Metamorphic enzyme assembly in polyketide diversification

Abstract

Natural product chemical diversity is fuelled by the emergence and ongoing evolution of biosynthetic pathways in secondary metabolism. However, co-evolution of enzymes for metabolic diversification is not well understood, especially at the biochemical level. Here, two parallel assemblies with an extraordinarily high sequence identity from Lyngbya majuscula form a beta-branched cyclopropane in the curacin A pathway (Cur), and a vinyl chloride group in the jamaicamide pathway (Jam). The components include a halogenase, a 3-hydroxy-3-methylglutaryl enzyme cassette for polyketide beta-branching, and an enoyl reductase domain. The halogenase from CurA, and the dehydratases (ECH(1)s), decarboxylases (ECH(2)s) and enoyl reductase domains from both Cur and Jam, were assessed biochemically to determine the mechanisms of cyclopropane and vinyl chloride formation. Unexpectedly, the polyketide beta-branching pathway was modified by introduction of a gamma-chlorination step on (S)-3-hydroxy-3-methylglutaryl mediated by Cur halogenase, a non-haem Fe(ii), alpha-ketoglutarate-dependent enzyme. In a divergent scheme, Cur ECH(2) was found to catalyse formation of the alpha,beta enoyl thioester, whereas Jam ECH(2) formed a vinyl chloride moiety by selectively generating the corresponding beta,gamma enoyl thioester of the 3-methyl-4-chloroglutaconyl decarboxylation product. Finally, the enoyl reductase domain of CurF specifically catalysed an unprecedented cyclopropanation on the chlorinated product of Cur ECH(2) instead of the canonical alpha,beta C = C saturation reaction. Thus, the combination of chlorination and polyketide beta-branching, coupled with mechanistic diversification of ECH(2) and enoyl reductase, leads to the formation of cyclopropane and vinyl chloride moieties. These results reveal a parallel interplay of evolutionary events in multienzyme systems leading to functional group diversity in secondary metabolites.

Figure 1. Comparison of enzyme assemblies in the Cur and Jam pathways

a, Formation of cyclopropane and vinyl chloride functional groups. b, Comparative sequence identities of the enzymes encoded by the two highly similar regions in the Cur and Jam pathways. The aligned DNA sequences are located at the boundaries of these two regions. c, Formation of 3-ACP₃ in the Cur pathway, and hypothesized reactions for 4-ACP₃, 5-ACP₃ and 6-ACP₃. The hypothetic chlorinated intermediates are shown along with the non-chlorinated ones. The β-branching carbon atoms are highlighted in red.

Figure 2. Halogenation and cyclopropanation in the Cur pathway

a-h, Partial FTICR mass spectra (12+ charge state of ACP_II) for Cur ECH₁, ECH₂ and ER reactions excluding (a-d) or including (e-h) the Cur Hal chlorination step. 1-ACP_II was incubated with Cur Hal for 2 h to generate the γ-Cl-1-ACP_II substrate. Reactions were incubated at 30°C for 2 h for the 1-ACP_II substrate and 30 min for the γ-Cl-1-ACP_II substrate. Asterisks denote unidentified species. i, GC-MS analysis of the enzyme products after butylamine cleavage, and comparison with authentic standards. For optimal sensitivity, the chromatograms were recorded at selective ion mode (SIM) by monitoring 55, 57, 83, 115, 155 and 157 atomic mass unit (amu). Retention times of the products were confirmed by coinjection with the authentic standards.

Figure 3. Comparison of ECH₂s and ERs in Cur and Jam pathways

a-f, Partial FTICR mass spectra (12+ charge state of ACP_II) for Cur and Jam ECH₁, ECH₂ and ER reactions with the γ-Cl-1-ACP_II substrate. The reactions were incubated at 30°C for 30 min. g, Comparison of catalytic efficiencies for cyclopropanation and saturation by Cur and Jam ERs. The product yields in the time-course studies were measured by IRMPD-based quantification. 3-ACP_II was used as substrate for Cur ER saturation, and γ-Cl-3-ACP_II was used as substrate for Cur ER cyclopropanation and Jam ER saturation. Assays were performed in triplicate, and standard deviation error bars are shown. h, GC-MS analysis to identify the structures of Cur and Jam ECH₂ products. The chromatograms were recorded at SIM by monitoring 57, 117, 154 and 189 amu. The retention times of products were confirmed by coinjection with the authentic standards.

Figure 4. Loss of Cur ECH₂-mediated regiochemical control by site-directed mutagenesis

a, The hypervariable region (in magenta) of Cur ECH₂ and the active site chamber modeled with the chlorinated substrate. The S-configuration of the HMG γ-carbon is preferred based on modeling results. b, Activity and regiochemical control of ECH₂ WT and Cur ECH₂ mutants. γ-Cl-1-ACP_II was used as the substrate for all reactions. (Left) HPLC analysis for ECH₁/ECH₂ coupled dehydration and decarboxylation. All reactions were quenched after 10 min incubation at 30°C. (Right) IRMPD-based quantification to measure the percentage of β,γ C=C products. The coupled ECH₁/ECH₂ reactions were incubated for 45 min before treated with Jam ER for 45 min at 30°C. Assays were performed in triplicate, and standard deviation error bars are shown.

Figure 5. Impact of enzyme assembly evolution on β-branching chemical diversity

a, Proposed ancestral forms of the enzyme assemblies in Cur and Jam pathways. b, The functional diversification of ERs. c, Differential regiochemical control by ECH₂s.