Macrodiolide Formation by the Thioesterase of a Modular Polyketide Synthase

Abstract

Elaiophylin is an unusual C2-symmetric antibiotic macrodiolide produced on a bacterial modular polyketide synthase assembly line. To probe the mechanism and selectivity of diolide formation, we sought to reconstitute ring formation in vitro by using a non-natural substrate. Incubation of recombinant elaiophylin thioesterase/cyclase with a synthetic pentaketide analogue of the presumed monomeric polyketide precursor of elaiophylin, specifically its N-acetylcysteamine thioester, produced a novel 16-membered C2-symmetric macrodiolide. A linear dimeric thioester is an intermediate in ring formation, which indicates iterative use of the thioesterase active site in ligation and subsequent cyclization. Furthermore, the elaiophylin thioesterase acts on a mixture of pentaketide and tetraketide thioesters to give both the symmetric decaketide diolide and the novel asymmetric hybrid nonaketide diolide. Such thioesterases have potential as tools for the in vitro construction of novel diolides.

Iterative Mechanism of Macrodiolide Formation in the Anticancer Compound Conglobatin

“…The chemical competence of 5 as an intermediate in conglobatin formation in vitro strongly supports an iterative mechanism for TE catalysis, as we recently proposed for biosynthesis of the symmetrical 16-membered diolide elaiophylin ( Zhou et al., 2015 ). In this mechanism, the full-length conglobatin monomer attached to the TE active site is attacked by the distal hydroxy group of an identical monomer on the adjacent ACP.…”

“…The related 20-membered symmetrical oxazole macrodiolides known as samroiyotmycins ( Figure 1 , 3 ) ( Dramae et al., 2013 ) are reported to be active antimalarial compounds. We have recently investigated the biosynthesis of the macrocyclic diolide elaiophylin ( Figure 1 ) and have shown that the thioesterase of the elaiophylin polyketide synthase acts by an iterative mechanism, first ligating two monomers and then re-loading the linear dimer onto the thioesterase active site for dimerization ( Zhou et al., 2015 ). This same mechanism has been previously demonstrated for the thioesterase/cyclase (TE) domain that catalyzes formation of typical non-ribosomal peptide synthetases ( Shaw-Reid et al., 1999; Hoyer et al., 2007; Robbel et al., 2009 ).…”

Biomimetic Thioesters as Probes for Enzymatic Assembly Lines: Synthesis, Applications, and Challenges

“…As such, thioesterases have received considerable attention. Beyond common hydrolysis reactions (Sharma and Boddy, 2007), they are capable of selectively transforming thioester substrates into highly complex macrocyclic esters (He et al, 2006), amides (Kudo et al, 2006;Trauger et al, 2000), and dimers (macrodiolides) (Zhou et al, 2015). Whereas all of these transformations are typically intramolecular, only recently has the first example of an intermolecular thioesterification been reported (A) Activation of carboxylic acids as thioesters and subsequent transformations in biochemistry.…”

“…Nonetheless, this finding suggests that the Corey-Seebach methodology (Yus et al, 2003) can be employed in NAC thioester synthesis routes. Furthermore, para-methoxybenzyl ethers (PMB) (Zhou et al, 2015) and the related formacetal 3,4-dimethoxybenzyloxymethyl (DMBOM) (Mortison et al, 2009) were successfully cleaved by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.…”

“…Crossmetathesis reactions have been carried out using Grubbs' second-generation catalyst (Gao et al, 2013;Hahn et al, 2014;Heine et al, 2014;Kandziora et al, 2014). If desired, it is noteworthy that a,b-unsaturated thioesters can also be selectively TFA, DCM, 0 C (Lohr et al, 2013) 4 N HCl, dioxane (Tao et al, 2003) HF pyr, THF (Frenzel et al, 2006) HF, CH 3 CN, overnight (Aldrich et al, 2005) THF/HCOOH/H 2 O 6/3/1, room temperature, 2 d (Hahn et al, 2014) BF 3 OEt 2 , DCM, 0 C (Liu et al, 1998) BF 3 OEt 2 , 4 Å mol sieve, DCM (Kudo et al, 2006) TsOH H 2 O, MeOH, overnight (Harvey et al, 2012) HF, CH 3 CN, 4 C, overnight (Fiers et al, 2015) HF pyr, THF/pyridine (Hill et al, 2003) TFA (Deska et al, 2011) TsOH H 2 O, MeOH (Bretschneider et al, 2013) 0.1 N HCl (He et al, 2014) pig liver esterase (Hahn et al, 2014) HF pyr, THF (Frenzel et al, 2006) DDQ, DCM/buffer pH 7, 0 C to room temperature (Mortison et al, 2009;Zhou et al, 2015) hy (Hansen et al, 2015) (Tsantrizos et al, 1999) MeI, CaCO 3 , CH 3 CN, H 2 O, 40 C (Le Sann et al, 1999) TFA, trifluoroacetic acid; DCM, dichloromethane; HF, hydrogen fluoride; THF, tetrahydrofuran; TsOH, tosylic acid; MeOH, methanol; DDQ, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.…”