Protoporphyrinogen oxidase (PPO)-inhibiting herbicides are used to control weeds in a variety of crops. These herbicides inhibit heme and photosynthesis in plants. PPO-inhibiting herbicides are used to control Amaranthus palmeri (Palmer amaranth) especially those with resistance to glyphosate and acetolactate synthase (ALS) inhibiting herbicides. While investigating the basis of high fomesafen-resistance in A. palmeri , we identified a new amino acid substitution of glycine to alanine in the catalytic domain of PPO2 at position 399 (G399A) (numbered according to the protein sequence of A. palmeri ). G399 is highly conserved in the PPO protein family across eukaryotic species. Through combined molecular, computational, and biochemical approaches, we established that PPO2 with G399A mutation has reduced affinity for several PPO-inhibiting herbicides, possibly due to steric hindrance induced by the mutation. This is the first report of a PPO2 amino acid substitution at G399 position in a field-selected weed population of A. palmeri . The mutant A. palmeri PPO2 showed high-level in vitro resistance to different PPO inhibitors relative to the wild type. The G399A mutation is very likely to confer resistance to other weed species under selection imposed by the extensive agricultural use of PPO-inhibiting herbicides.

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Target-based approaches toward new antimalarial treatments are highly valuable to prevent resistance development. We report several series of pyrazolopyran-based inhibitors targeting the enzyme serine hydroxymethyltransferase (SHMT), designed to improve microsomal metabolic stability and to identify suitable candidates for in vivo efficacy evaluation. The best ligands inhibited Plasmodium falciparum (Pf) and Arabidopsis thaliana (At) SHMT in target assays and PfNF54 strains in cell-based assays with values in the low nanomolar range (3.2-55 nM). A set of carboxylate derivatives demonstrated markedly improved in vitro metabolic stability (t > 2 h). A selected ligand showed significant in vivo efficacy with 73% of parasitemia reduction in a mouse model. Five new cocrystal structures with PvSHMT were solved at 2.3-2.6 Å resolution, revealing a unique water-mediated interaction with Tyr63 at the end of the para-aminobenzoate channel. They also displayed the high degree of conformational flexibility of the Cys364-loop lining this channel.

Main ConclusionThis is a first report of an Ala-205-Phe substitution in acetolactate synthase conferring resistance to imidazolinone, sulfonylurea, triazolopyrimidines, sulfonylamino-carbonyl-triazolinones, and pyrimidinyl (thio) benzoate herbicides.Resistance to acetolactate synthase (ALS) and photosystem II inhibiting herbicides was confirmed in a population of allotetraploid annual bluegrass (Poa annua L.; POAAN-R3) selected from golf course turf in Tennessee. Genetic sequencing revealed that seven of eight POAAN-R3 plants had a point mutation in the psbA gene resulting in a known Ser-264-Gly substitution on the D1 protein. Whole plant testing confirmed that this substitution conferred resistance to simazine in POAAN-R3. Two homeologous forms of the ALS gene (ALSa and ALSb) were detected and expressed in all POAAN-R3 plants sequenced. The seven plants possessing the Ser-264-Gly mutation conferring resistance to simazine also had a homozygous Ala-205-Phe substitution on ALSb, caused by two nucleic acid substitutions in one codon. In vitro ALS activity assays with recombinant protein and whole plant testing confirmed that this Ala-205-Phe substitution conferred resistance to imidazolinone, sulfonylurea, triazolopyrimidines, sulfonylamino-carbonyl- triazolinones, and pyrimidinyl (thio) benzoate herbicides. This is the first report of Ala-205-Phe mutation conferring wide spectrum resistance to ALS inhibiting herbicides.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-015-2399-9) contains supplementary material, which is available to authorized users.

A monofunctional prephenate dehydrogenase (PD) from Aquifex aeolicus was expressed as a His-tagged protein in Escherichia coli and was purified by nickel affinity chromatography allowing the first biochemical and biophysical characterization of a thermostable PD. A. aeolicus PD is susceptible to proteolysis. In this report, the properties of the full-length PD are compared with one of these products, an N-terminally truncated protein variant (D19PD) also expressed recombinantly in E. coli. Both forms are dimeric and show maximum activity at 95°C or higher. D19PD is more sensitive to temperature effects yielding a half-life of 55 min at 95°C versus 2 h for PD, and values of k cat and K m for prephenate, which are twice those determined for PD at 80°C. Low concentrations of guanidine-HCl activate enzyme activity, but at higher concentrations activity is lost concomitant with a multi-state pathway of denaturation that proceeds through unfolding of the dimer, oligomerization, then unfolding of monomers. Measurements of steady-state fluorescence intensity and its quenching by acrylamide in the presence of Gdn-HCl suggest that, of the two tryptophan residues per monomer, one is buried in a hydrophobic pocket and does not become solvent exposed until the protein unfolds, while the less buried tryptophan is at the active site. Tyrosine is a feedback inhibitor of PD activity over a wide temperature range and enhances the cooperativity between subunits in the binding of prephenate. Properties of this thermostable PD are compared and contrasted with those of E. coli chorismate mutaseprephenate dehydrogenase and other mesophilic homologs.