Fluoropyruvate: An unusual substrate for Escherichia coli pyruvate dehydrogenase

Leung, Lim S.; Frey, Perry A.

doi:10.1016/0006-291x(78)91529-2

Cited by 28 publications

(6 citation statements)

References 5 publications

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“…DXPS activity is not suppressed in a time-dependent manner (over the course of 0.5–20 min) in the presence of 0.5 mM HPA (Figure S7b). In addition, acetate, the product of acetyl-ThDP hydrolysis, − was not detected by 1 H NMR [5 μM DXPS, 25 mM HPA, 25 °C (data not shown)]. Taken together, these results suggest that HPA does not cause time-dependent inactivation of DXPS via acetyl-ThDP formation.…”

Section: Resultsmentioning

confidence: 84%

Revealing Donor Substrate-Dependent Mechanistic Control on DXPS, an Enzyme in Bacterial Central Metabolism

Johnston

Meyers

2021

Biochemistry

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The thiamin diphosphate-dependent enzyme 1-deoxy- d -xylulose 5-phosphate synthase (DXPS) catalyzes the formation of DXP from pyruvate (donor) and d -glyceraldehyde 3-phosphate ( d -GAP, acceptor). DXPS is essential in bacteria but absent in human metabolism, highlighting it as a potential antibacterial drug target. The enzyme possesses unique structural and mechanistic features that enable development of selective inhibition strategies and raise interesting questions about DXPS function in bacterial pathogens. DXPS distinguishes itself within the ThDP enzyme class by its exceptionally large active site and random sequential mechanism in DXP formation. In addition, DXPS displays catalytic promiscuity and relaxed acceptor substrate specificity, yet previous studies have suggested a preference for pyruvate as the donor substrate when d -GAP is the acceptor substrate. However, such donor specificity studies are potentially hindered by a lack of knowledge about specific, alternative donor–acceptor pairs. In this study, we exploited the promiscuous oxygenase activity of DXPS to uncover alternative donor substrates for DXPS. Characterization of glycolaldehyde, hydroxypyruvate, and ketobutyrate as donor substrates revealed differences in stabilization of enzyme-bound intermediates and acceptor substrate usage, illustrating the influence of the donor substrate on reaction mechanism and acceptor specificity. In addition, we found that DXPS prevents abortive acetyl-ThDP formation from a DHEThDP carbanion/enamine intermediate, similar to transketolase, supporting the potential physiological relevance of this intermediate on DXPS. Taken together, these results offer clues toward alternative roles for DXPS in bacterial pathogen metabolism.

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Section: Resultsmentioning

confidence: 84%

Revealing Donor Substrate-Dependent Mechanistic Control on DXPS, an Enzyme in Bacterial Central Metabolism

Johnston

Meyers

2021

Biochemistry

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“…Similarly, 4-fluoroglutamate after deamination to 2-keto-4-fluoroglutarate could be metabolized via the tricarboxylic acid cycle and defluorinated by a similar mechanism. The pyruvate decarboxylase component of the pyruvate dehydrogenase complex is capable of defluorination of 3-fluoropyruvate in a number of species (Leung & Frey, 1978;Gish et a/., 1988;Darland e t al., 1986;Meyer & O'Hagan, 1992a). This reaction would explain the defluorination of both 3-fluoropyruvate and 3-fluoroalanine by S. cattlya.…”

Section: Fluorination By Streptomjces C a T T H Ymentioning

confidence: 99%

Biosynthesis of fluorinated secondary metabolites by Streptomyces cattleya

et al. 1995

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The biosynthesis of organof luorine compounds by Streptomyces cattleye NRRL 8057 was examined using 19F N M R spectroscopy. The organism produced 1.2 mM fluoroacetate and 0 5 mM 4-fluorothreonine as secondary metabolites when cultured for 28 d on a chemically defined medium containing 2 mM fluoride. Cell suspensions from batch cultures harvested at the growth maximum of 4 d were not capable of fluoride uptake or fluorometabolite biosynthesis, but by 6 d had developed an efficient fluoride-uptake system and biosynthesized the two f luorometabolites in almost equal proportions. As the harvest age increased, the proportion of f luoroacetate to 4-f luorothreonine formed by cell suspensions rose progressively so that 1640ld cells showed a ratio of 76:26 for the two compounds. Fluoride uptake and fluorometabolite production by cell suspensions were highly dependent on pH, with both processes showing a maximum rate a t pH 6 9 but declining rapidly at higher pH values. This decrease was particularly marked in the case of fluoroacetate biosynthesis which was barely detectable a t pH 7.5. Fluoroacetate and 4-f luorothreonine showed only low levels of interconversion by cell suspensions, suggesting that the carbon skeleton of neither was derived by metabolism of the other. The limited interconversion observed is explicable in terms of a small degree of biological def luorination occurring with each compound, followed by reincorporation of the resulting fluoride ion into the organic form by the active fluorinating system, a phenomenon also noted on incubation of cell suspensions with a number of other fluorinated biochemical intermediates. Cell suspensions were supplemented with a variety of amino acids and tricarboxylic acid cycle intermediates to determine the identity of the carbon substrate of the fluorinating system. No compound tested significantly increased the total amount of fluorometabolites formed or altered their relative proportions. However, in studies with 14C-labelled precursors, the highest level of incorporation into f luoroacetate by cell suspensions was recorded with [U-14C]glycolate, suggesting that this compound or an activated derivative may be the substrate for the fluorinating system in the biosynthesis of f luoroacetate.

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“…7). The former can be converted to acetyl CoA and introduced to intermediary metabolism, and the latter can be transformed to fluorolactate via lactate dehydrogenase or defluorinated by pyruvate dehydrogenase (Leung and Frey, 1978), -250 -240 -230 -220 -210 -200 -190 -180 -170 -160 -150 -140 -130 -120 -110 -100 f1 ( Fig. 6 19 F NMR spectra of (a) supernatant from cultures of P. pseudoalcaligenes KF707 incubated with difluorobiphenyl and (b) after the fluorometabolite with a resonance at -136 ppm was semi-purified.…”

Section: Resultsmentioning

confidence: 99%

Biodegradation of polyfluorinated biphenyl in bacteria

2010

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Fluorinated aromatic compounds are significant environmental pollutants, and microorganisms play important roles in their biodegradation. The effect of fluorine substitution on the transformation of fluorobiphenyl in two bacteria was investigated. Pseudomonas pseudoalcaligenes KF707 and Burkholderia xenovorans LB400 used 2,3,4,5,6-pentafluorobiphenyl and 4,4'-difluorobiphenyl as sole sources of carbon and energy. The catabolism of the fluorinated compounds was examined by gas chromatography-mass spectrometry and fluorine-19 nuclear magnetic resonance spectroscopy (19F NMR), and revealed that the bacteria employed the upper pathway of biphenyl catabolism to degrade these xenobiotics. The novel fluorometabolites 3-pentafluorophenyl-cyclohexa-3,5-diene-1,2-diol and 3-pentafluorophenyl-benzene-1,2-diol were detected in the supernatants of biphenyl-grown resting cells incubated with 2,3,4,5,6-pentafluorobiphenyl, most likely as a consequence of the actions of BphA and BphB. 4-Fluorobenzoate was detected in cultures incubated with 4,4'-difluorobiphenyl and 19F NMR analysis of the supernatant from P. pseudoalcaligenes KF707 revealed the presence of additional water-soluble fluorometabolites.

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Fluoropyruvate: An unusual substrate for Escherichia coli pyruvate dehydrogenase

Cited by 28 publications

References 5 publications

Revealing Donor Substrate-Dependent Mechanistic Control on DXPS, an Enzyme in Bacterial Central Metabolism

Revealing Donor Substrate-Dependent Mechanistic Control on DXPS, an Enzyme in Bacterial Central Metabolism

Biosynthesis of fluorinated secondary metabolites by Streptomyces cattleya

Biodegradation of polyfluorinated biphenyl in bacteria

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