Synthesis of 6-phosphofructose aspartic acid and some related Amadori compounds

Hansen, Alexandar L.; Behrman, E. J.

doi:10.1016/j.carres.2016.05.003

Cited by 17 publications

(22 citation statements)

References 15 publications

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“…We first used biochemical assays to measure FraD and FraB activities in crude lysates prepared from wild-type Salmonella and in mutants lacking either fraB, fraD or the entire fra island, all grown for a short duration in 5 mM F-Asn (see Methods ). These measurements were made possible due to in-house synthesis of F-Asp and 6-P-F-Asp, the substrates of FraD and FraB, respectively17, and design of coupled assays that enabled facile colorimetric readouts (Sengupta and Gopalan, manuscript in preparation). Comparing the wild type and fra island deletion mutant offers a positive and negative control cohort (Table 1).…”

Section: Resultsmentioning

confidence: 99%

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A metabolic intermediate of the fructose-asparagine utilization pathway inhibits growth of a Salmonella fraB mutant

Sabag-Daigle

Blunk

Sengupta

et al. 2016

Sci Rep

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Insertions in the Salmonella enterica fra locus, which encodes the fructose-asparagine (F-Asn) utilization pathway, are highly attenuated in mouse models of inflammation (>1000-fold competitive index). Here, we report that F-Asn is bacteriostatic to a fraB mutant (IC50 19 μM), but not to the wild-type or a fra island deletion mutant. We hypothesized that the presence of FraD kinase and absence of FraB deglycase causes build-up of a toxic metabolite: 6-phosphofructose-aspartate (6-P-F-Asp). We used biochemical assays to assess FraB and FraD activities, and mass spectrometry to confirm that the fraB mutant accumulates 6-P-F-Asp. These results, together with our finding that mutants lacking fraD or the fra island are not attenuated in mice, suggest that the extreme attenuation of a fraB mutant stems from 6-P-F-Asp toxicity. Salmonella FraB is therefore an excellent drug target, a prospect strengthened by the absence of the fra locus in most of the gut microbiota.

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Section: Resultsmentioning

confidence: 99%

“…6-Phosphofructose-aspartate (6-P-F-Asp) could be made only by the glycerol (or ethylene glycol) procedure because the starting material, glucose-6-phosphate, is insoluble in methanol. 6-P-F-Asp was characterized by mass spectrometry and by both proton and 13 C NMR, and these details are provided elsewhere17.…”

Section: Methodsmentioning

confidence: 99%

A metabolic intermediate of the fructose-asparagine utilization pathway inhibits growth of a Salmonella fraB mutant

Sabag-Daigle

Blunk

Sengupta

et al. 2016

Sci Rep

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“…Synthesis of Fructose-Aspartate and its analogues: Fructose-D-aspartate, fructose-L-glutamate, and tagatose-L-aspartate were made under the same conditions as those described for fructose-L-aspartate (Hansen and Behrman, 2016). Some modifications were required for the synthesis of fructose-L-cysteic acid, xylulose-L-aspartate and ribulose-L-aspartate.…”

Section: Methodsmentioning

confidence: 99%

Characterization of a Salmonella sugar kinase essential for the utilization of fructose-asparagine

Biswas

Behrman

Gopalan

2017

Biochem. Cell Biol.

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Salmonella can utilize fructose-asparagine (F-Asn), a naturally occurring Amadori product, as its sole carbon and nitrogen source. Conversion of F-Asn to the common intermediates glucose-6-phosphate, aspartate, and ammonia was predicted to involve the sequential action of an asparaginase, a kinase, and a deglycase. Mutants lacking the deglycase are highly attenuated in mouse models of intestinal inflammation owing to the toxic build-up of the deglycase substrate. The limited distribution of this metabolic pathway in the animal gut microbiome raises the prospects for antibacterial discovery. We report the biochemical characterization of the kinase that was expected to transform fructose-aspartate to 6-phosphofructose-aspartate during F-Asn utilization. In addition to confirming its anticipated function, we determined through studies of fructose-aspartate analogues that this kinase exhibits a substrate-specificity with greater tolerance to changes to the amino acid (including the d-isomer of aspartate) than to the sugar.

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“…F-Asp and F-Asn were synthesized and purified as described elsewhere (17). In the case of F-Asn, the final preparation from this synthesis still contains between 0.1 and 0.2 mol of ammonium ion per mole (i.e., 85% zwitterion and 15% ammonium salt).…”

Section: Methodsmentioning

confidence: 99%

Salmonella FraE, an Asparaginase Homolog, Contributes to Fructose-Asparagine but Not Asparagine Utilization

et al. 2017

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Salmonella enterica can utilize fructose-asparagine (F-Asn) as a source of carbon and nitrogen. This capability has been attributed to five genes in the fra locus. Previously, we determined that mutations in fraB (deglycase), fraD (kinase), or fraA (transporter) eliminated the ability of Salmonella to grow on F-Asn, while a mutation in fraE allowed partial growth. We hypothesized that FraE, a putative periplasmic fructose-asparaginase, converts F-Asn to NH4 + and fructose-aspartate (F-Asp). FraA could then transport F-Asp into the cytoplasm for subsequent catabolism. Here, we report that growth of the fraE mutant on F-Asn is caused by a partially redundant activity provided by AnsB, a periplasmic asparaginase. Indeed, a fraE ansB double mutant is unable to grow on F-Asn. Moreover, biochemical assays using periplasmic extracts of mutants that express only FraE or AnsB confirmed that each of these enzymes converts F-Asn to F-Asp and NH4 +. However, FraE does not contribute to growth on asparagine. We tested and confirmed the hypothesis that a fraE ansB mutant can grow on F-Asp, while mutants lacking fraA, fraD, or fraB cannot. This finding provides strong evidence that FraA transports F-Asp but not F-Asn from the periplasm to the cytoplasm. Previously, we determined that F-Asn is toxic to a fraB mutant due to the accumulation of the FraB substrate, 6-phosphofructose-aspartate (6-P-F-Asp). Here, we found that, as expected, a fraB mutant is also inhibited by F-Asp. Collectively, these findings contribute to a better understanding of F-Asn utilization by Salmonella. IMPORTANCE Salmonella is able to utilize fructose-asparagine (F-Asn) as a nutrient. We recently reported that the disruption of a deglycase enzyme in the F-Asn utilization pathway inhibits the growth of Salmonella in mice and recognized this pathway as a novel and specific drug target. Here, we characterize the first step in the pathway wherein FraE hydrolyzes F-Asn to release NH4 + and F-Asp in the periplasm of the cell. A fraE mutant continues to grow slowly on F-Asn due to asparaginase activity encoded by ansB.

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Synthesis of 6-phosphofructose aspartic acid and some related Amadori compounds

Abstract: We describe the synthesis and characterization of 6-phosphofructose-aspartic acid, an intermediate in the metabolism of fructose-asparagine by Salmonella. We also report improved syntheses of fructose-asparagine itself and of fructose-aspartic acid.

Cited by 17 publications

References 15 publications

A metabolic intermediate of the fructose-asparagine utilization pathway inhibits growth of a Salmonella fraB mutant

A metabolic intermediate of the fructose-asparagine utilization pathway inhibits growth of a Salmonella fraB mutant

Characterization of a Salmonella sugar kinase essential for the utilization of fructose-asparagine

Salmonella FraE, an Asparaginase Homolog, Contributes to Fructose-Asparagine but Not Asparagine Utilization

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