Towards novel herbicide modes of action by inhibiting lysine biosynthesis in plants.

Weeds are becoming increasingly resistant to our current herbicides, posing a significant threat to agricultural production. Therefore, new herbicides with novel modes of action are urgently needed. In this study, we exploited a novel herbicide target, dihydrodipicolinate synthase (DHDPS), which catalyses the first and rate-limiting step in lysine biosynthesis. The first class of plant DHDPS inhibitors with micromolar potency against Arabidopsis thaliana DHDPS was identified using a high-throughput chemical screen. We determined that this class of inhibitors binds to a novel and unexplored pocket within DHDPS, which is highly conserved across plant species. The inhibitors also attenuated the germination and growth of A. thaliana seedlings and confirmed their pre-emergence herbicidal activity in soil-grown plants. These results provide proof-of-concept that lysine biosynthesis represents a promising target for the development of herbicides with a novel mode of action to tackle the global rise of herbicide-resistant weeds.

Differential lysine-mediated allosteric regulation of plant dihydrodipicolinate synthase isoforms.

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 6VVH and 6VVI.

Review: amino acid biosynthesis as a target for herbicide development.

There are three amino acid biosynthesis pathways that are targeted by current herbicides, namely those leading to the production of aromatic amino acids, branched chain amino acids and glutamine. However, their efficacy is diminishing as a result of the increasing number of resistant weeds. Indeed, resistance to most classes of herbicides is on the rise, posing a significant threat to the utility of current herbicides to sustain effective weed management. This review provides an overview of potential herbicide targets within amino acid biosynthesis that remain unexploited commercially, and recent inhibitor discovery efforts. Despite contemporary approaches to herbicide discovery, such as chemical repurposing and the use of omics technologies, there have been no new products introduced to the market that inhibit amino acid biosynthesis over the past three decades. This highlights the chasm that exists between identifying a potent inhibitor and introducing a commercial herbicide. The unpredictability of a mode of action at the systemic level, as well as poor physicochemical properties, often contribute to a lack of progression beyond the target inhibition stage. Nevertheless, it will be important to overcome these obstacles for the development of new herbicides to protect our agricultural industry and ensure food security for an increasing world population. © 2020 Society of Chemical Industry.

Identification of two dihydrodipicolinate synthase isoforms from Pseudomonas aeruginosa that differ in allosteric regulation.

Pseudomonas aeruginosa is one of the leading causes of nosocomial infections, accounting for 10% of all hospital-acquired infections. Current antibiotics against P. aeruginosa are becoming increasingly ineffective due to the exponential rise in drug resistance. Thus, there is an urgent need to validate and characterize novel drug targets to guide the development of new classes of antibiotics against this pathogen. One such target is the diaminopimelate (DAP) pathway, which is responsible for the biosynthesis of bacterial cell wall and protein building blocks, namely meso-DAP and lysine. The rate-limiting step of this pathway is catalysed by the enzyme dihydrodipicolinate synthase (DHDPS), typically encoded for in bacteria by a single dapA gene. Here, we show that P. aeruginosa encodes two functional DHDPS enzymes, PaDHDPS1 and PaDHDPS2. Although these isoforms have similar catalytic activities (kcat  = 29 s-1 and 44 s-1 for PaDHDPS1 and PaDHDPS2, respectively), they are differentially allosterically regulated by lysine, with only PaDHDPS2 showing inhibition by the end product of the DAP pathway (IC50  = 130 µm). The differences in allostery are attributed to a single amino acid difference in the allosteric binding pocket at position 56. This is the first example of a bacterium that contains multiple bona fide DHDPS enzymes, which differ in allosteric regulation. We speculate that the presence of the two isoforms allows an increase in the metabolic flux through the DAP pathway when required in this clinically important pathogen. DATABASES: PDB ID: 6P90.