH4 from the ambruticin pathway catalyzes sequential dehydration, epimerization and isomerization reactions on a (3 S ,4 S )-3-hydroxy-4-methyl substrate in which epimerization of the 4-methyl occurs after dehydration to a 4-methyl-2-enoyl intermediate and before double bond shift to the 4-methyl-3-enoyl product (Scheme S1) 8 . The Amb DH4 is most similar to non-isomerizing DHs with His, Asp, Pro and Tyr amino acids in the active site (Figure S9).…”

“…Introduction of a branched side chain in the active site led to selective 2-methyl epimerization relative to the (2 R ,3 R )-2-methyl-3-hydroxy product of the module KR. The presence of a branched-chain amino acid in the PKS DH active site is correlated with a capability for double-bond migration 27 , although the Amb DH4 is an exception 8 .…”

“…Olefin rearrangement has been characterized in only two cis -AT PKS systems 8, 29 . In the ansamitocin pathway, feeding studies implicated DH-containing module 3 in the formation of a conjugated 2,4-dienoyl product via a proposed vinylogous dehydration 29 .…”

An Unusual Fatty Acyl:Adenylate Ligase (FAAL)–Acyl Carrier Protein (ACP) Didomain in Ambruticin Biosynthesis

“…The ambruticins are a group of unusual myxobacterial polyketides that contain several cyclic structural elements that are introduced during excessive tailoring processes (Scheme ) . Reeves et al.…”

Hot off the press

“…ambruticin VS3 22) has been delineated. 18 The process initially involves a multifunctional domain AmbDH4 that catalyses a dehydration, epimerisation and enoyl isomerisation sequence. The unfavourable equilibrium of this process is then trapped by a C-methyltransferase catalysed α-methylation leading to the pentadiene fragment of the ambruticins.…”