An enzymatic fluorescent assay for the quantification of phosphite in a microtiter plate format

Berkowitz, Oliver; Jost, Ricarda; Pearse, Stuart J.; Lambers, Hans; Finnegan, Patrick M.; Hardy, G.; O’Brien, P.A.

doi:10.1016/j.ab.2011.01.014

Cited by 15 publications

(25 citation statements)

References 26 publications

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“…Current evidence suggests that plants do not assimilate Ph i into organic compounds and that oxidation of Ph i to P i as occurs in microbes is unlikely (Carswell et al ., 1997; Danova-Alt et al ., 2008; Berkowitz et al ., 2011). However, this does not rule out the possibility that Ph i affects metabolism by mimicking P i , for example by competing for catalytic sites or in the allosteric regulation of enzymatic reactions.…”

Section: Resultsmentioning

confidence: 99%

“…After clearing the lysate by centrifugation at 14 000 g and 4 °C for 15min, the supernatant was assayed for phosphate and phosphite as described previously (Ames, 1966; Berkowitz et al ., 2011). …”

Section: Methodsmentioning

confidence: 99%

“…Ph i is metabolically inert in plants, does not enter the organic P pool and thus accumulates in the tissues (Carswell et al ., 1996, 1997; Danova-Alt et al ., 2008; Berkowitz et al ., 2011). The treatment of severely P i -starved plants with Ph i suppresses the activation of many PSR genes and it is therefore assumed that Ph i is recognized by the same sensing mechanism as P i .…”

Section: Introductionmentioning

confidence: 99%

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Acclimation responses of Arabidopsis thaliana to sustained phosphite treatments

Berkowitz

Jost

Kollehn

et al. 2013

Journal of Experimental Botany

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Phosphite () induces a range of physiological and developmental responses in plants by disturbing the homeostasis of the macronutrient phosphate. Because of its close structural resemblance to phosphate, phosphite impairs the sensing, membrane transport, and subcellular compartmentation of phosphate. In addition, phosphite induces plant defence responses by an as yet unknown mode of action. In this study, the acclimation of Arabidopsis thaliana plants to a sustained phosphite supply in the growth medium was investigated and compared with plants growing under varying phosphate supplies. Unlike phosphate, phosphite did not suppress the formation of lateral roots in several Arabidopsis accessions. In addition, the expression of well-documented phosphate-starvation-induced genes, such as miRNA399d and At4, was not repressed by phosphite accumulation, whilst the induction of PHT1;1 and PAP1 was accentuated. Thus, a mimicking of phosphate by phosphite was not observed for these classical phosphate-starvation responses. Metabolomic analysis of phosphite-treated plants showed changes in several metabolite pools, most prominently those of aspartate, asparagine, glutamate, and serine. These alterations in amino acid pools provide novel insights for the understanding of phosphite-induced pathogen resistance.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Acclimation responses of Arabidopsis thaliana to sustained phosphite treatments

Berkowitz

Jost

Kollehn

et al. 2013

Journal of Experimental Botany

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“…PTDH with dual cofactor specificity has been fused to flavin monooxygenases and the resulting self-sufficient biocatalysts have excellent activities for enantioselective catalysis [6]–[9]. PTDH has also found use in fluorescence assays to quantify phosphite concentrations in plants [10].…”

Section: Introductionmentioning

confidence: 99%

Chemical Rescue and Inhibition Studies to Determine the Role of Arg301 in Phosphite Dehydrogenase

et al. 2014

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Phosphite dehydrogenase (PTDH) catalyzes the NAD+-dependent oxidation of phosphite to phosphate. This reaction requires the deprotonation of a water nucleophile for attack on phosphite. A crystal structure was recently solved that identified Arg301 as a potential base given its proximity and orientation to the substrates and a water molecule within the active site. Mutants of this residue showed its importance for efficient catalysis, with about a 100-fold loss in k cat and substantially increased K m,phosphite for the Ala mutant (R301A). The 2.35 Å resolution crystal structure of the R301A mutant with NAD+ bound shows that removal of the guanidine group renders the active site solvent exposed, suggesting the possibility of chemical rescue of activity. We show that the catalytic activity of this mutant is restored to near wild-type levels by the addition of exogenous guanidinium analogues; Brønsted analysis of the rates of chemical rescue suggests that protonation of the rescue reagent is complete in the transition state of the rate-limiting step. Kinetic isotope effects on the reaction in the presence of rescue agents show that hydride transfer remains at least partially rate-limiting, and inhibition experiments show that K i of sulfite with R301A is ∼400-fold increased compared to the parent enzyme, similar to the increase in K m for phosphite in this mutant. The results of our experiments indicate that Arg301 plays an important role in phosphite binding as well as catalysis, but that it is not likely to act as an active site base.

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“…As expected, no signal of the PTXD protein was observed in the WT control, whereas in both CrB‐1 and CrP‐13 transgenic lines, the signals corresponding to PTXD were present (Figure b). We determined PTXD activity in several C. reinhardtii transgenic strains, using an optimized fluorometric protocol previously reported (Berkowitz et al ., ). We found that strains CrB‐1, CrP‐6, CrP‐13, CrX‐3 and CrX‐9 showed significant levels of PTXD activity, which correlated with their capability to metabolize Phi (Figure c).…”

Section: Resultsmentioning

confidence: 97%

A novel genetic engineering platform for the effective management of biological contaminants for the production of microalgae

Loera-Quezada

Leyva‐González

Velázquez‐Juárez

et al. 2016

Plant Biotechnology Journal

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SummaryMicroalgal cultivation that takes advantage of solar energy is one of the most cost‐effective systems for the biotechnological production of biofuels, and a range of high value products, including pharmaceuticals, fertilizers and feed. However, one of the main constraints for the cultivation of microalgae is the potential contamination with biological pollutants, such as bacteria, fungi, zooplankton or other undesirable microalgae. In closed bioreactors, the control of contamination requires the sterilization of the media, containers and all materials, which increases the cost of production, whereas open pond systems severely limits the number of species that can be cultivated under extreme environmental conditions to prevent contaminations. Here, we report the metabolic engineering of Chlamydomonas reinhardtii to use phosphite as its sole phosphorus source by expressing the ptxD gene from Pseudomonas stutzeri WM88, which encodes a phosphite oxidoreductase able to oxidize phosphite into phosphate using NAD as a cofactor. Engineered C. reinhardtii lines are capable of becoming the dominant species in a mixed culture when fertilized with phosphite as a sole phosphorus source. Our results represent a new platform for the production of microalgae, potentially useful for both closed photobioreactors and open pond systems without the need for using sterile conditions nor antibiotics or herbicides to prevent contamination with biological pollutants.

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An enzymatic fluorescent assay for the quantification of phosphite in a microtiter plate format

Cited by 15 publications

References 26 publications

Acclimation responses of Arabidopsis thaliana to sustained phosphite treatments

Acclimation responses of Arabidopsis thaliana to sustained phosphite treatments

Chemical Rescue and Inhibition Studies to Determine the Role of Arg301 in Phosphite Dehydrogenase

A novel genetic engineering platform for the effective management of biological contaminants for the production of microalgae

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