AvmM catalyses macrocyclization through dehydration/Michael-type addition in alchivemycin A biosynthesis

Abstract

Macrocyclization is an important process that affords morphed scaffold in biosynthesis of bioactive natural products. Nature has adapted diverse biosynthetic strategies to form macrocycles. In this work, we report the identification and characterization of a small enzyme AvmM that can catalyze the construction of a 16-membered macrocyclic ring in the biosynthesis of alchivemycin A (1). We show through in vivo gene deletion, in vitro biochemical assay and isotope labelling experiments that AvmM catalyzes tandem dehydration and Michael-type addition to generate the core scaffold of 1. Mechanistic studies by crystallography, DFT calculations and MD simulations of AvmM reveal that the reactions are achieved with assistance from the special tenuazonic acid like moiety of substrate. Our results thus uncover an uncharacterized macrocyclization strategy in natural product biosynthesis.

Fig. 1. Macrocyclic reactions involved in natural product biosynthesis.

a Representative macrocyclic natural products formed through diverse strategies. New bonds formed through macrocyclizations are highlighted in red. The macrocycles are formed by TE-tethering in daptomycin and 6-deoxyerythronolide B, a cytochrome P450 in vancomycin, a rieske oxygenase in metacloprodigiosin, a radical SAM in streptide, a dual function enzyme catalyzing an amide oxidation and Mannich reaction in lankacidin C, a Diels-Alderase in versipelostatin, and a new enzyme catalyzing a β-elimination and Michael addition in alchivemycin A. b Proposed macrocyclic mechanism for AVM (1). The configuration of C23 hydroxyl group was proposed based on sequence alignment of the corresponding KR domain and the existence of conserved “W” motif. The cis-geometry of C23-24 double bond was confirmed by the coupling constant.

Fig. 2. Characterization of AvmM as a macrocyclase.

a HPLC analysis of metabolic extracts from S. sp. TP-A0867 wild-type and mutant strains. b In vitro enzyme assays of 3 or 4 incubated with the wild-type or mutant AvmM constructs. The conversion rates from 3 or 4 to 2 are as follow: ii) ~90%; iii) 0%; iv) ~100%; v) 0%; vi) ~11%; vii) 15%; viii) ~100%. c Time course in vitro assays of 3 with L182A mutant AvmM. The newly identified intermediate 4 is colored as red. d LC-MS comparison of 2 and D₂O labeled ²H-2. e Proton NMR comparison of 2 and D₂O labeled ²H-2. These experiments are repeated at least twice with similar results.

Fig. 3. Crystal structures of AvmM.

a Cartoon representations of the homotrimeric AvmM. b Close-up active-site view of co-crystal structure of the AvmM active site with product 2. The hydrogen bonding and π–π stacking interaction are presented by gold dash line and green dash line. c Relative activity of AvmM and its site-specific mutants on enzymatic reactions. n = 3 biologically independent experiments; Data are presented as mean values + /− SD and error bars represent SD (standard deviation).

Fig. 4. MD and DFT analysis.

a, b Close-up view of MD representative snapshots of AvmM active site complexed with 3 and 4. Water molecules are shown in red spheres and hydrogen bonds are in gold dash lines. c DFT-computed Gibbs free energies (in kcal mol⁻¹) at the CPCM(water)-B3LYP-D3/6-311 + +G(2d,p)//CPCM(water)-B3LYP-D3/6-31 + G(d) level of theory and transition state structures (carbon: gray, hydrogen: white, oxygen: red, nitrogen: blue, and distances are shown in Å).

Fig. 5. Proposed reaction mechanism for AvmM.

The bold dashed line indicates the putative hydrogen bonds. E104 and H108 are linked by three water molecules and only one is shown for clarity.