On the Mechanism of 5-Enolpyruvylshikimate 3-Phosphate Synthetase

Bondinell, William E.; Vnek, J; Knowles, Peter F.; Sprecher, Milon; Sprinson, David B.

doi:10.1016/s0021-9258(18)61774-0

Cited by 88 publications

(53 citation statements)

References 26 publications

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“…Pi is produced by either elimination, in the case of 5-enolpyruvylshikimate-3-phosphate synthase and UDP-Nacetylglucosamine enol pyruvate transferase, or nucleophilic displacement, as in the case of DAR 7-P synthase with the resulting cleavage of the c-o enolic bond of PEP. The classical methodology used to determine the fate of the PEP enolic oxygen is to utilize PEP labeled with 18 0 in the enoIic oxygen as the substrate in the enzymatic reaction (13). The biosynthetic products are then isolated from the enzymatic mixture, derivatized, and subjected to mass spectral analysis to determine the 18 0 content.…”

Section: Discussionmentioning

confidence: 99%

“…The [2-13CJ3-bromopyruvic acid synthesized as above was converted into [2-13C,180J3-bromopyruvic acid by the exchange method of Bondinell et al (13) utilizing a mixture of 2H 2l80 (un-normalized; 98% 180, 90% 2H) (350 JLl) and H 20 (120 JLl). The [2_ l3C,180J3-bromopyruvic acid was then converted into the title compound as described above (m.p.…”

Section: Synthesis Of [2_ 13 C 1801phosphoenolpyruvatementioning

confidence: 99%

“…This was determined by integration of the C-2 resonance due to [2_ l3C,lBOJPEP and the lBO-shifted carbon resonance due to [2_ l3C,180JPEP, in the subspectra created by multiple-quantum filtration of the l3C NMR spectra (14). [2][3][4][5][6][7][8][9][10][11][12][13] C1Phosp hoenolpyruvate to [2-13 C13_ Deoxyoctulosonate B-Phosphate KDO 8-P synthase (1 unit) was added to a 10-ml Erlenmeyer flask containing the following: [2.l 3CJphosphoenolpyruvate monocyclohexylamine salt (6.7 mg, 0.025 mmol), A 5-P disodium salt (7.0 mg, 0.025 mmol), Tris-HCI (pH 7.5) (0.5 mmol), and H 20, in a final volume of2.5 ml. This reaction mixture was incubated at 37°C for 2 h in a shaking water bath.…”

Section: Synthesis Of [2_ 13 C 1801phosphoenolpyruvatementioning

confidence: 99%

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Overproduction and One-step Purification of Escherichia coli 3-Deoxy-D-manno-octulosonic Acid 8-Phosphate Synthase and Oxygen Transfer Studies during Catalysis Using Isotopic-shifted Heteronuclear NMR

Dotson

Dua

Clemens

et al. 1995

Journal of Biological Chemistry

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The enzyme 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase catalyzes the condensation of D-arabinose 5-phosphate with phosphoenolpyruvate to give the unique 8-carbon acidic sugar 3-deoxy-D-manno-octulosonic acid 8-phosphate (KDO 8-P) found only in Gram-negative bacteria and required for lipid A maturation and cellular growth. The Escherichia coli gene kdsA that encodes KDO 8-P synthase has been amplified by polymerase chain reaction methodologies and subcloned into the expression vector, pT7-7. A simple one-step purification yields 200 mg of homogeneous KDO 8-P synthase per liter of cell culture. [2-13C,18O]Phosphoenolpyruvate (PEP) was prepared by first, exchange of [2-13C]-3-bromopyruvate with 2H2 18O followed by reaction of the labeled bromopyruvate with trimethylphosphite. The fate of the enolic oxygen in this multilabeled PEP, during the course of the KDO 8-P synthase-catalyzed reaction with D-arabinose 5-phosphate, was monitored by 13C and 31P NMR spectroscopy. The inorganic phosphate formed during the reaction was further analyzed via mass spectral analysis of its trimethyl ester derivative. The 13C NMR spectrum of an incubation mixture of [2-13C]PEP and D-arabinose 5-phosphate in 2H2 18O in the presence of KDO 8-P synthase was also recorded. [2-13C]KDO 8-P was utilized to determine the extent of nonenzymatic incorporation of 18O into the C-2 position of KDO 8-P. The results indicate that the enolic oxygen of the PEP is recovered with the inorganic phosphate, and the C-2 oxygen of KDO 8-P originates from the solvent, H2O.

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Section: Discussionmentioning

confidence: 99%

Section: Synthesis Of [2_ 13 C 1801phosphoenolpyruvatementioning

confidence: 99%

Section: Synthesis Of [2_ 13 C 1801phosphoenolpyruvatementioning

confidence: 99%

See 1 more Smart Citation

Overproduction and One-step Purification of Escherichia coli 3-Deoxy-D-manno-octulosonic Acid 8-Phosphate Synthase and Oxygen Transfer Studies during Catalysis Using Isotopic-shifted Heteronuclear NMR

Dotson

Dua

Clemens

et al. 1995

Journal of Biological Chemistry

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“…The reaction catalyzed by MurA (Fig. 1a) proceeds via an addition-elimination mechanism [16], analogous to that catalyzed by EPSP synthase [17][18][19]. A noncovalentlybound tetrahedral ketal intermediate is formed by direct attack of the 3′-OH of UDP-N-acetylglucosamine (UDP-GlcNAc) on the C-2 position of phosphoenolpyruvate (PEP), inorganic phosphate is then eliminated to form the product, UDP-N-acetylenolpyruvylglucosamine.…”

Section: Introductionmentioning

confidence: 99%

Structure of UDP-N-acetylglucosamine enolpyruvyl transferase, an enzyme essential for the synthesis of bacterial peptidoglycan, complexed with substrate UDP-N-acetylglucosamine and the drug fosfomycin

et al. 1996

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The present structure reveals the mode of binding of the natural substrate UDP-GlcNAc and of the drug fosfomycin, and provides information on the residues involved in catalysis. These results should aid the design of inhibitors which would interfere with enzyme-catalyzed reactions in the early stage of the bacterial cell wall biosynthesis. Furthermore, the crystal structure of MurA provides a model for predicting active-site residues in EPSP synthase that may be involved in catalysis and substrate binding.

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“…EPSP synthase accelerates THI breakdown by a factor of >10 5 , relative to nonenzymatic breakdown . The biochemical reaction catalyzed by this enzyme is unusual, since the enzymatic synthesis of EPSP from PEP and S3P involves C–O cleavage of the pyruvate ester . Normally, enzymes that utilize phosphate as a substrate break the high-energy P–O bond.…”

Section: Introductionmentioning

confidence: 99%

Unraveling the Addition–Elimination Mechanism of EPSP Synthase through Computer Modeling

et al. 2017

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Enolpyruvyl transfer from phosphoenolpyruvate (PEP) to the hydroxyl group of shikimate-5-OH-3-phosphate (S3P) is catalyzed by 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase in a reaction that involves breaking the C-O bond of PEP. Catalysis involves an addition-elimination mechanism with the formation of a tetrahedral intermediate (THI). Experiments have elucidated the mechanism of THI formation and breakdown. However, the catalytic action of EPSP synthase and the individual roles of catalytic residues Asp313 and Glu341 remains unclear. We have used a hybrid quantum mechanical/molecular mechanical (QM/MM) approach to explore the free energy surface in a reaction catalyzed by EPSP synthase. The Glu341 was the most favorable acid/base catalyst. Our results indicate that the protonation of PEP C3 precedes the nucleophilic attack on PEP C2 in the addition mechanism. Also, the breaking of the C-O bond of THI to form an EPSP cation intermediate must occur before proton transfer from PEP C3 to Glu341 in the elimination mechanism. Analysis of the FES supports cationic intermediate formation during the reaction catalyzed by EPSP synthase. Finally, the computational model indicates a proton transfer shift (Hammond shift) from Glu341 to C3 for an enzyme-based reaction with the shifted transition state, earlier than in the reference reaction in water.

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On the Mechanism of 5-Enolpyruvylshikimate 3-Phosphate Synthetase

Cited by 88 publications

References 26 publications

Overproduction and One-step Purification of Escherichia coli 3-Deoxy-D-manno-octulosonic Acid 8-Phosphate Synthase and Oxygen Transfer Studies during Catalysis Using Isotopic-shifted Heteronuclear NMR

Overproduction and One-step Purification of Escherichia coli 3-Deoxy-D-manno-octulosonic Acid 8-Phosphate Synthase and Oxygen Transfer Studies during Catalysis Using Isotopic-shifted Heteronuclear NMR

Structure of UDP-N-acetylglucosamine enolpyruvyl transferase, an enzyme essential for the synthesis of bacterial peptidoglycan, complexed with substrate UDP-N-acetylglucosamine and the drug fosfomycin

Unraveling the Addition–Elimination Mechanism of EPSP Synthase through Computer Modeling

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