Summary of Study ST003202

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001996. The data can be accessed directly via it's Project DOI: 10.21228/M8QH90 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003202
Study Title	Integrated multi-omics unveil the impact of the phosphinic compounds, desmethylphosphinothricin and its keto-analogue, on Escherichia coli metabolism
Study Summary	Desmethylphosphinothricin (L-Glu-γ-PH) is the phosphinic analogue of glutamate with a carbon-phosphorus (C-P) bond. In L-Glu-γ-PH the phosphinic group acts as a bioisostere of glutamate γ-carboxyl group allowing the molecule to be a substrate of Escherichia coli glutamate decarboxylase, a pyridoxal 5’-phosphate (PLP)-dependent α-decarboxylase. In addition, the L-Glu-γ-PH decarboxylation product, GABA-PH, is further metabolized by bacterial GABA-transaminase, another PLP-dependent enzyme, and succinic semialdehyde dehydrogenease, a NADP+ -dependent enzyme. The product of these consecutive reactions, the so-called GABA shunt, lead to the formation of succinate-PH, the phosphinic analogue of succinate, a TCA cycle intermediate. Notably, L-Glu-γ-PH displays an antibacterial activity of the same order of well-established antibiotics in E. coli. The dipeptide L-Leu-Glu-γ-PH was shown to display a higher efficacy, likely as a consequence of an improved penetration into the bacteria. Herein, with the aim of further understanding the intracellular effects of L-Glu-γ-PH, 1H NMR-based metabolomics and LC-MS-based shotgun proteomics were used. This study included also the keto-analogue of L-Glu-γ-PH, α-ketoglutarate-γ-PH (α-KG-γ-PH), which also exhibits antimicrobial activity. L-Glu-γ-PH and α-KG-γ-PH were found to similarly impact the bacterial metabolism, though the overall effect of α-KG-γ-PH is more pervasive, and not exclusively because of its intracellular conversion into L-Glu-γ-PH. Notably, both molecules impact the pathways where aspartate, glutamate and glutamine are used as precursors for the biosynthesis of related metabolites, activate the acid stress response and deprive cells of nitrogen. This work highlights the multi-target drug potential of L-Glu-γ-PH and α-KG-γ-PH and paves the way for their exploitation as antimicrobials.
Institute	ITQB NOVA
Last Name	Gonçalves
First Name	Luís
Address	Avenida Republica, Oeiras, Not USCanada, 2780-157 Oeiras, Portugal
Email	lgafeira@itqb.unl.pt
Phone	214469464
Submit Date	2024-05-08
Raw Data Available	Yes
Raw Data File Type(s)	fid
Analysis Type Detail	NMR
Release Date	2024-08-08
Release Version	1

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Project:

Project ID:	PR001996
Project DOI:	doi: 10.21228/M8QH90
Project Title:	Integrated multi-omics unveil the impact of the phosphinic compounds, desmethylphosphinothricin and its keto-analogue, on Escherichia coli metabolism
Project Summary:	Desmethylphosphinothricin (L-Glu-γ-PH) is the phosphinic analogue of glutamate with a carbon-phosphorus (C-P) bond. In L-Glu-γ-PH the phosphinic group acts as a bioisostere of glutamate γ-carboxyl group allowing the molecule to be a substrate of Escherichia coli glutamate decarboxylase, a pyridoxal 5’-phosphate (PLP)-dependent α-decarboxylase. In addition, the L-Glu-γ-PH decarboxylation product, GABA-PH, is further metabolized by bacterial GABA-transaminase, another PLP-dependent enzyme, and succinic semialdehyde dehydrogenease, a NADP+ -dependent enzyme. The product of these consecutive reactions, the so-called GABA shunt, lead to the formation of succinate-PH, the phosphinic analogue of succinate, a TCA cycle intermediate. Notably, L-Glu-γ-PH displays an antibacterial activity of the same order of well-established antibiotics in E. coli. The dipeptide L-Leu-Glu-γ-PH was shown to display a higher efficacy, likely as a consequence of an improved penetration into the bacteria. Herein, with the aim of further understanding the intracellular effects of L-Glu-γ-PH, 1H NMR-based metabolomics and LC-MS-based shotgun proteomics were used. This study included also the keto-analogue of L-Glu-γ-PH, α-ketoglutarate-γ-PH (α-KG-γ-PH), which also exhibits antimicrobial activity. L-Glu-γ-PH and α-KG-γ-PH were found to similarly impact the bacterial metabolism, though the overall effect of α-KG-γ-PH is more pervasive, and not exclusively because of its intracellular conversion into L-Glu-γ-PH. Notably, both molecules impact the pathways where aspartate, glutamate and glutamine are used as precursors for the biosynthesis of related metabolites, activate the acid stress response and deprive cells of nitrogen. This work highlights the multi-target drug potential of L-Glu-γ-PH and α-KG-γ-PH and paves the way for their exploitation as antimicrobials.
Institute:	ITQB NOVA
Last Name:	Gonçalves
First Name:	Luís G
Address:	Avenida Republica, Oeiras, Not USCanada, 2780-157 Oeiras, Portugal
Email:	lgafeira@itqb.unl.pt
Phone:	214469464

Subject:

Subject ID:	SU003321
Subject Type:	Bacteria
Subject Species:	Escherichia coli
Taxonomy ID:	562

Factors:

Subject type: Bacteria; Subject species: Escherichia coli (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Condition
SA348696	N	E. coli	a-Ketoglutarate-PH
SA348697	S	E. coli	a-Ketoglutarate-PH
SA348698	T	E. coli	a-Ketoglutarate-PH
SA348699	M	E. coli	a-Ketoglutarate-PH
SA348700	AL	E. coli	a-Ketoglutarate-PH
SA348701	AB	E. coli	a-Ketoglutarate-PH
SA348702	AC	E. coli	a-Ketoglutarate-PH
SA348703	AI	E. coli	a-Ketoglutarate-PH
SA348704	E	E. coli	a-Ketoglutarate-PH
SA348705	F	E. coli	a-Ketoglutarate-PH
SA348677	G	E. coli	Control
SA348678	H	E. coli	Control
SA348679	AO	E. coli	Control
SA348680	A	E. coli	Control
SA348681	AP	E. coli	Control
SA348682	AN	E. coli	Control
SA348683	B	E. coli	Control
SA348684	AF	E. coli	Control
SA348685	AE	E. coli	Control
SA348686	AM	E. coli	Control
SA348687	I	E. coli	L-Glutamate-y-PH
SA348688	Q	E. coli	L-Glutamate-y-PH
SA348689	R	E. coli	L-Glutamate-y-PH
SA348690	Z	E. coli	L-Glutamate-y-PH
SA348691	D	E. coli	L-Glutamate-y-PH
SA348692	C	E. coli	L-Glutamate-y-PH
SA348693	AG	E. coli	L-Glutamate-y-PH
SA348694	AA	E. coli	L-Glutamate-y-PH
SA348695	AH	E. coli	L-Glutamate-y-PH

Showing results 1 to 29 of 29

Showing results 1 to 29 of 29

Collection:

Collection ID:	CO003314
Collection Summary:	E. coli K12 strain MG1655 cultures, were started from from overnight pre-cultures. Three growths conditions were performed: control, in the presence of L-Glu-γ-PH and in the presence of α-KG-γ-PH. Phosphinic compounds concentrations chosen were close to MIC60, i.e. 3.5 µg/ml and 8.5 µg/ml, respectively. Bacterial cultures were incubated at 37 °C, 150 rpm until late-exponential phase (OD600 = 1.0), which was reached after 17.4 ± 0.9, 24.0 ± 2.5 and 26.0 ± 3.0 hours for the untreated cultures, the L-Glu-γ-PH-treated and the α-KG-γ-PH-treated cultures, respectively.
Collection Protocol Filename:	LG_protocol.txt
Sample Type:	E. coli

Treatment:

Treatment ID:	TR003330
Treatment Summary:	Samples were split in two 50-ml falcon tubes and immediately transferred to a -35 °C bath (Julabo FP50‐HE) for 2.5 minutes, for metabolism quenching. Then, the tubes were centrifuged for 5 min at 5,000 x g at 4 °C, to obtain the bacterial pellets to which two different protocols were applied in order to obtain samples for metabolomic and proteomic analysis. The cell pellets were washed in 1.5 ml of cold physiological solution, then transferred in a 2-ml eppendorf tube and centrifuged for 5 min at 5,000 x g at 4 °C; the supernatants were discarded, and each pellet was resuspended in 1 ml of cold physiological solution. The OD600 was measured using a 5-µl aliquot diluted 1:200. Based on the OD600 readings, the volume corresponding to OD600 = 25 (~350 µl) was transferred to another 2 ml eppendorf tube and centrifuged for 5 min at 5,000 x g, at 4 °C. The physiological solution was discarded, and the pellets immediately frozen in liquid nitrogen and stored at -20 °C.

Treatment ID:

TR003330

Treatment Summary:

Samples were split in two 50-ml falcon tubes and immediately transferred to a -35 °C bath (Julabo FP50‐HE) for 2.5 minutes, for metabolism quenching. Then, the tubes were centrifuged for 5 min at 5,000 x g at 4 °C, to obtain the bacterial pellets to which two different protocols were applied in order to obtain samples for metabolomic and proteomic analysis. The cell pellets were washed in 1.5 ml of cold physiological solution, then transferred in a 2-ml eppendorf tube and centrifuged for 5 min at 5,000 x g at 4 °C; the supernatants were discarded, and each pellet was resuspended in 1 ml of cold physiological solution. The OD600 was measured using a 5-µl aliquot diluted 1:200. Based on the OD600 readings, the volume corresponding to OD600 = 25 (~350 µl) was transferred to another 2 ml eppendorf tube and centrifuged for 5 min at 5,000 x g, at 4 °C. The physiological solution was discarded, and the pellets immediately frozen in liquid nitrogen and stored at -20 °C.

Sample Preparation:

Sampleprep ID:	SP003328
Sampleprep Summary:	Pellets stored for metabolomics studies were thawed on ice, resuspended in 750 µl of ice-cold methanol (60% v/v), submitted to three freeze‐thaw cycles using liquid nitrogen, and centrifuged for 5 minutes at 12,900 x g, at 4 °C. The supernatants were transferred into clean 1.5-ml eppendorf tubes, and the extraction procedure was repeated a second time on the residual pellet. Samples were then concentrated down to dryness using a speed‐vacuum concentrator (Vacufuge® plus, Eppendorf) and stored at -20 °C until use. Dried samples were dissolved in 250 µl of potassium phosphate buffer 0.35 M, pH 7.0, 451.5 µl D2O with 2 mM sodium azide and 50 µl D2O with 3.2 mM TSP (final volume 751.5 µl per sample). Subsequently, 600 µl of each sample was transferred to a 5 mm NMR tube for 1H NMR spectra acquisition. For 31P NMR, after 1H NMR acquisition, samples were pooled, concentrated by speed-vacuum concentrator (as above) and dissolved in 1 ml of D2O and transferred to a new 5 mm tube. Prior to 31P NMR spectra acquisition, samples were measured for pH, which ranged between 7.1-7.4.

Sampleprep ID:

SP003328

Sampleprep Summary:

Pellets stored for metabolomics studies were thawed on ice, resuspended in 750 µl of ice-cold methanol (60% v/v), submitted to three freeze‐thaw cycles using liquid nitrogen, and centrifuged for 5 minutes at 12,900 x g, at 4 °C. The supernatants were transferred into clean 1.5-ml eppendorf tubes, and the extraction procedure was repeated a second time on the residual pellet. Samples were then concentrated down to dryness using a speed‐vacuum concentrator (Vacufuge® plus, Eppendorf) and stored at -20 °C until use. Dried samples were dissolved in 250 µl of potassium phosphate buffer 0.35 M, pH 7.0, 451.5 µl D2O with 2 mM sodium azide and 50 µl D2O with 3.2 mM TSP (final volume 751.5 µl per sample). Subsequently, 600 µl of each sample was transferred to a 5 mm NMR tube for 1H NMR spectra acquisition. For 31P NMR, after 1H NMR acquisition, samples were pooled, concentrated by speed-vacuum concentrator (as above) and dissolved in 1 ml of D2O and transferred to a new 5 mm tube. Prior to 31P NMR spectra acquisition, samples were measured for pH, which ranged between 7.1-7.4.

Analysis:

Analysis ID:	AN005252
Analysis Type:	NMR
Analysis Protocol File:	LG_protocol.txt
Num Factors:	3
Num Metabolites:	62
Units:	nmol

NMR:

NMR ID:	NM000281
Analysis ID:	AN005252
Instrument Name:	Bruker Avance II+ 800 MHz
Instrument Type:	FT-NMR
NMR Experiment Type:	1D-1H
Spectrometer Frequency:	800