Discovery of α-Linolenic Acid 16(S)-Lipoxygenase: Cucumber (Cucumis sativus L.) Vegetative Lipoxygenase 3

Abstract

The GC-MS profiling of the endogenous oxylipins (Me/TMS) from cucumber (Cucumis sativus L.) leaves, flowers, and fruit peels revealed a remarkable abundance of 16-hydroxy-9,12,14-octadecatrienoic acid (16-HOT). Incubations of homogenates from these organs with α-linolenic acid yielded 16(S)-hydroperoxide (16-HPOT) as a predominant product. Targeted proteomic analyses of these tissues revealed the presence of several highly homologous isoforms of the putative "9S-lipoxygenase type 6". One of these isoenzymes (CsLOX3, an 877 amino acid polypeptide) was prepared by heterologous expression in E. coli and exhibited 16(S)- and 13(S)-lipoxygenase activity toward α-linolenic and linoleic acids, respectively. Furthermore, α-linolenate was a preferred substrate. The molecular structures of 16(S)-HOT and 16(S)-HPOT (Me or Me/TMS) were unequivocally confirmed by the mass spectral data, ¹H-NMR, 2D ¹H-¹H-COSY, TOCSY, HMBC, and HSQC spectra, as well as enantiomeric HPLC analyses. Thus, the vegetative CsLOX3, biosynthesizing 16(S)-HPOT, is the first 16(S)-LOX and ω3-LOX ever discovered. Eicosapentaenoic and hexadecatrienoic acids were also specifically transformed to the corresponding ω3(S)-hydroperoxides by CsLOX3.

Keywords: cucumber (Cucumis sativus L.); fatty acid hydroperoxides; lipoxygenase; molecular cloning; ω3 fatty acids; ω3(S) dioxygenation.

Figure 1

GC-MS analyses of NaBH₄-reduced products (Me/TMS) of α-linolenic acid oxidation by LOX preparation from cucumber flowers. (A) The selected ion GC-MS chromatograms at m/z 131, 183, 223, and 311. (B) Mass spectrum for product 1; inset, mass fragmentation scheme.

Figure 2

The observed ¹H-¹H-COSY (bold lines) and ¹H-¹³C-HMBC (curved arrows) correlations of compounds 1 and 2.

Figure 3

Normal phase HPLC (NP-HPLC) and chiral phase HPLC (CP-HPLC) analyses of NaBH₄-reduced products (Me esters) of α-linolenic acid incubation with LOX preparation from cucumber flowers. (A) The NP-HPLC chromatogram of total NaBH₄-reduced products (Me). (B) the CP-HPLC chromatogram of 16-HOT fraction collected from NP-HPLC column. The ultraviolet absorption (190–370 nm) was monitored by diode array detector, 235 nm chromatograms are shown. Full details of analyses are described in Section 4.

Figure 4

Normal phase HPLC (NP-HPLC) and chiral phase HPLC (CP-HPLC) analyses of NaBH₄-reduced products (Me esters) of linoleic acid incubation with LOX preparation from cucumber flowers. (A) The NP-HPLC chromatogram of total NaBH₄-reduced products (Me). (B) The CP-HPLC chromatogram of 13-HOD (Z,E) fraction collected from NP-HPLC column. (C) The CP-HPLC chromatogram of 9-HOD (Z,E) fraction collected from NP-HPLC column. The ultraviolet absorption (190–370 nm) was monitored by diode array detector, 235 nm chromatograms are shown. Full details of analyses are described in Section 4.

Figure 5

GC-MS total ion current (TIC) chromatograms of products (Me/TMS) of α-linolenic acid (A) or linoleic acid (C) conversions by the recombinant CsLOX3 after preliminary NaBH₄ reduction and hydrogenation over PtO₂; (B,D,E) mass spectra of major (totally reduced and hydrogenated) products (totally reduced and hydrogenated, Me/TMS); insets are the mass fragmentation schemes. (B,D,E) Spectra of 16-HSA, 13-HSA, and 9-HSA and 16-, 13-, and 9-hydroxystearic acid (Me/TMS) formed via the derivatization of 16-HPOT, 13-HPOD, and 9-HPOD, respectively. 16-HSA—16-hydroxystearic acid; 13-HSA—13-hydroxystearic acid; 9-HSA—9-hydroxystearic acid.

Figure 6

Mass spectra of products (Me/TMS) of (7Z,10Z,13Z)-hexadecatrienoic acid (A) or (5Z,8Z,11Z,14Z,17Z)-eicosapentaenoic acid (B) conversions by the recombinant CsLOX3 after preliminary NaBH₄ reduction and hydrogenation over PtO₂. Insets (A,B) are the mass fragmentation schemes for 14-hydroxypalmitic and 18-hydroxyeicosanoic acids (Me/TMS), respectively.

Figure 7

Specificity of α-linolenic acid oxygenation by conventional LOXs (A) and LOXs attacking the prochiral center C14 (B). Two prochiral centers (C11 and C14) of α-linolenic acid and the LOX specificity depending on selectivity of the initial hydrogen abstraction from C11 or C14.

Figure 8

Selected multiple alignments of CsLOX3 and related cucumber proteins. All genes encoding proteins shown are localized in chromosome 2 of C. sativus. Most of them (except XP_004150982.1) share 90–98% identity.

Figure 9

Phylogenetic tree of amino acid sequences of the selected studied LOXs from red algae, cyanobacteria, fungi, plants, and animals. Numbers 9 and 13 indicate the substrate specificity of LOXs determined using linoleic or linolenic acid. The numbers with an asterisk indicate enzymes characterized using both linoleic and linolenic acids. The black arrow indicates the CsLOX3 studied in the present work.