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

2021 ◽  
Author(s):  
Tatiana P. Soares da Costa ◽  
Cody J. Hall ◽  
Santosh Panjikar ◽  
Jessica A. Wyllie ◽  
Rebecca M. Christoff ◽  
...  
Keyword(s):  
Herbicidal Activity ◽  
Lysine Biosynthesis ◽  
Proof Of Concept ◽  
Herbicide Resistant ◽  
X Ray Crystallography ◽  
Rate Limiting Step ◽  
Promising Target ◽  
Chemical Screen ◽  
Germination And Growth ◽  
Rate Limiting

Weeds are becoming increasingly resistant to our current herbicides, posing a significant threat to agricultural production. Therefore, new herbicides 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. Using a high throughput chemical screen, we identified the first class of plant DHDPS inhibitors that have micromolar potency against Arabidopsis thaliana DHDPS isoforms. Employing X-ray crystallography, we determined that this class of inhibitors binds to a novel and unexplored pocket within DHDPS, which is highly conserved across plant species. We also demonstrated that the inhibitors 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.

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

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Tatiana P Soares da Costa ◽  
Cody J Hall ◽  
Santosh Panjikar ◽  
Jessica A Wyllie ◽  
Rebecca M Christoff ◽  
...  
Keyword(s):  
Herbicidal Activity ◽  
Lysine Biosynthesis ◽  
Proof Of Concept ◽  
Herbicide Resistant ◽  
Modes Of Action ◽  
Rate Limiting Step ◽  
Promising Target ◽  
Chemical Screen ◽  
Germination And Growth ◽  
Rate Limiting

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 were 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.

scholarly journals Structural, functional, and behavioral insights of dopamine dysfunction revealed by a deletion inSLC6A3

2019 ◽  
Vol 116 (9) ◽  
pp. 3853-3862 ◽  
Author(s):  
Nicholas G. Campbell ◽  
Aparna Shekar ◽  
Jenny I. Aguilar ◽  
Dungeng Peng ◽  
Vikas Navratna ◽  
...  
Keyword(s):  
Autism Spectrum ◽  
High Time Resolution ◽  
Paramagnetic Resonance ◽  
X Ray Crystallography ◽  
Fine Grained ◽  
Resonance Analysis ◽  
Rate Limiting Step ◽  
Abnormal Behaviors ◽  
Rate Limiting ◽  
Electron Paramagnetic

The human dopamine (DA) transporter (hDAT) mediates clearance of DA. Genetic variants in hDAT have been associated with DA dysfunction, a complication associated with several brain disorders, including autism spectrum disorder (ASD). Here, we investigated the structural and behavioral bases of an ASD-associated in-frame deletion in hDAT at N336 (∆N336). We uncovered that the deletion promoted a previously unobserved conformation of the intracellular gate of the transporter, likely representing the rate-limiting step of the transport process. It is defined by a “half-open and inward-facing” state (HOIF) of the intracellular gate that is stabilized by a network of interactions conserved phylogenetically, as we demonstrated in hDAT by Rosetta molecular modeling and fine-grained simulations, as well as in its bacterial homolog leucine transporter by electron paramagnetic resonance analysis and X-ray crystallography. The stabilization of the HOIF state is associated both with DA dysfunctions demonstrated in isolated brains ofDrosophila melanogasterexpressing hDAT ∆N336 and with abnormal behaviors observed at high-time resolution. These flies display increased fear, impaired social interactions, and locomotion traits we associate with DA dysfunction and the HOIF state. Together, our results describe how a genetic variation causes DA dysfunction and abnormal behaviors by stabilizing a HOIF state of the transporter.

scholarly journals Chemical clustering and visualization applied to macromolecular crystallography

2014 ◽  
Vol 70 (a1) ◽  
pp. C1145-C1145
Author(s):  
Andrew Bruno ◽  
Amanda Ruby ◽  
Joseph Luft ◽  
Thomas Grant ◽  
Jayaraman Seetharaman ◽  
...  
Keyword(s):  
Hierarchical Clustering ◽  
High Throughput ◽  
Active Sites ◽  
X Ray Crystallography ◽  
Rate Limiting Step ◽  
Potential Applications ◽  
Crystallization Conditions ◽  
Electron Density Map ◽  
Distance Coefficient ◽  
Rate Limiting

Many bioscience fields employ high-throughput methods to screen multiple biochemical conditions. The analysis of these becomes tedious without a degree of automation. Crystallization, a rate limiting step in biological X-ray crystallography, is one of these fields. Screening of multiple potential crystallization conditions (cocktails) is the most effective method of probing a proteins phase diagram and guiding crystallization but the interpretation of results can be consuming. To aid this empirical approach a cocktail distance coefficient was developed to quantitatively compare macromolecule crystallization conditions and outcome. These coefficients were evaluated against an existing similarity metric developed for crystallization, the C6 metric, using both virtual crystallization screens and by comparison of two related 1,536-cocktail high-throughput crystallization screens. Hierarchical clustering was employed to visualize one of these screens and the crystallization results from an exopolyphosphatase-related protein from Bacteroides fragilis, (BfR192) overlaid on this clustering. This demonstrated a strong correlation between certain chemically related clusters and crystal lead conditions. While this analysis was not used to guide the initial crystallization optimization, it led to the re-evaluation of unexplained peaks in the electron density map of the protein and the insertion and correct placement of a sodium, potassium and phosphate atoms in the structure. With these in place, the resulting structure of the putative active site demonstrated features consistent with active sites of other phosphatases which are involved in binding the phosphoryl moieties of nucleotide triphosphates. The new distance coefficient appears to be robust in this application and coupled with hierarchical clustering and the overlay of crystallization outcome reveals information of biological relevance. While tested with a single example the potential applications appear promising.

scholarly journals Revealing a hidden intermediate of rotatory catalysis with X-ray crystallography and Molecular simulations

2021 ◽  
Author(s):  
Mrinal Shekhar ◽  
Chitrak Gupta ◽  
Kano Suzuki ◽  
Abhishek Singharoy ◽  
Takeshi Murata
Keyword(s):  
Molecular Motors ◽  
Atp Hydrolysis ◽  
Structural Information ◽  
Hydrolysis Product ◽  
X Ray ◽  
X Ray Crystallography ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Dynamics Simulations ◽  
Rate Limiting

The mechanism of rotatory catalysis in ATP-hydrolyzing molecular motors remain an unresolved puzzle in biological energy transfer. Notwithstanding the wealth of available biochemical and structural information inferred from years of experiments, knowledge on how the coupling between the chemical and mechanical steps within motors enforces directional rotatory movements remains fragmentary. Even more contentious is to pinpoint the rate-limiting step of a multi-step rotation process. Here, using Vacuolar or V1-type hexameric ATPase as an exemplary rotational motor, we present a model of the complete 4-step conformational cycle involved in rotatory catalysis. First, using X-ray crystallography a new intermediate or 'dwell' is identified, which enables the release of an inorganic phosphate (or Pi) after ATP hydrolysis. Using molecular dynamics simulations, this new dwell is placed in a sequence with three other crystal structures to derive a putative cyclic rotation path. Free-energy simulations are employed to estimate the rate of the hexameric protein transfor-mations, and delineate allosteric effects that allow new reactant ATP entry only after hydrolysis product exit. An analysis of transfer entropy brings to light how the sidechain level interactions transcend into larger scale reorganizations, highlighting the role of the ubiquitous arginine-finger residues in coupling chemical and mechanical information. Inspection of all known rates encompassing the 4-step rotation mechanism implicates overcoming of the ADP interactions with V1-ATPase to be the rate-limiting step of motor action.

Improving photosynthesis through the enhancement of Rubisco carboxylation capacity

2021 ◽  
Author(s):  
Concepción Iñiguez ◽  
Pere Aguiló-Nicolau ◽  
Jeroni Galmés
Keyword(s):  
Active Sites ◽  
Arable Land ◽  
Small Subunit ◽  
Resource Use Efficiency ◽  
Rate Limiting Step ◽  
Promising Target ◽  
Use Efficiency ◽  
Co2 Concentrating Mechanisms ◽  
Near Future ◽  
Rate Limiting

Rising human population, along with the reduction in arable land and the impacts of global change, sets out the need for continuously improving agricultural resource use efficiency and crop yield (CY). Bioengineering approaches for photosynthesis optimization have largely demonstrated the potential for enhancing CY. This review is focused on the improvement of Rubisco functioning, which catalyzes the rate-limiting step of CO2 fixation required for plant growth, but also catalyzes the ribulose-bisphosphate oxygenation initiating the carbon and energy wasteful photorespiration pathway. Rubisco carboxylation capacity can be enhanced by engineering the Rubisco large and/or small subunit genes to improve its catalytic traits, or by engineering the mechanisms that provide enhanced Rubisco expression, activation and/or elevated [CO2] around the active sites to favor carboxylation over oxygenation. Recent advances have been made in the expression, assembly and activation of foreign (either natural or mutant) faster and/or more CO2-specific Rubisco versions. Some components of CO2 concentrating mechanisms (CCMs) from bacteria, algae and C4 plants has been successfully expressed in tobacco and rice. Still, none of the transformed plant lines expressing foreign Rubisco versions and/or simplified CCM components were able to grow faster than wild type plants under present atmospheric [CO2] and optimum conditions. However, the results obtained up to date suggest that it might be achievable in the near future. In addition, photosynthetic and yield improvements have already been observed when manipulating Rubisco quantity and activation degree in crops. Therefore, engineering Rubisco carboxylation capacity continues being a promising target for the improvement in photosynthesis and yield.

scholarly journals A beginner’s guide to macromolecular crystallization

2021 ◽  
Vol 43 (1) ◽  
pp. 36-43
Author(s):  
Fabrice Gorrec
Keyword(s):  
Crystal Nucleation ◽  
Trial And Error ◽  
Bioinformatics Analyses ◽  
X Ray ◽  
X Ray Crystallography ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Crystallization Conditions ◽  
Macromolecular Crystallization ◽  
Rate Limiting

Obtaining diffraction-quality crystals is currently the rate-limiting step in macromolecular X-ray crystallography of proteins, DNA, RNA or their complexes, in the vast majority of cases. Since each sample has different and specific characteristics – which is the reason for wanting to study every single one of them in the first place – crystallization conditions cannot be predicted. Hence, researchers must enable crystal nucleation and growth through experimentation and screening. The size, shape and surface of the sample or complexes of interest are often altered through genetic and biochemical manipulation to facilitate crystallization, based on bioinformatics analyses and trial and error. Pure samples are trialled against a very broad range of crystallization conditions. The currently predominant method to achieve crystallization is sitting drop vapour diffusion with nanolitre-class robotic liquid handlers. Once initial screening yields crystals, further optimization experiments are usually required to obtain larger and diffraction-quality crystals.

scholarly journals Iterative screen optimization maximizes the efficiency of macromolecular crystallization

2019 ◽  
Vol 75 (2) ◽  
pp. 123-131 ◽  
Author(s):  
Harrison G. Jones ◽  
Daniel Wrapp ◽  
Morgan S. A. Gilman ◽  
Michael B. Battles ◽  
Nianshuang Wang ◽  
...  
Keyword(s):  
Narrow Range ◽  
Three Dimensional ◽  
X Ray ◽  
X Ray Crystallography ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Crystallization Experiment ◽  
Automated Process ◽  
Macromolecular Crystallization ◽  
Rate Limiting

Advances in X-ray crystallography have streamlined the process of determining high-resolution three-dimensional macromolecular structures. However, a rate-limiting step in this process continues to be the generation of crystals that are of sufficient size and quality for subsequent diffraction experiments. Here, iterative screen optimization (ISO), a highly automated process in which the precipitant concentrations of each condition in a crystallization screen are modified based on the results of a prior crystallization experiment, is described. After designing a novel high-throughput crystallization screen to take full advantage of this method, the value of ISO is demonstrated by using it to successfully crystallize a panel of six diverse proteins. The results suggest that ISO is an effective method to obtain macromolecular crystals, particularly for proteins that crystallize under a narrow range of precipitant concentrations.

Ornithine Decarboxylase Activity in Cultured Endothelial Cells Stimulated by Serum, Thrombin and Serotonin

1978 ◽  
Vol 39 (02) ◽  
pp. 496-503 ◽  
Author(s):  
P A D’Amore ◽  
H B Hechtman ◽  
D Shepro
Keyword(s):  
Endothelial Cells ◽  
Ornithine Decarboxylase ◽  
Decarboxylase Activity ◽  
Aortic Endothelial Cells ◽  
Ornithine Decarboxylase Activity ◽  
Rate Limiting Step ◽  
Bovine Aortic Endothelial Cells ◽  
Limiting Step ◽  
Rate Limiting ◽  
Serum Serotonin

SummaryOrnithine decarboxylase (ODC) activity, the rate-limiting step in the synthesis of polyamines, can be demonstrated in cultured, bovine, aortic endothelial cells (EC). Serum, serotonin and thrombin produce a rise in ODC activity. The serotonin-induced ODC activity is significantly blocked by imipramine (10-5 M) or Lilly 11 0140 (10-6M). Preincubation of EC with these blockers together almost completely depresses the 5-HT-stimulated ODC activity. These observations suggest a manner by which platelets may maintain EC structural and metabolic soundness.

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