Differential lysine‐mediated allosteric regulation of plant dihydrodipicolinate synthase isoforms

Abstract

Lysine biosynthesis in plants occurs via the diaminopimelate pathway. The first committed and rate‐limiting step of this pathway is catalysed by dihydrodipicolinate synthase (DHDPS), which is allosterically regulated by the end product, l‐lysine (lysine). Given that lysine is a common nutritionally limiting amino acid in cereal crops, there has been much interest in probing the regulation of DHDPS. Interestingly, knockouts in Arabidopsis thaliana of each isoform (AtDHDPS1 and AtDHDPS2) result in different phenotypes, despite the enzymes sharing > 85% protein sequence identity. Accordingly, in this study, we compared the catalytic activity, lysine‐mediated inhibition and structures of both A. thaliana DHDPS isoforms. We found that although the recombinantly produced enzymes have similar kinetic properties, AtDHDPS1 is 10‐fold more sensitive to lysine. We subsequently used X‐ray crystallography to probe for structural differences between the apo‐ and lysine‐bound isoforms that could account for the differential allosteric inhibition. Despite no significant changes in the overall structures of the active or allosteric sites, we noted differences in the rotamer conformation of a key allosteric site residue (Trp116) and proposed that this could result in differences in lysine dissociation. Microscale thermophoresis studies supported our hypothesis, with AtDHDPS1 having a ~ 6‐fold tighter lysine dissociation constant compared to AtDHDPS2, which agrees with the lower half minimal inhibitory concentration for lysine observed. Thus, we highlight that subtle differences in protein structures, which could not have been predicted from the primary sequences, can have profound effects on the allostery of a key enzyme involved in lysine biosynthesis in plants. Databases Structures described are available in the Protein Data Bank under the accession numbers https://doi.org/10.2210/pdb6VVH/pdb and https://doi.org/10.2210/pdb6VVI/pdb.

Towards Novel Herbicide Modes of Action by Inhibiting Lysine Biosynthesis in Plants

“…The mode of inhibition was shown to be via a novel binding pocket adjacent to the lysine binding site, which results in the allosteric inhibition of the enzyme. Lysine has recently been shown to differentially inhibit the AtDHDPS isoforms, with AtDHDPS1 being 10-fold more sensitive to the allosteric inhibitor (Hall et al, 2021). In this study, we demonstrate that the MBDTA compounds have similar inhibitory effects against both enzymes.…”

“…The inhibitory activity of MBDTA-1 and MBDTA-2 against both recombinant A. thaliana DHDPS proteins was quantitated using a DHDPS-DHDPR coupled assay (Atkinson et al, 2013). This was achieved by titrating different concentrations of each compound with substrates fixed at previously determined Michaelis-Menten constant values (Griffin et al, 2012;Hall et al, 2021). The IC50 values of MBDTA-1 and MBDTA-2 against AtDHDPS1 were determined to be 126 ± 6.50 µM and 63.3 ± 1.80 µM, respectively (Figure 4A).…”

“…To probe the molecular determinants for inhibition, recombinant AtDHDPS1 was cocrystallised with MBDTA-2 using the same crystallisation conditions as for the apo enzyme with the addition of inhibitor (Hall et al, 2021). Diffraction data were obtained at a maximal resolution of 2.29 Å, phases solved by molecular replacement and repeating rounds of model building and refinement were performed that allowed us to generate an atomic inhibitor-bound model (Figure 5A, Table 1).…”

“…AtDHDPS1 was co-crystallised as previously described in the presence of MBDTA-2 (Hall et al, 2021). Briefly, protein (8.5 mg•mL -1 ) was incubated at 20 °C with MBDTA-2 at a final (in 2% (v/v) DMSO) and 20% (v/v) glycerol) and flash frozen in liquid nitrogen.…”

“…Double knockouts of DHDPS1 and DHDPS2 result in non-viable embryos even after exogenous supplementation with lysine, indicating that DHDPS activity is essential (Jones-Held et al, 2012). AtDHDPS enzymes exist as homotetramers (Figure 2A), with the active site located within the (β/α)8-barrel (Figure 2B) and the allosteric cleft in the tight-dimer interface located in the interior of the structure (Figure 2C) (Griffin et al, 2012;Hall et al, 2021). interspacing residues.…”