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. 2019 Apr 10;141(14):5659-5663.
doi: 10.1021/jacs.9b02204. Epub 2019 Mar 26.

Enzyme-Catalyzed Inverse-Electron Demand Diels-Alder Reaction in the Biosynthesis of Antifungal Ilicicolin H

Zhuan Zhang, Cooper S Jamieson, Yi-Lei Zhao  1 , Dehai Li  2 , Masao Ohashi, K N Houk, Yi Tang
Affiliations

Enzyme-Catalyzed Inverse-Electron Demand Diels-Alder Reaction in the Biosynthesis of Antifungal Ilicicolin H

Zhuan Zhang et al. J Am Chem Soc. .

Abstract

The pericyclases are a growing superfamily of enzymes that catalyze pericyclic reactions. We report a pericyclase IccD catalyzing an inverse-electron demand Diels-Alder (IEDDA) reaction with a rate acceleration of 3 × 105-fold in the biosynthesis of fungal natural product ilicicolin H. We demonstrate IccD is highly periselective toward the IEDDA cycloaddition over a competing normal electron demand Diels-Alder (NEDDA) reaction from an ambimodal transition state. A predicted flavoenzyme IccE was identified to epimerize the IEDDA product 8- epi-ilicicolin H to ilicicolin H, a step that is critical for the observed antifungal activity of ilicicolin H. Our results reveal the ilicicolin H biosynthetic pathway and add to the collection of pericyclic reactions that are catalyzed by pericyclases.

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Figures

Figure 1
Figure 1
NEDDA and IEDDA reactions. (A) General scheme of both reactions. EWG: electron withdrawing group; EDG: electron donating group; (B) Proposed enzyme-catalyzed IEDDA reaction in ilicicolin H (1) biosynthesis, as demonstrated in total synthetic effort towards 1.
Figure 2
Figure 2
Characterization of ilicicolin H (1) biosynthesis. A) The icc cluster encodes a PKS-NRPS (KS-AT-DH-MT-KR-ERo-ACP-C-A-T-R. KS, ketosynthase; AT, acyltransferase; DH, dehydratase; MT, methyltransferase; KR, ketoreductase; ACP, acyl carrier protein; C, condensation; A, adenylation; PCP, peptidyl carrier protein; R, reductase) IccA, a trans-ER IccB, a ring expansion P450 IccC, a putative C-methyltransferase IccD and a putative old yellow enzyme IccE; B) Product profiles from heterologous expression of different combinations of icc cluster in A. nidulans A1145. Control in trace i is A. nidulans transformed with empty vectors only; C) Proposed biosynthetic pathway of 1; D) In vitro characterization of IccE-catalyzed epimerization. The assays were conducted in 50 mM Tris-HCl at pH 7.0, in the presence of 0.2 mM 2 and 0.1 μM IccE.
Figure 3
Figure 3
Characterization of IccD-catalyzed pericyclic reaction. (A) HPLC profile of time-course reaction of IccD. The assays were conducted at 28°C from 0 to 120 min; (B) Comparison of relative IEDDA and NEDDA product ratio starting from 4; (C) Calculated reaction surface energies and dynamics results from 4 converting to 2 and 5. RDY: reaction dynamics yield from TS-1.
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