Stilbene epoxidation and detoxification in a Photorhabdus luminescens-nematode symbiosis

Abstract

Members of the gammaproteobacterial Photorhabdus genus share mutualistic relationships with Heterorhabditis nematodes, and the pairs infect a wide swath of insect larvae. Photorhabdus species produce a family of stilbenes, with two major components being 3,5-dihydroxy-4-isopropyl-trans-stilbene (compound 1) and its stilbene epoxide (compound 2). This family of molecules harbors antimicrobial and immunosuppressive activities, and its pathway is responsible for producing a nematode "food signal" involved in nematode development. However, stilbene epoxidation biosynthesis and its biological roles remain unknown. Here, we identified an orphan protein (Plu2236) from Photorhabdus luminescens that catalyzes stilbene epoxidation. Structural, mutational, and biochemical analyses confirmed the enzyme adopts a fold common to FAD-dependent monooxygenases, contains a tightly bound FAD prosthetic group, and is required for the stereoselective epoxidation of compounds 1 and 2. The epoxidase gene was dispensable in a nematode-infective juvenile recovery assay, indicating the oxidized compound is not required for the food signal. The epoxide exhibited reduced cytotoxicity toward its producer, suggesting this may be a natural route for intracellular detoxification. In an insect infection model, we also observed two stilbene-derived metabolites that were dependent on the epoxidase. NMR, computational, and chemical degradation studies established their structures as new stilbene-l-proline conjugates, prolbenes A (compound 3) and B (compound 4). The prolbenes lacked immunosuppressive and antimicrobial activities compared with their stilbene substrates, suggesting a metabolite attenuation mechanism in the animal model. Collectively, our studies provide a structural view for stereoselective stilbene epoxidation and functionalization in an invertebrate animal infection model and provide new insights into stilbene cellular detoxification.

Keywords: Gram-negative bacteria; crystal structure; natural product; secondary metabolism; symbiosis.

Figure 1.

X-ray crystallographic structure of FAD-dependent epoxidase. A, schematic representation of overall fold of PlFMO with the FAD and NADPH domains shown in salmon and cyan, respectively, along with the long helix α11, stabilizing the FAD and NADPH domains, shown in red. The β-strands, α-helices, and 3₁₀-helices are marked sequentially in black, blue, and pink numbers, respectively. B, mF_o − DF_c omit-map shown at a contour level of 2.5σ along with the final refined model of FAD. C, LIGPLOT representation, created using the PDBsum (53) server, showing both hydrogen bonding and hydrophobic interactions between the FAD and PlFMO. D, superposition of PlFMO (PDB code 4HB9) and BtTetX (PDB code 2XDO) structures using the DALI server (54). A schematic representation of PlFMO is colored as in D and that of BtTetX is colored in gray. All images were created using the PyMOL molecular graphics system, version 1.5 Schrödinger, LLC.

Figure 2.

HPLC/MS analysis for production of stilbene 1 and stilbene epoxide 2. A, in P. luminescens culture broth (top, P. luminescens WT; bottom, Δplu2236). B, in G. mellonella insect infected with P. luminescens (top, P. luminescens WT-infected G. mellonella; bottom, Δplu2236-infected G. mellonella). The 10 mg of crude ethyl acetate-soluble materials were dissolved in 200 μl of methanol, and 10 μl of samples were subsequently injected for HPLC/MS analysis. The m/z 255 and m/z 271 correspond to protonated molecular ions of 1 and 2 in the positive ion mode, respectively. Peak intensity of the metabolites was determined by extracted ion counts in the same scale of y axis. Shown in C are the chemical structures of the P. luminescens metabolites, stilbene (1), and stilbene epoxide (2), respectively.

Figure 3.

In vitro reconstitution of PlFMO. A, UV-visible spectra of purified PlFMO (red) and a free FAD standard (blue). B and C, in vitro production of the stilbene epoxide (2) in the presence of NADPH (B) or NADH (C), respectively. Intensity of the peak shown in B and C was determined by ion extraction and is shown on the same scale, with m/z 271.1334 corresponding to stilbene epoxide (2) within a 10 ppm window, and intensity of standard 2 indicates 2 × 10⁷ counts. All experiments were prepared in triplicate. HPLC/MS data were analyzed using a gradient from 40 to 100% aqueous acetonitrile containing 0.1% formic acid over 15 min with a 0.7 ml/min solvent flow rate.

Figure 4.

HPLC/MS was used for the detection of prolbene metabolites 3 and 4 from insect model G. mellonella larvae infected with P. luminescens WT (B) and Δplu2236 (C). Control indicates non-infected insect larvae (A). Peaks were extracted with m/z 385 ion signal corresponding to positively charged ion of the metabolites 3 and 4. Inset in B shows time-dependent production behavior of metabolites 1-4. The relative intensity of the metabolites in the inset was determined by integration values of peaks extracted with corresponding m/z.

Figure 5.

Structural characterization of prolbene metabolites 3 and 4. Shown in A are key 2D NMR correlations deduced from the interpretation of the ¹H-¹H COSY (bold) and ¹H-¹³C HMBC (arrow) NMR spectra of compounds 3 and 4. B, Marfey's analysis: FDAA derivatives of proline were analyzed by HPLC/MS with reversed-phase C₁₈ column, and HPLC/MS was established by extracted ion method with corresponding m/z 368 of derivatives. C, observed HR-ESI-QTOFMS spectra of 3 (top) and 4 (bottom).

Figure 6.

Absolute configuration determination of prolbene metabolites 3 and 4. A, calculated ECD spectra of 3 (top) and 4 (bottom) in the gas phase at the B3LYP/6–31+G(d) level and its experimental ECD in methanol (black). B, DP4 probabilities obtained from the two possible diastereomers A (left) and B (right) of prolbenes 3 and 4. NMR chemical shift calculations were performed using the Gaussian 09 package (Gaussian Inc.) in B3LYP/6–31G(d,p) theory level.

Figure 7.

Proposed biosynthesis of stilbene epoxidation and l-proline functionalization in P. luminescens.

Figure 8.

Growth inhibitory comparison of stilbene 1 and stilbene epoxide 2. Producing strains P. luminescens wild type (A), Δplu2236 (B), and ΔhexA (C) were used for bacterial host cytotoxicity measurements of the stilbene metabolites 1 and 2. Optical density (A₆₀₀) was measured and collected over three different time points (12, 24, and 48 h in triplicate).