Biosynthesis of the Unusual Carbon Skeleton of Nocuolin A

Abstract

Nocuolin A is a cytotoxic cyanobacterial metabolite that is proposed to be produced by enzymes of the noc biosynthetic gene cluster. Nocuolin A features a 1,2,3-oxadiazine moiety, a structural feature unique among natural products and, so far, inaccessible through organic synthesis, suggesting that novel enzymatic chemistry might be involved in its biosynthesis. This heterocycle is substituted with two alkyl chains and a 3-hydroxypropanoyl moiety. We report here our efforts to elucidate the origin of the carbon skeleton of nocuolin A. Supplementation of cyanobacterial cultures with stable isotope-labeled fatty acids revealed that the central C₁₃ chain is assembled from two medium-chain fatty acids, hexanoic and octanoic acids. Using biochemical assays, we show that a fatty acyl-AMP ligase, NocH, activates both fatty acids as acyl adenylates, which are loaded onto an acyl carrier protein domain and undergo a nondecarboxylative Claisen condensation catalyzed by the ketosynthase NocG. This enzyme is part of a phylogenetically well-defined clade within similar genomic contexts. NocG presents a unique combination of characteristics found in other ketosynthases, namely in terms of substrate specificity and reactivity. Further supplementation experiments indicate that the 3-hydroxypropanoyl moiety of 1 originates from methionine, through an as-yet-uncharacterized mechanism. This work provides ample biochemical evidence connecting the putative noc biosynthetic gene cluster to nocuolin A and identifies the origin of all its carbon atoms, setting the stage for elucidation of its unusual biosynthetic chemistry.

Figure 1

Schematic highlighting the structure of nocuolin A, the noc BGC, and the proposed biosynthetic steps. (a) Chemical structures of nocuolin A (1) and geralcin D, the natural product with the closest structural resemblance to the nocuolin A scaffold. (b) noc locus and its proposed relationship with nocuolin A (1), the chlorosphaerolactylates (e.g., 2) and nocuolactylates (e.g., 3). (c) Representation of the proposed biosynthesis of 1. (d) Detailed annotation of noc-encoded biosynthetic enzymes (some have previously been included in the cly BGC) with a potential role in the biosynthesis of 1.

Figure 2

Supplementation of Nodularia sp. LEGE06071 with d₁₁-hexanoic acid and d₁₅-octanoic acid leads to d₁₁-1/d₂₂-1 and d₁₃-1, respectively. LC-HRMS-derived extracted ion chromatograms (EICs) of 1 and labeled version with (a) d₁₁-hexanoic acid and (b) d₁₅-octanoic acid in Nodularia sp. LEGE 06071. (c) HRMS/MS spectrum of d₁₃-1 and 1 ([M + H]⁺) highlighting the diagnostic fragmentations supporting the structural proposal.

Figure 3

FAALs NocH and ClyA (NocM) activate hexanoic/octanoic acids and dodecanoic acid as acyl-ACP thioesters, respectively, in competition assays. Shown are LC-HRMS-derived EICs of a trypsinated ACP fragment loaded with each fatty acid.

Figure 4

NocG belongs to a new class of KSs. (a) LC-HRMS-derived EICs of the β-ketoacid 4 and ketone 5 produced by NocG upon enzymatic assays with hexanoyl- and octanoyl-thioesters. (b) LC-HRMS-derived EICs of 6 upon reduction of 4 with sodium borohydride proving the nondecarboxylative nature of NocG reaction. (c) Phylogenetic tree composed of NocG (*), its homologs and other known ketosynthases (the scale bar indicates the degree of divergence as substitutions per site).

Figure 5

3-Hydroxypropanoyl moiety of 1 is derived from C-2 to C-4 and N-1 of l-methionine. (a) HRMS spectra of 1 and its stable isotope labeled versions following supplementation with d₈-l-methionine, ¹³C₅-l-methionine, and ¹⁵N,¹³C₅-l-methionine, confirming incorporation of this precursor into 1. (b) HRMS/MS spectrum of ¹³C₃-1 confirming methionine incorporation into the 3-hydroxypropanoyl moiety. (c) Fine structure of the M peak in the isotope clusters of [M + H]⁺ ions of key isotopologues of 1 (values next to each peak correspond to peak areas, and their ratios are tabulated), supporting incorporation of a nitrogen atom from methionine into 1. (d) HRMS spectra of 1 and its stable isotope labeled versions following supplementation with 1-¹³C-l-methionine, supporting a nonspecific incorporation of C-1 from l-methionine. (e) Schematic representation of incorporation of C and N atom-derived l-methionine into the 3-hydroxypropanoyl moiety of 1.