Flavin-linked oxidase catalyzes pyrrolizine formation of dichloropyrrole-containing polyketide extender unit in chlorizidine A

Abstract

The marine alkaloid chlorizidine A contains chlorinated pyrroloisoindolone and pyrrolizine rings that are rare chemical features in bacterial natural products. Herein, we report the biosynthetic logic of their construction in Streptomyces sp. CNH-287 based on the identification of the chlorizidine A biosynthetic gene cluster. Using whole pathway heterologous expression and genetic manipulations, we show that chlorizidine A is assembled by a polyketide synthase that uniquely incorporates a fatty acid synthase-derived dichloropyrrolyl extender unit into the pyrroloisoindolone enzymatic product. We further provide the first biochemical characterization of a flavoenzyme associated with the oxidative formation of chlorizidine A's distinctive pyrrolizine ring. This work illuminates new enzymatic assembly line processes leading to rare nitrogen-containing rings in nature.

Figure 1

Structures of chlorizidine A (1) and other dichloropyrrole-containing natural products and their general biogeneses from a common intermediate. The full numbering scheme of 1 is shown in SI (page S11).

Figure 2

Molecular basis for chlorizidine A biosynthesis. (a) Gene organization of the clz biosynthetic cluster from Streptomyces sp. CNH-287. See Table S1 for proposed function assignments. (b) Proposed polyketide biosynthesis of chlorizidine A (1). The conversion of 4 to the Clz6 AT-2 substrate 8 is proposed to involve a divergent FAS pathway (blue) via the FabF ketosynthase Clz11 and primary FAS reducing enzymes encoded elsewhere in the genome. Compounds 5a and 7a correspond to the free carboxylic acid of intermediates 5 and 7, respectively. Abbreviations: ACP, acyl carrier protein; AT, acyl transferase; CoA, coenzyme A; DH, dehydratase; ER, enoyl reductase; KR, ketoreductase; KS, ketoacyl synthase; R, thioester reductase.

Figure 3

HPLC profiles at 254 nm of the in vitro conversion of 10 to 1 catalyzed by Clz9. (a) Control reaction using heat-inactivated enzyme. (b) 15 min reaction. (c) 16 hr reaction.

Scheme 1

Proposed mechanisms for the stereoselective cyclization of 10 to (S)-1. Deprotonotation of the phenolic hydroxyl facilitates abstraction of the hydride by the FAD cofactor and generation of the intermediate 11. Further nucleophilic attack from the pyrrole-nitrogen yields (S)-1. The presence of molecular oxygen can restore the reduced FADH₂ cofactor.