Crystal Structures of Escherichia coli KDO8P Synthase Complexes Reveal the Source of Catalytic Irreversibility

Vainer, Radion; Belakhov, V. V.; Rabkin, Emilia; Baasov, Timor; Adir, Noam

doi:10.1016/j.jmb.2005.06.021

Cited by 16 publications

(18 citation statements)

References 40 publications

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“…Crystallographic studies and solid state rotational-echo double resonance (REDOR) NMR made on binary complexes of the synthase with each of the substrate, A5P and PEP, and the product, Kdo8P [119,120], allowed the identification of the active site position. According to collected data, the active site on each monomer is near to, but not in direct contact with, an adjacent monomer.…”

Section: Enzyme Structure and Catalytic Mechanismmentioning

confidence: 99%

“…In fact, Kdo8P rebinds the enzyme active site preferentially with an altered orientation; in this way, residues that are necessary for the correct positioning of the reverse reaction substrates, Kdo8P and Pi, are not aligned with the reactive site and the catalysis can not take place [120]. According to Vainer and co-workers, when the Kdo8PS.Kdo8P structure is superimposed onto the apoKdo8PS structure, the positions of the His202 are identical.…”

Section: Enzyme Structure and Catalytic Mechanismmentioning

confidence: 99%

“…Regarding the enzymatic catalytic cycle, a lot of investigations have been performed employing a variety of methods among which rapid chemical quench flow [128], mass spectrometry [129], and NMR [120,130]. One of the most important contributions to the study of the catalytic cycle has been the synthesis of various analogues of PEP, A5P and of putative transient intermediates [131][132][133].…”

Section: Enzyme Structure and Catalytic Mechanismmentioning

confidence: 99%

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New Targets for Antibacterial Design: Kdo Biosynthesis and LPS Machinery Transport to the Cell Surface

Cipolla¹,

Polissi²,

Airoldi

et al. 2011

CMC

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Lipopolysaccharide (LPS), which constitutes the lipid portion of the outer leaflet of Gram-negative bacteria, is essential for growth. It is also responsible for the variety of biological effects associated with Gram-negative sepsis. Recent advances have elucidated the exact chemical structure of this highly complex macromolecule and much of the enzymology involved in its biosynthesis. Enzymes involved in LPS biogenesis are optimal targets for the development of novel therapeutics since they are sufficiently conserved among diverse, clinically-relevant bacteria and no analogue counterpart is present in humans. During the last thirty years a number of inhibitors of LPS biosynthesis have been developed: some of these compounds have antibacterial properties, while others show excellent in vitro activity and are undergoing further investigation. The main focus of this review will be the biology of LPS in bacteria summarizing the knowledge about structure and enzymatic catalysis, as well as chemical efforts towards the synthesis of inhibitors of the key enzymes involved in the biosynthesis of Kdo, toward the minimal conserve structure Kdo(2)-LipA. In addition, very recent advances in deciphering the molecular mechanisms of LPS transport to the cell surface, as a new target to develop novel antibacterials, will be reported. Future directions and perspectives will also be outlined.

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Section: Enzyme Structure and Catalytic Mechanismmentioning

confidence: 99%

Section: Enzyme Structure and Catalytic Mechanismmentioning

confidence: 99%

Section: Enzyme Structure and Catalytic Mechanismmentioning

confidence: 99%

See 1 more Smart Citation

New Targets for Antibacterial Design: Kdo Biosynthesis and LPS Machinery Transport to the Cell Surface

Cipolla¹,

Polissi²,

Airoldi

et al. 2011

CMC

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“…EcoKDO8PS and the engineered metal-independent AaeKDO8PS enzymes are reported to bind substrates in more than one orientation, indicating that the enzymic reaction requires significant movement. 23,30 Intriguingly, the substitution that has the most dramatic effect on metal-activated activity, Cys246-Ser, also has a significant effect on K m A5P . This mutation alone in the metal-independent enzyme results in a substantial elevation of the parameter; this effect is most pronounced on the metalindependent activity of the NmeN23C/C246S double mutation.…”

Section: Effect Of Mutations On Kinetic Parametersmentioning

confidence: 99%

“…To determine the temperature optimum of NmeK-DO8PS, BTP buffers (50 mM, pH 7.5) were made at temperatures of 20,25,30,35,40,45,50, 60, and 70°C. Reaction mixtures containing the two substrates in the appropriate BTP buffer were initiated with 0.017 unit of NmeKDO8PS after incubation for 6 min.…”

Section: Temperature and Phmentioning

confidence: 99%

Reversing Evolution: Re-establishing Obligate Metal Ion Dependence in a Metal-independent KDO8P Synthase

Cochrane

Cookson

Jameson

et al. 2009

Journal of Molecular Biology

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Role of UDP‐N‐acetylmuramic acid in the regulation of MurA activity revealed by molecular dynamics simulations

de Oliveira,

da Costa,

Silva

et al. 2024

Protein Science

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The peptidoglycan biosynthesis pathway plays a vital role in bacterial cells, and facilitates peptidoglycan layer formation, a fundamental structural component of the bacterial cell wall. The enzymes in this pathway are candidates for antibiotic development, as most do not have mammalian homologues. The UDP‐N‐acetylglucosamine (UNAG) enolpyruvyl transferase enzyme (MurA) in the peptidoglycan pathway cytoplasmic step is responsible for the phosphoenolpyruvate (PEP)–UNAG catalytic reaction, forming UNAG enolpyruvate and inorganic phosphate. Reportedly, UDP‐N‐acetylmuramic acid (UNAM) binds tightly to MurA forming a dormant UNAM–PEP–MurA complex and acting as a MurA feedback inhibitor. MurA inhibitors are complex, owing to competitive binding interactions with PEP, UNAM, and UNAG at the MurA active site. We used computational methods to explore UNAM and UNAG binding. UNAM showed stronger hydrogen‐bond interactions with the Arg120 and Arg91 residues, which help to stabilize the closed conformation of MurA, than UNAG. Binding free energy calculations using end‐point computational methods showed that UNAM has a higher binding affinity than UNAG, when PEP is attached to Cys115. The unbinding process, simulated using τ–random acceleration molecular dynamics, showed that UNAM has a longer relative residence time than UNAG, which is related to several complex dissociation pathways, each with multiple intermediate metastable states. This prevents the loop from opening and exposing the Arg120 residue to accommodate UNAG and potential new ligands. Moreover, we demonstrate the importance of Cys115‐linked PEP in closed‐state loop stabilization. We provide a basis for evaluating novel UNAM analogues as potential MurA inhibitors.Public significanceMurA is a critical enzyme involved in bacterial cell wall biosynthesis and is involved in antibiotic resistance development. UNAM can remain in the target protein's active site for an extended time compared to its natural substrate, UNAG. The prolonged interaction of this highly stable complex known as the ‘dormant complex’ comprises UNAM–PEP–MurA and offers insights into antibiotic development, providing potential options against drug‐resistant bacteria and advancing our understanding of microbial biology.

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Crystal Structures of Escherichia coli KDO8P Synthase Complexes Reveal the Source of Catalytic Irreversibility

Cited by 16 publications

References 40 publications

New Targets for Antibacterial Design: Kdo Biosynthesis and LPS Machinery Transport to the Cell Surface

New Targets for Antibacterial Design: Kdo Biosynthesis and LPS Machinery Transport to the Cell Surface

Reversing Evolution: Re-establishing Obligate Metal Ion Dependence in a Metal-independent KDO8P Synthase

Role of UDP‐N‐acetylmuramic acid in the regulation of MurA activity revealed by molecular dynamics simulations

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