Regio- and stereodivergent antibiotic oxidative carbocyclizations catalysed by Rieske oxygenase-like enzymes

Abstract

Oxidative cyclizations, exemplified by the biosynthetic assembly of the penicillin nucleus from a tripeptide precursor, are arguably the most synthetically powerful implementation of C-H activation reactions in nature. Here, we show that Rieske oxygenase-like enzymes mediate regio- and stereodivergent oxidative cyclizations to form 10- and 12-membered carbocyclic rings in the key steps of the biosynthesis of the antibiotics streptorubin B and metacycloprodigiosin, respectively. These reactions represent the first examples of oxidative carbocyclizations catalysed by non-haem iron-dependent oxidases and define a novel type of catalytic activity for Rieske enzymes. A better understanding of how these enzymes achieve such remarkable regio- and stereocontrol in the functionalization of unactivated hydrocarbon chains will greatly facilitate the development of selective man-made C-H activation catalysts.

Figure 1

Key oxidative cyclization reactions in the biosynthesis of clinically-used natural products. Hydrogen atoms removed in the reactions are highlighted in blue and new bonds formed are highlighted in red. (A) Isopenicillin N synthase (IPNS) is a non-heme iron-dependent enzyme that catalyzes two oxidative cyclization reactions within the tripeptide 1 to form the bicyclic nucleus of isopenicillin N 2 at the expense of a molecule of oxygen. (B) Clavaminate synthase 2 (CAS2) is a non-heme iron-dependent enzyme that catalyzes the oxidative cyclization of the monocyclic β-lactam 3 to form (3S, 5S)-dihydroclavaminic acid 4, an intermediate in the biosynthesis of the bicyclic β-lactamase inhibitor clavulanic acid, utilizing a molecule of oxygen together with two electrons from α-ketoglutarate. (C) HppE is a non-heme iron dependent enzyme that catalyzes the oxidative cyclization of the β-hydroxyphosphonate 5 using molecular oxygen and two electrons from NADH to form the key biologically-active epoxide group in the antibiotic fosfomycin 6. (D) OxyB is a cytochrome P450 that has been shown to catalyze the oxidative cyclization of an acyl carrier protein-bound synthetic analogue of a nonribosomally biosynthesized peptide thioester to form an analogue of a key cross-linked vancomycin precursor at the expense of a molecule of oxygen and two electrons from NADPH.

Figure 2

Roles of Rieske non-heme iron-dependent oxygenases and oxygenase-like enzymes, together with associated enzymes, in the biosynthesis of natural products and the degradation of organic compounds. (A) Oxidative carbocyclization reactions proposed to be mediated by Rieske oxygenase-like enzymes in metacycloprodigiosin 7, streptorubin B 9, prodigiosin R1 10 and roseophilin 11 biosynthesis. The hydrogen atoms removed in the reactions are highlighted in blue and the new bonds formed are highlighted in red. A Rieske oxygenase is also proposed to effect the replacement of a nitrogen atom with an oxygen atom (highlighted in red) in roseophilin 11 biosynthesis. (B) Organization of the red gene cluster that directs streptorubin B 9 biosynthesis in S. coelicolor. The redG, redH and redI genes discussed in this study are highlighted in orange. The mcpG, mcpH and mcpI genes, which are involved in the biosynthesis of metacycloprodigiosin 7 and are homologues of redG, redH and redI, are found in the same relative order on the chromosome of S. longispororuber (highlighted in blue). (C) Role of RedH in undecylprodigiosin 8 biosynthesis. (D) Sequence alignment of the Rieske oxygenase-like enzymes RedG and McpG that mediate oxidative carbocyclization reactions in streptorubin B 9 and metacycloprodigiosin 7 biosynthesis, respectively, with the structurally-characterized Rieske oxygenase naphthalene dioxygenase (NDO). Conserved residues within RedG and McpG that ligate the [2Fe-2S] cluster and Fe(II) atom in NDO are highlighted in yellow. An Asp residue of NDO (mutated to Glu in RedG and McpG) proposed to mediate electron transfer between the [2Fe-2S] cluster and the Fe(II) atom is highlighted in green. (E) Typical reactions catalyzed by Rieske oxygenases. Naphthalene dioxygenase (NDO) catalyzes the dihydroxylation of naphthalene 14 to form 15 using molecular oxygen and two electrons derived from NADH. PrnD catalyzes the oxidation of an amino group in 16 to afford the nitro group in pyrrolnitrin 17 utilizing molecular oxygen and electrons from NADPH. Oxygen atoms derived from molecular oxygen that are introduced into the products are highlighted in red.

Figure 3

Data that elucidate the role of RedG and McpG in streptorubin B 9 and metacycloprodigiosin 7 biosynthesis, respectively. (A) Extracted ion chromatograms (EICs) for m/z range 392–394 from LC-MS analyses in positive ion mode of extracts of S. coelicolor M511 (top trace), a ΔredG mutant of S. coelicolor M511 (middle trace) and the ΔredG mutant expressing redG in trans (bottom trace). (B) EICs for m/z range 392–394 from LC-MS analyses of extracts of S. venezuelae fed with MBC 12 and 2-undecylpyrrole 13 (bottom trace) and S. venezuelae expressing redH and redG fed with MBC 12 and 2-undecylpyrrole 13 (top trace). (C) EIC for m/z 380 (top trace), m/z 378 (middle trace) and m/z range 392–394 (bottom trace) from LC-MS analyses of extracts of a redI::oriT-apr mutant of S. coelicolor. (D) EICs for m/z range 392–394 from LC-MS analyses of extracts of S. venezuelae (bottom trace) and S. venezuelae expressing redH and redG (top trace), both fed with synthetic undecylprodigiosin 8. (E) EICs for m/z range 392–394 from LCMS analyses of extracts of S. coelicolor ΔredG mutant expressing mcpG in trans. (F) Top: CD spectra of streptorubin B 9 (red) and metacycloprodigiosin 7 (blue). Bottom: CD spectrum of the cyclic derivative of undecylprodigiosin isolated from the S. coelicolor ΔredG mutant expressing mcpG in trans (black). (G) Diagnostic regions of the ¹H NMR spectra of streptorubin B 9 (top trace) metacycloprodigiosin 7 (middle trace) and the cyclic derivative of undecylprodigiosin isolated from the S. coelicolor ΔredG mutant expressing mcpG in trans (bottom trace).