Recombinant Soybean Lipoxygenase 2 (GmLOX2) Acts Primarily as a ω6(S)-Lipoxygenase

Abstract

The lipoxygenase (LOX) cascade is a source of bioactive oxylipins that play a regulatory role in plants, animals, and fungi. Soybean (Glycine max (L.) Merr.) LOXs are the classical models for LOX research. Progress in genomics has uncovered a large diversity of GmLOX isoenzymes. Most of them await biochemical investigations. The catalytic properties of recombinant soybean LOX2 (GmLOX2) are described in the present work. The GmLOX2 gene has been cloned before, but only for nucleotide sequencing, while the recombinant protein was not prepared and studied. In the present work, the recombinant GmLOX2 behavior towards linoleic, α-linolenic, eicosatetraenoic (20:4), eicosapentaenoic (20:5), and hexadecatrienoic (16:3) acids was examined. Linoleic acid was a preferred substrate. Oxidation of linoleic acid afforded 94% optically pure (13S)-hydroperoxide and 6% racemic 9-hydroperoxide. GmLOX2 was less active on other substrates but possessed an even higher degree of regio- and stereospecificity. For example, it converted α-linolenic acid into (13S)-hydroperoxide at about 98% yield. GmLOX2 showed similar specificity towards other substrates, producing (15S)-hydroperoxides (with 20:4 and 20:5) or (11S)-hydroperoxide (with 16:3). Thus, the obtained data demonstrate that soybean GmLOX2 is a specific (13S)-LOX. Overall, the catalytic properties of GmLOX2 are quite similar to those of GmLOX1, but pH is optimum.

Keywords: fatty acid oxidation; lipoxygenase-2; regio- and stereospecificity; soybean; substrate specificity.

Figure 1

Effect of pH on activity of recombinant GmLOX2 with linoleic acid (140 μM) as substrate. Values represent the means of five replications.

Figure 2

GC-MS analyses of products of conversion of linoleic (A) and α-linolenic (B) acids by the recombinant GmLOX2 after preliminary NaBH₄ reduction and hydrogenation over PtO₂; mass spectra of products (Me/TMS) of NaBH₄ reduction and hydrogenation over PtO₂ of 13-hydroperoxides (C) and 9-hydroperoxides (D) of linoleic and α-linolenic acids. 9-HSA, 9-hydroxystearic acid; 13-HSA, 13-hydroxystearic acid.

Figure 3

GC-MS analyses of products of conversion of arachidonic (A) and eicosapentaenoic (B) acids by the recombinant GmLOX2 after preliminary NaBH₄ reduction and hydrogenation over PtO₂; mass spectra of products (Me/TMS) of NaBH₄ reduction and hydrogenation over PtO₂ of 15-hydroperoxides (C) and 5-hydroperoxides (D) of arachidonic and eicosapentaenoic acids. 5-HEA, 5-hydroxyeicosanoic acid; 15-HEA, 15-hydroxyeicosanoic acid.

Figure 4

GC-MS analyses of products of conversion of hexadecatrienoic acid (A) by the recombinant GmLOX2 after preliminary NaBH₄ reduction and hydrogenation over PtO₂; mass spectra of products (Me/TMS) of NaBH₄ reduction and hydrogenation over PtO₂ of 11-hydroperoxide (B) and 7-hydroperoxide (C) of hexadecatrienoic acid. 7-HHA, 7-hydroxyhexadecanoic acid; 11-HHA, 11-hydroxyhexadecanoic acid.

Figure 5

Phylogenetic analysis of G. max and A. thaliana LOXs. G. max LOXs: XP006585500.1, XP003521704.2, XP014634584.1, XP003521841.1, XP003528382.1, XP003536076.1, XP003546741.1, XP003554800.1, XP003531186.1, XP003531597.1, XP003528556.1, NP001237323.2 LOX9, AAB67732.1 VLXB, XP003556820.2, XP003537949.1, XP003519005.1, XP003555640.1, XP003555620.1, NP001237338.2 LOX-10, XP040866631.1, XP006599136.1, XP003537948.1, XP003539375.1, XP006606050.1, NP001238692.2 9(S)-specific LOX-4, XP003543820.4, NP001238676.1, XP006576654.1, XP003528455.2, NP001235189.1 VLXC, P09439.1 GmLOX2 (the object of the present research, highlighted in bold text and enlarged), XP003531596.1, P09186.1 LOX-3, NP001236153.2 13-LOX-1. A. thaliana LOXs (highlighted in bold and underlined text): Q06327.1 AtLOX1, P38418.1 AtLOX2, Q9LNR3.1 AtLOX3, Q9FNX8.1 AtLOX4, Q9LUW0.2 AtLOX5, Q9CAG3.1 AtLOX6. 9LOX(I)—9-LOXs type 1; 13LOX(I)—13-LOXs type 1; 13LOX(II)—13-lipoxygenases type 2. Blue branches—maximum bootstraps, yellow branches—medium bootstraps, red branches—minimum bootstraps.

Figure 6

Alignment of partial G. max LOX1 and LOX2 sequences. The 19 amino acid deletion near the N-end in GmLOX1 is highlighted with the frame. The PLAT domain is defined according to the NCBI database (highlighted in bold and underlined text).

Figure 7

Multiple alignments of partial G. max LOX sequences annotated in the NCBI database as hypothetical 9-specific LOXs.