Crystal structures of vegetative soybean lipoxygenase VLX-B and VLX-D, and comparisons with seed isoforms LOX-1 and LOX-3

Abstract

The lipoxygenase family of lipid-peroxidizing, nonheme iron dioxygenases form products that are precursors for diverse physiological processes in both plants and animals. In soybean (Glycine max), five vegetative isoforms, VLX-A, VLX-B, VLX-C, VLX-D, VLX-E, and four seed isoforms LOX-1, LOX-2, LOX-3a, LOX-3b have been identified. In this study, we determined the crystal structures of the substrate-free forms of two major vegetative isoforms, with distinct enzymatic characteristics, VLX-B and VLX-D. Their structures are similar to the two seed isoforms, LOX-1 and LOX-3, having two domains with similar secondary structural elements: a beta-barrel N-terminal domain containing highly flexible loops and an alpha-helix-rich C-terminal catalytic domain. Detailed comparison of the structures of these two vegetative isoforms with the structures of LOX-1 and LOX-3 reveals important differences that help explain distinct aspects of the activity and positional specificity of these enzymes. In particular, the shape of the three branches of the internal subcavity, corresponding to substrate-binding and O(2) access, differs among the isoforms in a manner that reflects the differences in positional specificities.

Fig. 1

Molecular mass determination of VLX-B and VLX-D and Dynamic light scattering data for VLX-B (solid line) and VLX-D (dotted line) (~2 mg/ml each). The calculated molecular radius and molecular weight are 4.14 nm and 95 kDa for VLX-B and 4.25 nm and 96 kDa for VLX-D, respectively.

Fig. 2

Amino acid sequence comparisons of VLX-B, VLX-D, LOX-1, LOX-3, Oryctolagus cuniculus 15-LOX (1LOX) and Plexaura homomalla 8R-LOX (1ZQ4). Secondary structural elements of VLX-B are highlighted in colored bars on top of the corresponding sequence. The N-terminal domain and C-terminal domain are separated by a blue vertical line. A vertical red dotted line is marked at every 20 amino acids, and every 100th amino acids are indicated by their corresponding residue numbers.

Fig. 3

Crystal structure of VLX-B (A) and VLX-D (B). The asymmetric unit in the lattice of VLX-D is composed of two molecules (inset of (B)). The N-terminal and C-terminal domains are colored in orange and light blue for VLX-B and violet and light green for VLX-D, respectively. Secondary structural elements have been numbered sequentially as α1–α23 and β1–β19 for the α-helices and β-strands, respectively. The Fe2⁺ ion is indicated by the red sphere. (C) Superimposed views of VLX-B (orange) and VLX-D (green).

Fig. 4

The shape of subcavity IIa of VLX-B, VLX-D, LOX-1, and LOX-3. The O₂ cavity (red arrow), extended cavity (blue arrow), and entrance site (green arrow) are indicated. The iron is depicted the by red ball (B). The view in (B) is ~45° rotated from the view in (A), along the horizontal axis. In LOX-1, the modeled linoleic acid is depicted in violet color. This figure was generated with the program PyMOL (v 0.99).

Fig. 5

(A) Residues constituting subcavity IIa that are conserved among the four soybean lipoxygenases. The shape of subcavity IIa of VLX-B, linoleic acid (violet stick model), and iron (pink ball) are depicted. (B) The residues constituting the cavity IIa; conserved residues are represented by gray ball and sticks together with their van der Waal nets. The nonconserved residues were represented by stick model (VLX-B, orange; VLX-D, blue; LOX-1, green; LOX-3, cyan). (C) Internal cavity of VLX-B. The residues are represented in the same format as in (B).