<i>Escherichia coli</i> chorismate synthase

Bornemann, Stephen; Lowe, David J.; Thorneley, R. N. F.

doi:10.1042/bst0240084

Cited by 19 publications

(17 citation statements)

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“…[11] The third mutant (ΔaroC) is missing the gene encoding key shikimate pathway enzyme chorismate synthase, and is unable to synthesize not only PABA but also the aromatic amino acids L-phenylalanine, L-tyrosine, and L-tryptophan, as well as the aromatic metabolites para-hydroxybenzoic acid (PHBA) and 2,3-hydroxybenzoic acid. [12] We confirmed that all three strains were unable to grow in PABA-free M9 glycerol minimal media and established each strain's inability to utilize substrate 1 in place of PABA. All media used for the ΔaroC mutant were supplemented with the additional required nutrients.…”

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confidence: 59%

Rescuing Auxotrophic Microorganisms with Nonenzymatic Chemistry

2013

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Chemical manipulations of small molecule metabolites take place in all living systems and are essential for sustaining life through cellular metabolism. [1] The vast majority of these transformations are carried out by macromolecular, enzymatic catalysts. In contrast to the natural world, most chemical reactions employed in laboratory syntheses utilize nonbiological reagents and catalysts. [2] Although non-biological catalysts and reagents have been shown to function in the presence of cells, [3] the question of whether their reactivity can interface with cellular metabolism and influence biological function remains underexplored. We are interested in further developing this aspect of reaction methodology, which we term biocompatible chemistry: non-enzymatic chemical transformations that can be used to manipulate the structures of small molecules in the presence of living organisms. Successful integration of biocompatible chemistry with cellular metabolism would have implications for biotechnology as it could expand the capabilities of biotransformation in ways that are not possible using enzymes. Establishing a link between non-enzymatic catalysis and metabolism could also have potential implications for prebiotic chemistry, as it would suggest that primitive cells could have relied upon non-enzymatic transformations occurring in the surrounding environment to generate essential metabolites. [4] Here we describe biocompatible chemical reactions that interact with microbial metabolism and rescue auxotrophy through the production of essential metabolites. This experimental approach enables us to directly connect the growth of a living organism to the success of a non-biological chemical transformation. *Prof. E. P. Balskus, balskus@chemistry.harvard.edu, http://scholar.harvard.edu/balskus. Supporting information for this article is available on the WWW under http://www.angewandte.org. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptAuxotrophs are organisms that lack the ability to synthesize key nutrients required for growth and survival. [5] The necessary compounds must instead be obtained from external sources, typically the diet or metabolic activities of other organisms living in close proximity. The numerous examples of auxtotrophs in Nature include humans, who are unable to synthesize many essential vitamins and metabolites. It is possible to rescue auxotrophy in bacteria by transformation with genes encoding enzymes that can replace or complement the missing metabolic activity. Restoration of growth in this way constitutes an extremely powerful strategy for selection of evolved biomolecules in the context of directed evolution. [6] Recently, Hecht and co-workers demonstrated that it is possible to rescue the growth of E. coli auxotrophs using de novo protein scaffolds not found in natural systems. [7] Significantly, this result implies that catalysts other than native enzymes can replace key metabolic functions in living cells.Our interest in merging non-enzymat...

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confidence: 59%

Rescuing Auxotrophic Microorganisms with Nonenzymatic Chemistry

2013

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“…Kinetic isotope effect studies using the Neurospora crassa enzyme are consistent with a nonconcerted mechanism (Balasubramanian et al, 1995). An X-group mechanism, involving nucleophilic attack at the C(1) position of EPSP (Floss et al, 1972), appears to be unlikely (Hawkes et al, 1990;Bornemann et al, 1996). Studies with (6S)-6-fluoro-EPSP (3) (Scheme 1) (Bornemann et al, 1995a) and (6R)-[6-2 H]EPSP (Bornemann et al, 1995b) suggest that a mechanism involving the initial deprotonation at C(6) to form an anionic allylic intermediate is also unlikely.…”

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confidence: 67%

The Transient Kinetics of Escherichia coli Chorismate Synthase: Substrate Consumption, Product Formation, Phosphate Dissociation, and Characterization of a Flavin Intermediate

1996

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Chorismate synthase is the seventh enzyme of the shikimate pathway and catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate. The reaction involves the 1,4-elimination of phosphate and the C-(6proR) hydrogen of the substrate with unusual anti stereochemistry and requires a reduced flavin cofactor. This paper describes the kinetics of the formation and decay of a flavin intermediate, EPSP consumption, chorismate and phosphate formation, and phosphate dissociation during single and multiple turnover experiments, determined using rapid reaction techniques. The kinetics of phosphate dissociation using the substrate analogues (6R)-[6-2H]EPSP and (6S)-6-fluoro-EPSP have also been determined. The observations are consistent with a nonconcerted chorismate synthase reaction. The flavin intermediate is not simply associated with the conversion of substrate to product because it forms before the substrate is consumed. The transient spectral changes must be associated primarily with events such as protonation of the reduced flavin, a charge transfer complex between reduced flavin and an aromatic amino acid, or a conformational change in the protein. This does not rule out the direct role of flavin in catalysis.

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confidence: 99%

“…The strict requirement for a reduced FMN cofactor (2-4) and the trans-1,4-elimination of the 3-phosphate and the 6R-H (5-7) are both unusual aspects of the chorismate synthase reaction. A number of non-concerted mechanisms have been proposed to account for these properties (1,8,9).Evidence for a non-concerted mechanism includes a secondary tritium kinetic isotope effect at C(3) (10), the slow conversion of (6S)-6-fluoro-EPSP to 6-fluoro-chorismate (11), transient kinetics studies (12), and more recently, a secondary ␤ deuterium kinetic isotope effect at C(4) (13). These studies collectively make the radical (Scheme 1A) and E1 (Scheme 1B) mechanisms involving the initial cleavage of the C(3)-O bond the most likely, and alternative mechanisms where the C(6)-H bond breaks first the least likely (8,14).…”

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confidence: 99%

Studies with Substrate and Cofactor Analogues Provide Evidence for a Radical Mechanism in the Chorismate Synthase Reaction

Osborne¹,

Thorneley²,

Abell³

et al. 2000

Journal of Biological Chemistry

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Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate. The strict requirement for a reduced FMN cofactor and a trans-1,4-elimination are unusual. (6R)-6-Fluoro-EPSP was shown to be converted to chorismate stoichiometrically with enzyme-active sites in the presence of dithionite. This conversion was associated with the oxidation of FMN to give a stable flavin semiquinone. The IC 50 of the fluorinated substrate analogue was 0.5 and 250 M with the Escherichia coli enzyme, depending on whether it was preincubated with the enzyme or not. The lack of dissociation of the flavin semiquinone and chorismate from the enzyme appears to be the basis of the essentially irreversible inhibition by this analogue. A dithionite-dependent transient formation of flavin semiquinone during turnover of (6S)-6-fluoro-EPSP has been observed. These reactions are best rationalized by radical chemistry that is strongly supportive of a radical mechanism occurring during normal turnover. The lack of activity with 5-deaza-FMN provides additional evidence for the role of flavin in catalysis by the E. coli enzyme.Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) 1 to chorismate (Scheme 1) (1). This enzyme is the seventh enzyme of the shikimate pathway and is present in bacteria, fungi, and plants but not in mammals. It is therefore a potential target for antimicrobial agents and herbicides. The strict requirement for a reduced FMN cofactor (2-4) and the trans-1,4-elimination of the 3-phosphate and the 6R-H (5-7) are both unusual aspects of the chorismate synthase reaction. A number of non-concerted mechanisms have been proposed to account for these properties (1,8,9).Evidence for a non-concerted mechanism includes a secondary tritium kinetic isotope effect at C(3) (10), the slow conversion of (6S)-6-fluoro-EPSP to 6-fluoro-chorismate (11), transient kinetics studies (12), and more recently, a secondary ␤ deuterium kinetic isotope effect at C(4) (13). These studies collectively make the radical (Scheme 1A) and E1 (Scheme 1B) mechanisms involving the initial cleavage of the C(3)-O bond the most likely, and alternative mechanisms where the C(6)-H bond breaks first the least likely (8,14). Evidence for the radical mechanism (Scheme 1A), which provides a role for the reduced flavin in catalysis, comes from the lack of activity of the bifunctional Neurospora crassa enzyme with 5-deaza-FMN (15) and the formation of a flavin semiquinone with the monofunctional Escherichia coli enzyme and the inhibitor (6R)-6-fluoro-EPSP (16).This paper describes new studies with the E. coli enzyme that are strongly supportive of the proposed radical mechanism (Scheme 1A). The product of the reaction with the inhibitor (6R)-6-fluoro-EPSP has been identified, allowing the formation of the stable flavin semiquinone radical in the presence of dithionite to be rationalized. The formation of a flavin semiquinone radical during turnover of (6S)-6-fluoro-EPSP has been observed in the prese...

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Escherichia coli chorismate synthase

Cited by 19 publications

References 17 publications

Rescuing Auxotrophic Microorganisms with Nonenzymatic Chemistry

Rescuing Auxotrophic Microorganisms with Nonenzymatic Chemistry

The Transient Kinetics of Escherichia coli Chorismate Synthase: Substrate Consumption, Product Formation, Phosphate Dissociation, and Characterization of a Flavin Intermediate

Studies with Substrate and Cofactor Analogues Provide Evidence for a Radical Mechanism in the Chorismate Synthase Reaction

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