Biochemical Characterization of a Eukaryotic Decalin-Forming Diels-Alderase

Abstract

The trans-decalin structure formed by intramolecular Diels-Alder cycloaddition is widely present among bioactive natural products isolated from fungi. We elucidated the concise three-enzyme biosynthetic pathway of the cytotoxic myceliothermophin and biochemically characterized the Diels-Alderase that catalyzes the formation of trans-decalin from an acyclic substrate. Computational studies of the reaction mechanism rationalize both the substrate and stereoselectivity of the enzyme.

Figure 1

Structures of decalin containing fungal polyketides and polyketide-amino acid hybrids.

Figure 2

Verification of the myceliothermophin gene cluster. (A) The myc cluster encodes the PKS-NRPS MycA (domain abbreviations: KS: ketosynthase; AT: acyltransferase; DH: dehydratase; MT: methyltransferase; KR: ketoreductase; ACP: acyl carrier protein; C: condensation; A: adenylation; PCP: peptidyl carrier protein; and R: reductase); the in trans enoylreductase (ER) MycC; and the putative DAase MycB; (B) Product profile of wild type and single–gene knockout strains of M. thermophila; and that of A. nidulans transformed with combinations of myc genes; (C) structures of metabolites isolated. The stereochemistries of 3, 4 and 5 are assumed to be similar to 1.

Figure 3

Activity of the DAase MycB. (A) MycB can catalyze the cycloaddition of the ketone 5 to 1, but is not reactive towards the enol 3. (B) Time course analysis of the conversion of 5 to 1. The starting concentrations for 5 and MycB are 200 µM and 1 µM, respectively. (C) Saturation kinetics of MycB. Each data point performed in triplicate.

Figure 4

The proposed biosynthetic pathway of 1 based on isolated natural products and biochemical characterization of MycB. DFT analysis were performed on model substrates (1’–8’) of which the isobutyl group in 1–8 is replaced by a methyl group. Calculations were performed at the CPCM (water)/M06-2X/6-311+G(d,p)//M06-2X/6-31G(d) level of theory. The computed free energies of model compounds are shown in parenthesis. Calculations of stereoselectivity under uncatalyzed and catalyzed conditions are shown in the dash box.