Discovery and Mechanism of a Diiron Enzyme in Ethylidene Azetidine Formation

Abstract

Azetidine is a strained four-membered N-heterocycle that has important applications in medicinal chemistry and organic synthesis. Despite its relevance to human health and agriculture, azetidine biosynthesis remains largely unexplored. Herein, the DUF6202 family enzyme PolF is shown to utilize a diiron center and O₂ to catalyze sequential desaturation and azetidination of l-isoleucine to yield the ethylidene azetidine moiety during the biosynthesis of polyoxin. Crystallographic analysis of PolF in complex with iron (or manganese) and l-isoleucine (or an alkene intermediate) reveals that it is a heme oxygenase-like diiron oxidase with a new iron-binding motif. Trapping an O₂-bound intermediate in crystallo along with a distinctive iron-to-protein ratio observed under anoxic and aerobic environments indicates that binding of the second iron occurs in an O₂-dependent manner. Mössbauer and stopped-flow optical absorption spectroscopy together with deuterium kinetic isotope effect measurements demonstrate that a μ-peroxodiiron(III) species is likely responsible for C-H bond activation. Mass Spectrometry analysis reveals that the decay of the μ-peroxodiiron(III) species is accompanied by the formation of a desaturated intermediate. Bioinformatic analysis identifies ca. 200 PolF homologues indicative of new biosynthetic pathways for azetidine-containing natural products.

Figure 1.. Azetidine ring biosynthesis proceeds through at least two mechanistically distinct pathways.

(A) Intramolecular cyclization of SAM results in azetidine-2-carboxylic acid formation. (B) The diiron containing enzyme PolF catalyzes desaturation followed by cyclization to furnish polyoximic acid (this study), which was originally proposed to be installed via the action of three enzymes PolC/E/F. Unlike the reaction catalyzed by AzeJ/VioH, which relies on the electrophilicity of the γ-carbon of the methylene moiety in SAM, the reaction catalyzed by PolF utilizes a redox-active diiron center for C–H activation during construction of the strained heterocycle in polyoximic acid.

Figure 2.. Biochemical characterization of the PolF catalyzed reaction.

(A) Liquid chromatography-mass spectrometry (LC-MS) analysis of enzymatic reactions demonstrated that PolF alone, PolC/PolF or PolC/PolE/PolF catalyzed the conversion of L-isoleucine into polyoximic acid (1), whereas assays with PolC/PolE did not yield this product. (B) The activity of PolF was found to depend on both iron(II) and molecular oxygen (O₂). A very weak 1-Dns peak was detected in reactions conducted without O₂, likely due to trace amounts of O₂ present in the glove box. (C) The PolF reaction was worked up using dansyl derivatization. (D) LC-MS analysis demonstrating that PolF accepts l-Ile, d-Ile, l-Val, and d-Val as substrates leading to the formation of the corresponding alkene azetidines. (E) The structures of the products from the enzymatic reactions were elucidated through detailed NMR analysis.

Figure 3.. Structure and multiple sequence alignment of PolF with other HDO enzymes.

(A) Asymmetric unit of the PolF crystal structure. (B) Overall structure of chain B, showing binding of two irons and the substrate l-isoleucine (PDB code: 9PJ2). (C) Active site of chain B. The 2F_o–F_c electron density map (gray mesh) is contoured at 1.3σ and the anomalous difference density map (orange mesh) is contoured at 3.0σ. (D) Multiple sequence alignment of PolF with other known HDO enzymes. Residues involved in coordinating Fe1 and Fe2 in PolF are highlighted in pink and blue, respectively. The glutamate residue highlighted in gray is conserved and used for Fe1 binding in other HDO enzymes, but is not involved in iron coordination in PolF. (E) Relative activity of PolF mutants targeting residues in the diiron-binding motif. The activity was defined by formation of product 1 in 2 h with substrate l-isoleucine. Error bars indicate the standard deviation from two independent measurements. (F) Active site of PolF in the presence of intermediate D-6 and manganese (PDB code: 9PJ3). The 2F_o–F_c electron density map (gray mesh) is contoured at 1.3σ and the anomalous difference density map (orange mesh) is contoured at 3.0σ.

Figure 4.. Spectroscopic evidence of formation of a peroxo-diferric intermediate in the PolF catalyzed reaction.

(A) Optical absorption spectra of the PolF reaction with O₂ in the presence of l-isoleucine at selected reaction time points. (B) The 605 nm kinetic traces of the PolF reaction with O₂ in the presence of l-isoleucine (black) or d₁₀-l-isoleucine (blue). (C) The 605 nm kinetic traces using l-valine (black) or d-valine (blue). In (B) and (C), the dashed lines are simulations using the three-step kinetic model, see details in the SI. (D) Freeze quench Mössbauer spectra (grey vertical bars) recorded on the samples generated at selected time points in the reaction with d₁₀-l-isoleucine. The black lines are overall simulations, and the blue lines are the simulations of the μ-1,2-peroxo-diferric intermediate. See Figure S47 for the full Mössbauer data and Tables S6–S7 for the Mössbauer simulation parameters and time dependent iron speciation.

Figure 5.. Identification of a reaction intermediate in the PolF catalyzed reaction.

(A) Quantification of p2 in chemical-quench samples by LC-MS (see LC-MS traces in Fig. S22). (B) Chemical structures of 3–6 produced in the PolF catalyzed reaction with l-valine as the substrate. (C) LC-MS analysis of samples quenched at 300 s showing the production of compounds 3, 4, 6 and 7 when l-valine is used as the substrate. Samples have been derivatized with Marfey’s reagent 1-fluoro-2,4-dinitrophenyl-5-l-alanine amide (FDAA). (D) Of the tested intermediates (i.e., l-3, l-4, l-5 and l-6), only l-6 was converted to the final product 2a.

Figure 6.

Proposed mechanism for PolF catalyzed sequential desaturation and azetidination.