Genome-Mined Diels-Alderase Catalyzes Formation of the cis-Octahydrodecalins of Varicidin A and B

Abstract

Pericyclases are an emerging family of enzymes catalyzing pericyclic reactions. A class of lipocalin-like enzymes recently characterized as Diels-Alderases (DAses) catalyze decalin formation through intramolecular Diels-Alder (IMDA) reactions between electron-rich dienes and electron-deficient dienophiles. Using this class of enzyme as a beacon for genome mining, we discovered a biosynthetic gene cluster from Penicillium variabile and identified that it encodes for the biosynthesis of varicidin A (1), a new antifungal natural product containing a cis-octahydrodecalin core. Biochemical analysis reveals a carboxylative deactivation strategy used in varicidin biosynthesis to suppress the nonenzymatic IMDA reaction of an early acyclic intermediate that favors trans-decalin formation. A P450 oxidizes the reactive intermediate to yield a relatively unreactive combination of an electron-deficient diene and an electron-deficient dienophile. The DAse PvhB catalyzes the final stage IMDA on the carboxylated intermediate to form the cis-decalin that is important for the antifungal activity.

Figure 1.

Lipocalin-like DAses catalyzes IMDA reactions in biosynthesis of decalin-containing fungal NPs. (A) Characterized DAses and their decalin containing products; (B) Genome mining of a DAse (PvhB) containing cryptic gene cluster from P. variable. Abbreviations: KS, ketosynthase; MAT, malonyl-CoA transferase; DH, dehydratase; MT, methyltransferase; KR, ketoreductase; ACP, acyl carrier protein; C, condensation; A, adenylation; T, thiolation; R, reductase. MFS: major facilitator superfamily; TF: transcription factor; N-MT: N-methyltransferase; ER: enoylreductase; DAse: Diels-Alderase.

Figure 2.

Biosynthesis of varicidin A 1. (A) Product profiles of A. nidulans transformed with combinations of pvh genes; (B) The proposed biosynthetic pathway of 1; (C) Biochemical characterization of the DAse PvhB in vitro. The traces are HPLC with λ = 280 nm. (D) Crystal structure of 2 (see CIF file as supporting information).

Figure 3.

DFT calculated transition states of NEDDA and IEDDA. (A) Transition state structures TS-1 and TS-2 with energies shown for nonenzymatic endo-cyclizations of 3 forming 4 and 5, respectively. (B) endo TS-3 and exo TS-4 of IEDDA reactions in solution as dianions. TS-4 leads to formation of the cis-decalin stereochemistry for varicidin A. (C) endo TS-5 and exo TS-6 of NEDDA reactions when tetramic acid is protonated.