Identification and characterization of the key enzyme in the biosynthesis of the neurotoxin β-ODAP in grass pea

Abstract

Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in Asia and Africa for food and forage. It is a highly nutritious and robust crop, capable of surviving both droughts and floods. However, it produces a neurotoxic compound, β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), which can cause a severe neurological disorder when consumed as a primary diet component. While the catalytic activity associated with β-ODAP formation was demonstrated more than 50 years ago, the enzyme responsible for this activity has not been identified. Here, we report on the identity, activity, 3D structure, and phylogenesis of this enzyme-β-ODAP synthase (BOS). We show that BOS belongs to the benzylalcohol O-acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase, deacetylvindoline 4-O-acetyltransferase superfamily of acyltransferases and is structurally similar to hydroxycinnamoyl transferase. Using molecular docking, we propose a mechanism for its catalytic activity, and using heterologous expression in tobacco leaves (Nicotiana benthamiana), we demonstrate that expression of BOS in the presence of its substrates is sufficient for β-ODAP production in vivo. The identification of BOS may pave the way toward engineering β-ODAP-free grass pea cultivars, which are safe for human and animal consumption.

Keywords: BAHD acyltransferase; Lathyrus sativus; beta-ODAP; beta-ODAP synthetase; diaminopropionic acid; food security; grass pea; hydroxycinnamoyl transferase; neurolathyrism; oxalyl-CoA.

Figure 1

Reaction scheme and catalytic efficiencies of BOS. A, a scheme of the reaction catalyzed by BOS. B, additional CoA substrates analyzed in this work. C, apparent catalytic efficiencies of BOS with different acyl-CoA substrates. The data for oxalyl-CoA were derived from a fit to the Michaelis–Menten equation; error bars denote SD of three independent repeats. Data for all other substrates were fitted to the linear regime of the Michaelis–Menten model, and k_cat/K_m was deduced from the slope; error bars denote SE of the fit. BOS, β-ODAP synthetase.

Figure 2

Secondary structure–based sequence alignment of BOS and HCT homologs. The sequences of hydroxycinnamoyl:CoA-shikimate hydroxycinnamoyl transferases (HCTs) from different species were aligned to that of BOS. Sequences are labeled by their PDB codes: Selaginella moellendorffii (6DD2); Plectranthus scutellarioides (5KJV); Coffea canephora (4G0B); Arabidopsis thaliana (5KJU); Panicum virgatum (5FAL); and Sorghum bicolor (4KE4). BOS secondary structure elements are noted above the sequences and those of Sorghum bicolor (4KE4) - at the bottom. α-Helices and η-helices are shown as spirals, and β-strands are indicated by arrows. Conserved residues are shown in red blocks. Green stars denote BOS His162 and Asp166, which are part of the conserved BAHD acyltransferase HXXXD motif. BAHD, Benzylalcohol O-acetyltransferase, Anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-Hydroxycinnamoyl/benzoyltransferase, Deacetylvindoline 4-O-acetyltransferase; BOS, β-ODAP synthetase.

Figure 3

Phylogenetic tree of BAHD superfamily proteins. Sequence names are prefixed by a two-letter species code and colored by species: L. sativus (Ls), blue; A. thaliana (At), green; M. truncutula (Mt), purple; P. trichocarpa (Pt), brown; other species are colored black. The outer ring and clades are colored as follows (clockwise from the open end on the left): putative clade IIIb (dark purple), clade II (red), clade IV (pink), clade Vb (light green), clade Va (dark green), clade IIIa (light purple), clade Ia (light blue), and clade Ib (dark blue). Sequences of proteins with known functions are shaded in light blue; BOS is marked by a red arrow and shaded in yellow. BAHD, Benzylalcohol O-acetyltransferase, Anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-Hydroxycinnamoyl/benzoyltransferase, Deacetylvindoline 4-O-acetyltransferase.

Figure 4

Structure of BOS and docked substrates. A, ribbon diagram of BOS (PDB ID: 6ZBS). The N- (cyan) and C-terminal (green) domains are connected by loop (residues 183–209; blue) and segment comprised of a β-strand and a short loop (residues 371–393, red). B, surface representation with oxalyl-CoA (magenta) docked into the active site. The conserved DFGWG motif is colored yellow. C, side view showing both oxalyl-CoA (magenta) and L-DAPA (cyan) docked into the active site. D, a close-up view showing oxalyl-CoA (magenta) and L-DAPA (green) docked into the active site along with the conserved catalytic residues His162 and Asp166 (orange). A rotamer of Asp166 that places its residue in hydrogen-bonding distance to His166 was chosen manually to demonstrate a possible interaction. H-bonds are denoted by dashed lines. BOS, β-ODAP synthetase; L-DAPA, L-α,β-diaminopropionic acid.