Characterization of recombinant dihydrodipicolinate synthase from the bread wheat Triticum aestivum

Abstract

Recombinant wheat DHDPS was produced for the first time in milligram quantities and shown to be an enzymatically active tetramer in solution using analytical ultracentrifugation and small angle X-ray scattering. Wheat is an important cereal crop with an extensive role in global food supply. Given our rapidly growing population, strategies to increase the nutritional value and production of bread wheat are of major significance in agricultural science to satisfy our dietary requirements. Lysine is one of the most limiting essential amino acids in wheat, thus, a thorough understanding of lysine biosynthesis is of upmost importance to improve its nutritional value. Dihydrodipicolinate synthase (DHDPS; EC 4.3.3.7) catalyzes the first committed step in the lysine biosynthesis pathway of plants. Here, we report for the first time the expression and purification of recombinant DHDPS from the bread wheat Triticum aestivum (Ta-DHDPS). The optimized protocol yielded 36 mg of > 98% pure recombinant Ta-DHDPS per liter of culture. Enzyme kinetic studies demonstrate that the recombinant Ta-DHDPS has a K (pyruvate) of 0.45 mM, K (l-aspartate-4-semialdehyde) of 0.07 mM, k of 56 s, and is inhibited by lysine (IC of 0.033 mM), which agree well with previous studies using labor-intensive purification from wheat suspension cultures. We subsequently employed circular dichroism spectroscopy, analytical ultracentrifugation and small angle X-ray scattering to show that the recombinant enzyme is folded with 60% α/β structure and exists as a 7.5 S tetrameric species with a R of 33 Å and D of 118 Å. This study is the first to report the biophysical properties of the recombinant Ta-DHDPS in aqueous solution and offers an excellent platform for future studies aimed at improving nutritional value and primary production of bread wheat.

Differential lysine‐mediated allosteric regulation of plant dihydrodipicolinate synthase isoforms

“…4B). The IC 50 reported for AtDHDPS2 is similar to that observed for other plant DHDPS enzymes (10–50 µ m ) [28,29], while AtDHDPS1 displays a much tighter affinity for the allosteric regulator. These data confirm previous speculation that these two isoforms differ in allosteric regulation [21,36].…”

“…The construct was subsequently commercially synthesised by Bioneer Pacific (Kew East, VIC, Australia) and ligated into pET11a (Merck, Bayswater, VIC, Australia) using BamHI and EcoRI restriction sites, before transformation into Escherichia coli BL21 (DE3) cells. The plasmid pET151/D‐Topo harbouring dapA2 was co‐transformed with pACYC184, containing the GroEL/ES chaperones, into E. coli BL21 (DE3) cells [28]. Expression was carried out by culturing transformed cells in Luria–Bertani media, containing 100 µg·mL −1 of ampicillin, at 25 °C with agitation (160 r.p.m.)…”

“…Allosteric regulation of the DAP pathway occurs via the binding of lysine to DHDPS. Unlike bacteria, plants have been shown to be highly sensitive to lysine inhibition with IC 50 values between 10 and 50 μ m [27,28]. Several residues have been implicated to be important for allosteric regulation (Figure 2).…”

From bacteria to plants: a repurposing strategy in the pursuit for novel herbicides

“…Furthermore, the high degree of conservation of DHDPR enzymes across plant species suggests that the specificity of these compounds for weeds is unlikely (Supplementary Figure S5). 31 Directed evolution experiments would therefore be of interest to identify mutations which may be used to engineer crops resistant to DHDPR active site inhibitors. Identifying such mutations would also facilitate the monitoring of weed populations for the emergence of resistance so that early intervention strategies may be implemented.…”

A F420-dependent single domain chemogenetic tool for protein de-dimerization

“…Sedimentation velocity experiments were performed in a XL-A analytical ultracentrifuge (Beckman Coulter) using an 8-hole An50-Ti rotor and double-sector cells containing synthetic quartz windows as previously described [66][67][68]. Cells were loaded with 380 µL of protein diluted in 1× PBS (pH 7.4) supplemented with 1 mM CaCl 2 and 0.5 mM MgCl 2 and 400 µL of reference solution.…”