Biosynthesis of ebelactone A: isotopic tracer, advanced precursor and genetic studies reveal a thioesterase-independent cyclization to give a polyketide β-lactone

Abstract

Macrocyclization of polyketides generates arrays of molecular architectures that are directly linked to biological activities. The four-membered ring in oxetanones (β-lactones) is found in a variety of bioactive polyketides (for example, lipstatin, hymeglusin and ebelactone), yet details of its molecular assembly have not been extensively elucidated. Using ebelactone as a model system, and its producer Streptomyces aburaviensis ATCC 31860, labeling with sodium [1-(13)C,(18)O2]propionate afforded ebelactone A that contains (18)O at all oxygen sites. The pattern of (13)C-(18)O bond retention defines the steps for ebelactone biosynthesis, and demonstrates that β-lactone ring formation occurs by attack of a β-hydroxy group onto the carbonyl moiety of an acyclic precursor. Reaction of ebelactone A with N-acetylcysteamine (NAC) gives the β-hydroxyacyl thioester, which cyclizes quantitatively to give ebelactone A in aqueous ethanol. The putative gene cluster encoding the polyketide synthase (PKS) for biosynthesis of 1 was also identified; notably the ebelactone PKS lacks a terminal thioesterase (TE) domain and no stand alone TE was found. Thus the formation of ebelactone is not TE dependent, supporting the hypothesis that cyclization occurs on the PKS surface in a process that is modeled by the chemical cyclization of the NAC thioester.

Operon for Biosynthesis of Lipstatin, the Beta-Lactone Inhibitor of Human Pancreatic Lipase

“…Therefore, it is plausible that LstD is involved in the reduction of the 3-keto group to a 3-hydroxyl group, which is probably also involved in the exchange of the H-2 proton with the solvent proton by an unknown mechanism. After the 3-keto is reduced, the resulting 3-hydroxy group may attack spontaneously on the carbonyl moiety of the ACP-tether acyl intermediate to give the ␤-lactone ring, in analogy with the formation of the ␤-lactone ring in the ebelactone biosynthesis pathway (55). On the other hand, the production of compound 4 in the ⌬lstE and ⌬lstF::aadA mutants implies that the LstD dehydrogenase might also reduce the 3-keto group to a 3-hydroxyl in the absence of a 5-O-formylleucine group to afford compound 4, or another, unknown enzyme in S. toxytricini contributes to this reduction.…”

Active Multienzyme Assemblies for Long-Chain Olefinic Hydrocarbon Biosynthesis

“…First, a multienzyme assembly of Ole proteins could protect the cell by sequestering the reactive Ole pathway ␤-lactone intermediate and preventing a nonspecific reaction with cytosolic proteins. The ␤-lactone moiety is common in microbial natural products and can function as an antibiotic by reacting with active-site residues in essential esterase and protease enzymes (12,13). The ␤-lactone tetrahydrolipstatin from Streptomyces toxytricini is very similar in structure to the ␤-lactone produced by OleC, and its biosynthesis is encoded by an ole-like gene cluster that lacks the oleB decarboxylase gene (10,12).…”

“…Homologous gene clusters lacking an oleB gene have been identified in Streptomyces, and these produce ␤-lactone natural products rather than olefins (10). Microbial secondary metabolites containing ␤-lactones often serve as antibiotics and are known as general esterase and protease inhibitors (12,13).…”

Evidence for an iterative module in chain elongation on the azalomycin polyketide synthase

“…However, an increasing number of PKS systems are now known in which the main product apparently requires iterative use of a module to accomplish two or even three successive rounds of chain extension. First noted in the stigmatellin PKS from Stigmatella aurantiaca [20], further examples have been uncovered in the PKSs for aureothin [21–22], borrelidin [23–24], lankacidin [25–26], neoaureothin [27], etnangien [28], crocacin [29], ebelactone [30] and thiolactomycin [31–32]. Given the close mechanistic analogy between fatty acid synthases and an iterative PKS module, it is instructive that a normally colinear extension module of the pikromycin PKS, when studied as a stand-alone protein in vitro, catalyses two rounds of polyketide chain extension [19].…”

“…Two of these are the near-identical PKSs (Figure S1, Supporting Information File 1) for the antifungal 36-membered marginolactones azalomycin 1a–c (from Streptomyces malaysiensis (formerly Streptomyces violaceusniger ) DSM4137) and kanchanamycin 1d [35–36] from Streptomyces olivaceus Tü4018; and the third is the PKS for the β-lactone ebelactone, a potent esterase inhibitor from Streptomyces aburaviensis 2a,b (Fig. 1) [30]. These examples are particularly interesting as potential model systems because the chemical outcome of the two successive extensions catalysed by the iterative module is predicted to be different (vide infra).…”