A Novel Single-Site Mutation in the Catalytic Domain of Protoporphyrinogen Oxidase IX (PPO) Confers Resistance to PPO-Inhibiting Herbicides.

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.

Potent Inhibitors of Plasmodial Serine Hydroxymethyltransferase (SHMT) Featuring a Spirocyclic Scaffold.

With the discovery that serine hydroxymethyltransferase (SHMT) is a druggable target for antimalarials, the aim of this study was to design novel inhibitors of this key enzyme in the folate biosynthesis cycle. Herein, 19 novel spirocyclic ligands based on either 2-indolinone or dihydroindene scaffolds and featuring a pyrazolopyran core are reported. Strong target affinities for Plasmodium falciparum (Pf) SHMT (14-76 nm) and cellular potencies in the low nanomolar range (165-334 nm) were measured together with interesting selectivity against human cytosolic SHMT1 (hSHMT1). Four co-crystal structures with Plasmodium vivax (Pv) SHMT solved at 2.2-2.4 Šresolution revealed the key role of the vinylogous cyanamide for anchoring ligands within the active site. The spirocyclic motif in the molecules enforces the pyrazolopyran core to adopt a substantially more curved conformation than that of previous non-spirocyclic analogues. Finally, solvation of the spirocyclic lactam ring of the receptor-bound ligands is discussed.

Conformational Aspects in the Design of Inhibitors for Serine Hydroxymethyltransferase (SHMT): Biphenyl, Aryl Sulfonamide, and Aryl Sulfone Motifs.

Malaria remains a major threat to mankind due to the perpetual emergence of resistance against marketed drugs. Twenty-one pyrazolopyran-based inhibitors bearing terminal biphenyl, aryl sulfonamide, or aryl sulfone motifs were synthesized and tested towards serine hydroxymethyltransferase (SHMT), a key enzyme of the folate cycle. The best ligands inhibited Plasmodium falciparum (Pf) and Arabidopsis thaliana (At) SHMT in target, as well as PfNF54 strains in cell-based assays in the low nanomolar range (18-56 nm). Seven co-crystal structures with P. vivax (Pv) SHMT were solved at 2.2-2.6 Šresolution. We observed an unprecedented influence of the torsion angle of ortho-substituted biphenyl moieties on cell-based efficacy. The peculiar lipophilic character of the sulfonyl moiety was highlighted in the complexes with aryl sulfonamide analogues, which bind in their preferred staggered orientation. The results are discussed within the context of conformational preferences in the ligands.

Antimalarial Inhibitors Targeting Serine Hydroxymethyltransferase (SHMT) with in Vivo Efficacy and Analysis of their Binding Mode Based on X-ray Cocrystal Structures.

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 (t1/2 > 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.

A new amino acid substitution (Ala-205-Phe) in acetolactate synthase (ALS) confers broad spectrum resistance to ALS-inhibiting herbicides.

MAIN CONCLUSION: This 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.

Fine tuning of a biological machine: DnaK gains improved chaperone activity by altered allosteric communication and substrate binding.

DnaK is a member of the Hsp70 family of molecular chaperones. This molecular machine couples the binding and hydrolysis of ATP to binding and release of substrate proteins. The switches that are involved in allosteric communication within this multidomain protein are mostly unknown. Previous insights were largely obtained by mutants, which displayed either wild-type activity or reduced folding assistance of substrate proteins. With a directed evolution approach for improved folding assistance we selected a DnaK variant characterized by a glycine to alanine substitution at position 384 (G384A); this resulted in a 2.5-fold higher chaperone activity in an in vitro DnaK-assisted firefly luciferase refolding assay. Quantitative biochemical characterization revealed several changes of key kinetic parameters compared to the wild type. Most pronounced is a 13-fold reduced rate constant for substrate release in the ATP-bound state, which we assume, in conjunction with the resulting increase in substrate affinity, to be related to improved chaperone activity. As the underlying mechanistic reason for this change we propose an altered interface of allosteric communication of mutant G384A, which is notably located at a hinge position between nucleotide and substrate binding domain.

Directed evolution of the DnaK chaperone: mutations in the lid domain result in enhanced chaperone activity.

We improved the DnaK molecular chaperone system for increased folding efficiency towards two target proteins, by using a multi-parameter screening procedure. First, we used a folding-deficient C-terminal truncated chloramphenicol acetyl transferase (CAT_Cd9) to obtain tunable selective pressure for enhanced DnaK chaperon function in vivo. Second, we screened selected clones in vitro for CAT_Cd9 activity after growth under selective pressure. We then analyzed how these variants performed as compared to wild type DnaK towards folding assistance of a second target protein; namely, chemically denatured firefly luciferase. A total of 11 single point DnaK mutants and 1 truncated variant were identified using CAT_Cd9 as the protein target, while 4 of the 12 selected variants showed improved luciferase refolding in vitro. This shows that improving the DnaK chaperone by using a certain target substrate protein, does not necessarily result in a loss or reduction in its ability to assist other proteins. Of the 12 identified mutations, half were clustered in the nucleotide binding domain, and half in the lid domain (LD) of DnaK. The truncated variant is characterized by a 35-residue C-terminal truncation (Cd35) and exhibited the highest improvement for luciferase refolding. Cd35 showed a 7-fold increase in initial refolding rate for denatured luciferase and resulted in a 5-fold increase in maximal luminescence as compared to wild type DnaK. Given that the best in vitro performing mutants contained LD substitutions, and that the LD is not involved in ATP binding, ATP hydrolysis or client protein association, but is involved in allosteric regulation of the chaperone cycle, we propose that improved DnaK variants result in changes to allosteric domain communication, ultimately retuning the ATP-dependent chaperone cycle.

Biochemical characterization of prephenate dehydrogenase from the hyperthermophilic bacterium Aquifex aeolicus.

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 (Delta19PD) also expressed recombinantly in E. coli. Both forms are dimeric and show maximum activity at 95 degrees C or higher. Delta19PD is more sensitive to temperature effects yielding a half-life of 55 min at 95 degrees C versus 2 h for PD, and values of kcat and Km for prephenate, which are twice those determined for PD at 80 degrees 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 mutase-prephenate dehydrogenase and other mesophilic homologs.