A Polyketide Cyclase That Forms Medium-Ring Lactones

Abstract

Medium-ring lactones are synthetically challenging due to unfavorable energetics involved in cyclization. We have discovered a thioesterase enzyme DcsB, from the decarestrictine C1 (1) biosynthetic pathway, that efficiently performs medium-ring lactonizations. DcsB shows broad substrate promiscuity toward linear substrates that vary in lengths and substituents, and is a potential biocatalyst for lactonization. X-ray crystal structure and computational analyses provide insights into the molecular basis of catalysis.

Figure 1.

Nonanolides and related natural products. (A) Compounds with 10-membered lactones. (B) Compounds that are proposed to be derived from 10-membered lactones.

Figure 2.

Fungal nonanolide biosynthesis. (A) The dcs and homologous biosynthetic gene clusters. The % sequence identities are shown in parenthesis; (B) Proposed biosynthetic pathway of 1. (C) LC-MS analysis of metabolites produced from expression of dcs genes in A. nidulans. (D) Assaying activity of DcsB using small molecule thioesters.

Figure 3.

Assaying the substrate promiscuity of DcsB. (A) Analogs 4b-4e used to probe functional group requirements; (B) simple alcohol-terminated substrates 4f-4l. ^a Isolated yield; ^b The cyclized product was not formed and only substrate hydrolysis was observed; ^c Yield determined by GC-MS analysis. Reaction conditions: 1 mM substrate, 0.5 μM DcsB except for 2k in which 2.4 μM DcsB was used.

Figure 4.

Crystal structures of DcsB with bound and modeled substrate complexes. (A) Overall structure of DcsB-substrate analogue 4a-2’ complex. The active site catalytic triad residues (S114, H276, and D247) are shown in blue sticks while the substrate analog 4a-2’ is shown in yellow. The β-hydroxyl group in 4a-2’ is epimerized compared to the natural substrate 4a-2 and is bound in an unproductive conformation; (B) Active site of DcsB with docked substrate 4a-2 that is shown in grey sticks. The thioester is 5.6 Å from the catalytic S114 residue; (C) Covalent docking of the Ser114-bound acyl-intermediate of 4a-2 shown in teal.