Enzymatic spiroketal formation via oxidative rearrangement of pentangular polyketides

Abstract

The structural complexity and bioactivity of natural products often depend on enzymatic redox tailoring steps. This is exemplified by the generation of the bisbenzannulated [5,6]-spiroketal pharmacophore in the bacterial rubromycin family of aromatic polyketides, which exhibit a wide array of bioactivities such as the inhibition of HIV reverse transcriptase or DNA helicase. Here we elucidate the complex flavoenzyme-driven formation of the rubromycin pharmacophore that is markedly distinct from conventional (bio)synthetic strategies for spiroketal formation. Accordingly, a polycyclic aromatic precursor undergoes extensive enzymatic oxidative rearrangement catalyzed by two flavoprotein monooxygenases and a flavoprotein oxidase that ultimately results in a drastic distortion of the carbon skeleton. The one-pot in vitro reconstitution of the key enzymatic steps as well as the comprehensive characterization of reactive intermediates allow to unravel the intricate underlying reactions, during which four carbon-carbon bonds are broken and two CO₂ become eliminated. This work provides detailed insight into perplexing redox tailoring enzymology that sets the stage for the (chemo)enzymatic production and bioengineering of bioactive spiroketal-containing polyketides.

Fig. 1. Overview of the proposed biosynthesis of bacterial rubromycin-type polyketides and final pathway products.

a Griseorhodin A biosynthetic gene cluster encoding, e.g., the minimal type II polyketide synthase (PKS), cyclases, and tailoring enzymes. b Initial steps afford a reactive acyl-carrier protein (ACP)-bound poly-β-ketone, which is subsequently cyclized and modified to 3. Compounds 3 and 11 were previously identified in the course of gene deletion experiments (ΔgrhO5 and ΔgrhO6, respectively, encoding flavin-dependent tailoring enzymes investigated in this work) and assigned as putative advanced intermediates. The conversion of 3 into 4 via 8 and 11 (dashed box) and additional intermediates was elucidated in this work. A ketoreductase (presumably GrhO10) then converts 4 into 13. c Examples of mature rubromycins likely formed from 13.

Fig. 2. Enzyme assays with substrate 3 in presence of O₂ (shown are the RP-HPLC chromatograms at λ = 254 nm).

Incubation times before reaction quenching are indicated to the left. The respective enzyme and cofactor composition is shown to the right. No conversion of 3 was observed in control assays lacking NADPH (trace 1) or GrhO5 (trace 2). Traces 3–8 show time points from the same, discontinuous assay with GrhO5, in which shunt product 9 accumulated aside from intermediate 10. Addition of GrhO1 boosted 10 formation and counteracted 9 formation (trace 9). Addition of GrhO6 (traces 10 and 11) lead to conversion of 10 into 4 (that rapidly forms ring-opened 12). Note that 7 is not observed due to poor separation resulting from polymerization and irreversible binding. The structures of the intermediates are shown above. The proposed enzymatic steps are presented in Fig. 3. All assays were at least conducted three times independently and representative examples are shown (for uncropped chromatograms, see Supplementary Fig. 8).

Fig. 3. Proposed spiroketal formation in rubromycin polyketide biosynthesis.

Redox steps catalyzed by GrhO5, GrhO1, and GrhO6 are highlighted with the respective enzyme symbols. Other steps may be catalyzed or occur spontaneously in the enzymes’ active sites, see text for details. The rings A–E of compounds that undergo modification in the ensuing biosynthetic step are color-coded according to the final product 4 (bottom left); important carbons are numbered for each step. Note that the carbon numbering of intermediates up to 8 is according to compound 3, whereas different carbon numberings are used for 10/11 and the final product 4. Oxygen atoms derived from O₂ and H₂O (based on isotope-labeling experiments) are highlighted in red and blue, respectively. Water-derived ¹⁸O is most likely also incorporated from spontaneous and reversible keto hydration as shown in step VI (gray arrows). a Proposed GrhO5-mediated conversion of 3 into 8. For secocollinone detection and structural characterization by NMR, derivatization to 6 by dimethyl sulfate (DMS) was required (gray dashed box). b Formation of on-pathway intermediate 10 (black arrows) and shunt product 9 (gray arrows) from 8. GrhO1 boosts 10 formation, while 9 production is minimized (see Fig. 2). c Proposed GrhO6-catalyzed formation of 4 from 10/11. Presumably, 3 and 11 are largely skipped as intermediates in the reducing environment of the cell. Dashed arrows indicate autooxidation steps. Electron-dependent steps (indicated by 2[H]) require NADPH as preferred reductant for both GrhO5 and GrhO6, see text. The spiroketal-configuration of 11 (racemic) and 4 (chiral) were analyzed by CD spectroscopy, see text. All intermediates in boxes were characterized by NMR and/or HRMS. Note that the final product 4 spontaneously converts to 12 (see below).

Fig. 4. Chemical preparation of 4 from 13 (isolated from S. albus KR7).

Synthesized 4 converts into 12 in aqueous solution at neutral pH, as also observed in the enzyme assays (Fig. 2). Note that the structure of 12 is supported by NMR and IR data, as well as DFT calculations, but a C6-ketohydrate instead of the shown ring-opened compound 12 cannot be ruled out (see Supplementary Fig. 48). Enzymatic steps are indicated with dashed arrows, non-enzymatic chemical steps with bold arrows. A ketoreductase may deter water addition in vivo en route to 1. The stereochemical configuration of 13 and 4/12 is depicted as previously determined for 1 and verified in this work. DMP: Dess–Martin periodinane, DCM: dichloromethane.

Fig. 5. Gene cluster comparison performed with MultiGeneBlast.

Same colors indicate high amino acid sequence similarities and predicted similar functions. The biosynthetic gene clusters for production of the spiroketal-containing compounds griseorhodin A (1, AF509565), heliquinomycin (NZ_SUMB01000006), hyaluromycin (NZ_BCFL01000018), and rubromycin (2, AF293355) are shown. These clusters harbor grhO5 (green), grhO1 (blue), and grhO6 (green) homologs. Corresponding homologs are connected with a shadow and the percentages of the amino acid sequence identities of the encoded proteins are shown. The grhO8 and grhO9 genes also show homology to grhO5 and grhO6 and encode FAD-dependent monooxygenases involved in early redox tailoring steps. The biosynthetic gene clusters of the non-spiroketal pentangular polyketides lack grhO1, grhO5, and grhO6 gene candidates necessary for spiroketal formation (see Supplementary Fig. 51 for full comparison).