Fig 1.
Products of CYP6DJ1 assays with (+)-(4R)-limonene and products detected in female beetles after treatment with (+)-(4R)-limonene.
Panels A-C show different regions of the same chromatograms of CYP6DJ1 assays and extracts of beetles treated with (+)-(4R)-limonene. (A) (4R,8R)-Limonene-8,9-epoxide (peak 3) and (4R,8S)-limonene-8,9-epoxide (peak 4) were produced by CYP6DJ1 in in vitro assays with (+)-(4R)-limonene as substrate and were detected in beetles treated with (+)-(4R)-limonene. (B) Unidentified product (peak 5), (+)-trans-(3R,4S)-isopiperitenol (peak 6), (+)-cis-(4S,6S)-carveol (peak 7) and (+)-trans-(4S,6R)-carveol (peak 8) were present in beetles treated with (+)-(4R)-limonene. (C) (4R)-Perilla alcohol (peak 9) was produced in CYP6DJ1 assays with (+)-(4R)-limonene and was detected in beetles treated with (+)-(4R)-limonene. Chromatograms are shown with the total of the extracted ions 91, 94, 108, 109, 119, 121, 137, 152 m/z. Retention indices and mass spectra of peaks 1–10 are shown in S2 Table and S3 Fig.
Fig 2.
Products of CYP6DJ1 assays with (–)-(4S)-limonene and products detected in female beetles after treatment with (–)-(4S)-limonene.
Panels A-D show different regions of the same chromatograms of CYP6DJ1 assays and extracts of beetles treated with (–)-(4S)-limonene. (A) (1S,2R,4S)-Limonene-1,2-epoxide (peak 1) and (1R,2S,4S)-limonene-1,2-epoxide (peak 2) were detected in beetles treated with (–)-(4S)-limonene. (4S,8S)-Limonene-8,9-epoxide (peak 3) and (4S,8R)-limonene-8,9-epoxide (peak 4) were produced by CYP6DJ1 in in vitro assays with (–)-(4S)-limonene and were detected in beetles treated with (–)-(4S)-limonene. (B) An unidentified product (peak 5), (–)-trans-(3S,4R)-isopiperitenol (peak 6), (–)-cis-(4R,6R)-carveol (peak 7) and (–)-trans-(4R,6S)-carveol (peak 8) were detected in beetles treated with (–)-(4S)-limonene. (C) (4S)-Perilla alcohol (peak 9) was produced in CYP6DJ1 assays with (–)-(4S)-limonene and was detected in beetles treated with (–)-(4S)-limonene. (D) (4S)-Limonene-1,2-diol was detected in beetles treated with (–)-(4S)-limonene. Chromatograms are shown with the total of the extracted ions 91, 94, 108, 109, 119, 121, 137, 152 m/z. Retention indices and mass spectra of peaks 1–10 are shown in S2 Table and S4 and S5 Figs.
Fig 3.
Products of CYP6DJ1 assays with terpinolene and products detected in female beetles after treatment with terpinolene.
Four unidentified terpinolene-derived metabolites are present in extracts of terpinolene treated beetles (peaks 11–14), three of which, (peaks 12–14) are found in CYP6DJ1 assays with terpinolene. Chromatograms are shown with the total of the extracted ions 91, 94, 108, 109, 119, 121, 137, 152 m/z. Retention indices and mass spectra of peaks 1–10 are shown in S2 Table and S6 Fig.
Fig 4.
Chemical structure of the epoxide products (peak 1–4) of (+)-(4R)-limonene and (–)-(4S)-limonene identified in CYP6DJ1 assays and extracts of beetles treated with these monoterpenes.
Enantiomers are shown directly across the dotted line from each other. (4R,8R)-Limonene-8,9-epoxide and (4S,8S)-limonene-8,9-epoxide are enantiomers and both eluted as peak 3 in an achiral column. (4R,8S)-Limonene-8,9-epoxide and (4S,8R)-limonene-8,9-epoxide are enantiomers and both eluted as peak 4.
Fig 5.
Chemical structure of the alcohol products (peak 6–10) products of (+)-(4R)-limonene and (–)-(4S)-limonene identified in CYP6DJ1 assays and extracts of beetles treated with these monoterpenes.
Enantiomers are shown directly across the dotted line from each other. (+)-trans-(3R,4S)-Isopiperitenol and (–)-trans-(3S,4R)-isopiperitenol are enantiomers and the proposed products of peak 6. (+)-cis-(4S,6S)-Carveol and (–)-cis-(4R,6R)-carveol are enantiomers and both eluted as peak 7. (+)-trans-(4S,6R)-Carveol and (–)-trans-(4R,6S)-carveol are enantiomers and both eluted as peak 8. (4R)-Perilla alcohol and (4S)-perilla alcohol are enantiomers and both eluted as peak 9.
Fig 6.
Product profiles of recombinant CYP6DJ1 with (+)-(4R)-limonene, (–)-(4S)-limonene or terpinolene as substrates and metabolites detected in extracts of beetles treated with (+)-(4R)-limonene, (–)-(4S)-limonene or terpinolene.
The relative abundance (percentage) across the profiles was calculated by peak area of the extracted ion chromatogram. Retention indices and mass spectra are shown in S2 Table and S3–S5 Figs. CYP6DJ1 profile N = 3, Beetle profile N = 3.
Fig 7.
Products of recombinant CYP6BW1 and CYP6BW3 with diterpene resin acids.
CYP6BW1 and CYP6BW3 assays with (A) isopimaric acid (B) palustric acid, (C) dehydroabietic acid, (D) levopimaric acid, (E) abietic acid, (F) neoabietic acid, as a substrate. Retention indices and mass spectra of peaks 15–21 are shown in S3 Table and S7 and S8 Figs.