Biochemical determination of enzyme-bound metabolites: preferential accumulation of a programmed octaketide on the enediyne polyketide synthase CalE8

Abstract

Despite considerable interest in the enediyne family of antitumor antibiotics, assembly of their polyketide core structures in nature remains poorly understood. Discriminating methods to access enzyme-bound intermediates are critical for elucidating unresolved polyketide and nonribosomal peptide biosynthetic pathways. Here, we describe the development of broadly applicable techniques for the mild chemical release and analysis of intermediates bound to carrier proteins (CPs), providing access to these species even in sensitive systems. These techniques were applied to CalE8, the polyketide synthase (PKS) involved in calicheamicin biosynthesis, facilitating the unambiguous identification of enzyme-bound polyketides on an enediyne PKS. Moreover, these methods enabled the preparation of fully unloaded CalE8, providing a "clean slate" for reconstituted activity and allowing us to demonstrate the preferential accumulation of a PKS-bound octaketide with evidence of programmed processing control by CalE8. This intermediate, which has the expected chain length for enediyne core construction, could previously only be indirectly inferred. These studies prove that this polyketide is an authentic product of CalE8 and may be a key precursor to the enediyne core of calicheamicin, as it is the only programmed, enzyme-bound species observed for any enediyne system to date. Our experimental advances into a generally inaccessible system illustrate the utility of these techniques for investigating CP-based biosynthetic pathways.

Figure 1. Enediyne antibiotics and related octaketides

(A) Representative structures of enediyne natural products from each of three structural families, showing incorporation patterns of isotopically labeled acetate (see inset) for the core structures. (B) In vitro production of heptaene 1 by the calicheamicin PKS CalE8 and TE CalE7. KS, β-ketoacyl synthase; AT, acyl transferase; CP, carrier protein; KR, ketoreductase; DH, dehydratase; PPT, phosphopantetheinyl transferase. Inset: 4'-phosphopantetheine tether installed post-translationally on the CP domain. (C) A biosynthetic proposal for divergence inspired by production of octaketide 2 during heterologous expression of CalE8 alone.

Figure 2. Chemical release of CalE8-bound polyketides

(A) Derivatized polyketides released upon cysteamine treatment of purified CalE8. HPLC traces (λ = 325 nm, gray; λ = 375 nm, black) of adducts extracted following treatment of CalE8 with 0.2M cysteamine are shown. (B) Extracted ion chromatograms, showing observed masses for 8a–e (ESI+). (C) Reaction cascade leading to cystamine adducts 8a–e. (D) Solvolysis and hydrolysis of polyketides bound to CalE8. HPLC traces (λ = 325 nm, gray; λ = 375 nm, black) of released products are shown; labelled peaks were confirmed by LCMS analysis. Within each panel, chromatogram intensity scales are identical to allow for a direct comparison.

Figure 3. The effect of light exposure on polyketides synthesized in vivo by CalE8 during heterologous expression

(A) PKS-bound intermediates that are carried by CalE8 through the purification process and released upon alkaline hydrolysis. Shown are HPLC traces (λ = 325 nm, gray; λ= 375 nm, black) of base hydrolysis products from CalE8 expressed for (i) 18 h under ambient light and (ii) 18 h in the dark. (B) Polyketides found as free metabolites in cultures expressing CalE8. Shown are HPLC traces (λ= 325 nm, gray; λ= 375 nm, black) of compounds extracted from clarified cell lysate from cultures expressing CalE8 for (i) 18 h under ambient light and (ii) 18 h in the dark. Gray boxes highlight notable effects of light exposure. Within each panel, chromatogram intensity scales are identical to allow for a direct comparison.

Figure 4. In vitro synthesis and accumulation of polyketides on CalE8

PKS-bound intermediates that are assembled on cysteamine-treated CalE8 during reconstitution reactions and released upon alkaline hydrolysis. Shown are HPLC traces (λ = 325 nm, gray; λ = 375 nm, black) of base hydrolysis products from in vitro reactions of CalE8 supplied with no substrate (trace i), 0.5 mM each MalCoA and NADPH (traces ii - iv), or 2 mM each MalCoA and NADPH (trace v). "N" = nonaketide overextension product. Chromatogram intensity scales are identical, allowing for a direct comparison.

Figure 5. The effect of light exposure and pre-treatment with cysteamine on in vitrosynthesis and accumulation of polyketides on CalE8

PKS-bound intermediates that are assembled on CalE8 during reconstitution reactions and released upon alkaline hydrolysis. Shown are HPLC traces (λ= 325 nm, gray; λ= 375 nm, black) of base hydrolysis products from in vitro reactions of CalE8 supplied with 0.5 mM each MalSNAc and NAPDH. Chromatogram intensity scales are identical, allowing for a direct comparison.

Scheme 1

A mechanistic proposal for the early steps of enediyne biosynthesis and divergence to the different subclasses. A radical pathway is suggested for this process, but cationic and electrocyclic mechanisms can be envisioned as well