Trehalose 6‐phosphate phosphatases of<i>Pseudomonas aeruginosa</i>

Cross, Megan; Biberacher, Sonja; Park, Suk-Youl; Rajan, Siji; Korhonen, Pasi K.; Gasser, Robin B.; Kim, Ji Soo; Coster, Mark J.; Hofmann, Andreas

doi:10.1096/fj.201800500r

Cited by 10 publications

(11 citation statements)

References 88 publications

(81 reference statements)

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“…EDTA has no clinical utility as an antibacterial, but inhibitors with greater specificity have been reported. For example, trehalose 6 phosphate has been reported to be an inhibitor of the P. aeruginosa OtsB [ 75 ]. In M. smegmatis and M. tuberculosis the antibiotics diumycin and moenomycin can inhibit OtsB activity, but the doses required for 50% inhibition are high (50 and 100 µg/ml) and these drugs were less active toward the M. tuberculosis enzyme [ 76 ].…”

Section: Introductionmentioning

confidence: 99%

Trehalose and bacterial virulence

Vanaporn

Titball

2020

Virulence

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Trehalose is a disaccharide of two D-glucose molecules linked by a glycosidic linkage, which plays both structural and functional roles in bacteria. Trehalose can be synthesized and degraded by several pathways, and induction of trehalose biosynthesis is typically associated with exposure to abiotic stress. The ability of trehalose to protect against abiotic stress has been exploited to stabilize a range of bacterial vaccines. More recently, there has been interest in the role of this molecule in microbial virulence. There is now evidence that trehalose or trehalose derivatives play important roles in virulence of a diverse range of Gram-positive and Gram-negative pathogens of animals or plants. Trehalose and/or trehalose derivatives can play important roles in host colonization and growth in the host, and can modulate the interactions with host defense mechanisms. However, the roles are typically pathogen-specific. These findings suggest that trehalose metabolism may be a target for novel pathogen-specific rather than broad spectrum interventions.

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Section: Introductionmentioning

confidence: 99%

Trehalose and bacterial virulence

Vanaporn

Titball

2020

Virulence

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“…Given the exquisite substrate specificity of TPPs 32–35 , there is a strict requirement for the original and rather expensive substrate trehalose-6-phosphate in enzyme assays testing the activity and inhibition of these proteins. Therefore, we employed an economical two-tiered screening approach that consisted of a first-stage screening of compound libraries with a ligand binding assay based on thermal protein denaturation and a second-stage validation of hits in enzyme activity assays 31,36 .…”

Section: Resultsmentioning

confidence: 99%

“…The reason for this susceptibility is not entirely clear, but we speculate that the conjugation of one or both cysteine residues specific to P. aeruginosa (Cys85, Cys231) might lead to conformational alterations that restrict access to the substrate binding site. In particular, it seems likely that modification of the side chain of Cys85 causes a rearrangement of the residues lining the rather narrow access to the active site 35 .…”

Section: Discussionmentioning

confidence: 99%

“…The TPPs investigated in this study included the enzymes from the nematodes A. ceylanicum , T. canis and H. contortus ; the bacteria M. tuberculosis , Stenotrophomonas maltophilia and Streptococcus pneumoniae ; and the extrachromosomal TPP from P. aeruginosa 35,45 . All proteins were expressed and purified as over-expressed recombinant proteins according to our previously published protocol 45 .…”

Section: Methodsmentioning

confidence: 99%

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A suicide inhibitor of nematode trehalose-6-phosphate phosphatases

Cross

York

Długosz

et al. 2019

Sci Rep

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Protein-based drug discovery strategies have the distinct advantage of providing insights into the molecular mechanisms of chemical effectors. Currently, there are no known trehalose-6-phosphate phosphatase (TPP) inhibitors that possess reasonable inhibition constants and chemical scaffolds amenable to convenient modification. In the present study, we subjected recombinant TPPs to a two-tiered screening approach to evaluate several diverse compound groups with respect to their potential as TPP inhibitors. From a total of 5452 compounds tested, N-(phenylthio)phthalimide was identified as an inhibitor of nematode TPPs with apparent Ki values of 1.0 μM and 0.56 μM against the enzymes from the zoonotic roundworms Ancylostoma ceylanicum and Toxocara canis, respectively. Using site-directed mutagenesis, we demonstrate that this compound acts as a suicide inhibitor that conjugates a strictly conserved cysteine residue in the vicinity of the active site of nematode TPPs. The anthelmintic properties of N-(phenylthio)phthalimide were assessed in whole nematode assays using larvae of the ascaroids T. canis and T. cati, as well as the barber’s pole worm Haemonchus contortus. The compound was particularly effective against each of the ascaroids with an IC50 value of 9.3 μM in the survival assay of T. cati larvae, whereas no bioactivity was observed against H. contortus.

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“…Most of the effort in these studies has been directed toward investigating the impact of diffusion in fractal media on the reaction kinetics (22), with little focus on characterizing the effect of crowding on the mean effective enzyme kinetics. However, because it has now been shown that in vitro, some enzymes might not be limited by their translational diffusion but by their apparent association rate constants (24)(25)(26)(27)(28), the reevaluation of crowding in these reactions is important. In this work, therefore, we introduced computational methods for studying spatial effects of any kind, applying our work to the effects of crowding on reaction-limited enzymes with the goal of bridging the discrepancy between the in vitro measurement of kinetic parameters and the actual in vivo conditions.…”

Section: Introductionmentioning

confidence: 99%

Particle-Based Simulation Reveals Macromolecular Crowding Effects on the Michaelis-Menten Mechanism

Weilandt

Hatzimanikatis

2019

Biophysical Journal

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Many computational models for analyzing and predicting cell physiology rely on in vitro data collected in dilute and controlled buffer solutions. However, this can mislead models because up to 40% of the intracellular volume-depending on the organism, the physiology, and the cellular compartment-is occupied by a dense mixture of proteins, lipids, polysaccharides, RNA, and DNA. These intracellular macromolecules interfere with the interactions of enzymes and their reactants and thus affect the kinetics of biochemical reactions, making in vivo reactions considerably more complex than the in vitro data indicates. In this work, we present a new, to our knowledge, type of kinetics that captures and quantifies the effect of volume exclusion and other spatial phenomena on the kinetics of elementary reactions. We further developed a framework that allows for the efficient parameterization of these kinetics using particle simulations. Our formulation, entitled generalized elementary kinetics, can be used to analyze and predict the effect of intracellular crowding on enzymatic reactions and was herein applied to investigate the influence of crowding on phosphoglycerate mutase in Escherichia coli, which exhibits prototypical reversible Michaelis-Menten kinetics. Current research indicates that many enzymes are reaction limited and not diffusion limited, and our results suggest that the influence of fractal diffusion is minimal for these reaction-limited enzymes. Instead, increased association rates and decreased dissociation rates lead to a strong decrease in the effective maximal velocities V max and the effective Michaelis-Menten constants K M under physiologically relevant volume occupancies. Finally, the effects of crowding were explored in the context of a linear pathway, with the finding that crowding can have a redistributing effect on the effective flux responses in the case of twofold enzyme overexpression. We suggest that this framework, in combination with detailed kinetics models, will improve our understanding of enzyme reaction networks under nonideal conditions. SIGNIFICANCE Kinetic models are essential for understanding and designing biochemical and biophysical processes in living organisms. Currently, kinetic models rely on the in vitro characterization of biochemical reactions, although intracellular reactions are taking place in crowded, nonideal conditions. The interactions of the enzymes and their reactants with other macromolecules in a cell alter the enzyme kinetics significantly, but little has been done to model and quantify the impact of these interactions on the in vivo reaction rates. We present a computational framework that allows us for the first time, to our knowledge, to estimate the in vivo apparent kinetic parameters of an enzyme that follows Michaelis-Menten kinetics. Interestingly, crowding conditions similar to those in Escherichia coli can reduce the maximal enzyme activity 10fold.

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Trehalose 6‐phosphate phosphatases ofPseudomonas aeruginosa

Cited by 10 publications

References 88 publications

Trehalose and bacterial virulence

Trehalose and bacterial virulence

A suicide inhibitor of nematode trehalose-6-phosphate phosphatases

Particle-Based Simulation Reveals Macromolecular Crowding Effects on the Michaelis-Menten Mechanism

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