Effect of analogues of diaminopimelic acid on the <i>meso</i>‐diaminopimelate‐adding enzyme from <i>Escherichia coli</i>

Auger, Geneviève; Heijenoort, Jean van; Vederas, John C.; Blanot, Didier

doi:10.1016/0014-5793(96)00619-9

Cited by 30 publications

(20 citation statements)

References 23 publications

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“…On the other hand, very close analogues of meso-A 2 pm, such as meso-lanthionine, can be added by MurE Ct , though with a lower efficiency. Sulfur analogues of meso-A 2 pm, such as meso-lanthionine and L-allo-cystathionine, can be incorporated by MurE Ec and can totally replace meso-A 2 pm in the PG of genetically engineered E. coli cells (3,34,36). The maximum velocity of MurE Ct reported here (320 nmol ⅐ min Ϫ1 ⅐ mg Ϫ1 ) differs those of MurE enzymes in other gram-negative bacteria, with 1.4 mol ⅐ min Ϫ1 ⅐ mg Ϫ1 (10) and 2.6 mol ⅐ min Ϫ1 ⅐ mg Ϫ1 (42) being reported for MurE Ec and MurE from Pseudomonas aeruginosa, respectively.…”

Section: Discussionmentioning

confidence: 99%

Functional and Biochemical Analysis of theChlamydia trachomatisLigase MurE

et al. 2009

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Chlamydiae are unusual obligately intracellular bacteria that do not synthesize detectable peptidoglycan. However, they possess genes that appear to encode products with peptidoglycan biosynthetic activity. Bioinformatic analysis predicts that chlamydial MurE possesses UDP-MurNAc-L-Ala-D-Glu:meso-diaminopimelic acid (UDP-MurNAc-L-Ala-D-Glu:meso-A 2 pm) ligase activity. Nevertheless, there are no experimental data to confirm this hypothesis. In this paper we demonstrate that the murE gene from Chlamydia trachomatis is capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-L-Ala-D-Glu:meso-A 2 pm ligase activity. Recombinant MurE from C. trachomatis (MurE Ct ) was overproduced in and purified from E. coli in order to investigate its kinetic parameters in vitro. By use of UDP-MurNAc-L-Ala-D-Glu as the nucleotide substrate, MurE Ct demonstrated ATP-dependent meso-A 2 pm ligase activity with pH and magnesium ion optima of 8.6 and 30 mM, respectively. Other amino acids (meso-lanthionine, the LL and DD isomers of A 2 pm, D-lysine) were also recognized by MurE Ct. However, the activities for these amino acid substrates were weaker than that for meso-A 2 pm. The specificity of MurE Ct for three possible C. trachomatis peptidoglycan nucleotide substrates was also determined in order to deduce which amino acid might be present at the first position of the UDP-MurNAc-pentapeptide. Relative k cat /K m ratios for UDP-MurNAc-L-Ala-D-Glu, UDPMurNAc-L-Ser-D-Glu, and UDP-MurNAc-Gly-D-Glu were 100, 115, and 27, respectively. Our results are consistent with the synthesis in chlamydiae of a UDP-MurNAc-pentapeptide in which the third amino acid is meso-A 2 pm. However, due to the lack of specificity of MurE Ct for nucleotide substrates in vitro, it is not obvious which amino acid is present at the first position of the pentapeptide.

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Section: Discussionmentioning

confidence: 99%

Functional and Biochemical Analysis of theChlamydia trachomatisLigase MurE

et al. 2009

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“…Diamino acid substrates such as meso-A 2 pm or L-lysine have two recognition sites: the proximal site, which participates in the peptide bond, and the distal site, which carries the nonreacting amino function (29). In the peptide subunit of peptidoglycan, the proximal recognition site of meso-A 2 pm and L-lysine consists in the L asymmetric center, and the distal recognition site in the D (meso-A 2 pm) or achiral (L-lysine) center (Fig.…”

Section: Discussionmentioning

confidence: 99%

The MurE Synthetase from Thermotoga maritima Is Endowed with an Unusual d-Lysine Adding Activity

Boniface¹,

Bouhss

Mengin‐Lecreulx

et al. 2006

Journal of Biological Chemistry

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The peptidoglycan of Thermotoga maritima, an extremely thermophilic eubacterium, was shown to contain no diaminopimelic acid and approximate amounts of both enantiomers of lysine (Huber, R., Langworthy, T. A., König, H., Thomm Peptidoglycan is a giant macromolecule composed of alternating N-acetylglucosamine and N-acetylmuramyl residues cross-linked by short peptides. Its biosynthesis is a complex two-stage process. The first stage consists of the formation of the disaccharide-pentapeptide monomer unit, whereas the second stage concerns the polymerization reactions (1). The assembly of the peptide part of the monomer unit is ensured by a series of enzymes (MurC, MurD, MurE, and MurF) called the Mur synthetases. It has been shown that the Mur synthetases constitute a family of enzymes with common mechanistic and structural features (2, 3).Synthetase MurE catalyzes the addition of the third amino acid residue of the peptide chain. This residue, generally a diamino acid, varies among the bacterial species: meso-diaminopimelic acid (meso-A 2 pm) 3 for most Gram-negative bacteria and bacilli, L-lysine for most Grampositive bacteria, L-ornithine, meso-lanthionine, LL-A 2 pm, L-diaminobutyric acid, L-homoserine, etc. in particular species (4). In many organisms, the third residue is involved in the cross-linking of the macromolecule; in those cases, the incorporation by MurE of a "wrong" amino acid results in cell lysis (5). Therefore, MurE must be endowed with a high specificity to select the "right" amino acid among closely related amino acids that coexist within the cell. Recently, the crystallization of MurE from Escherichia coli allowed Gordon et al. (6) to decipher the structural bases for this high specificity.Thermotoga maritima is a Gram-negative, extremely thermophilic bacterium isolated from geothermally heated sea floors (7). The analysis of its peptidoglycan revealed the absence of A 2 pm and the presence of both enantiomers of lysine. In this paper, we describe the properties of purified MurE from this bacterial species; in particular, we demonstrate that it is capable of adding L-and D-lysine to UDP-N-acetylmuramoyl (MurNAc)-L-Ala-D-Glu with comparable efficiencies but in different ways. The explanation of this finding in terms of known consensus sequences in MurE proteins is discussed. Moreover, we bring evidences of its physiological relevance by showing that: (i) the novel D-lysinecontaining nucleotide is a substrate for MraY from T. maritima in vitro and (ii) the overexpression of T. maritima murE in E. coli results in important lysine incorporation into the peptidoglycan of the host.

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“…These enzymes contain highly conserved regions [4–6] and they operate via a similar mechanism involving carboxyl activation of the nucleotide substrate to an acylphosphate intermediate; and followed by nucleophilic attack by the amino group of the condensing amino acids, with the formation of a peptide bond and the elimination of a phosphate group [7–12]. Inhibitors have been designed for MurD [9,13–15] and MurE [16,17]. Mechanistic and structural studies of the Mur enzymes and screening of these enzymes for inhibitors are severely hampered because of the lack of pathway intermediates [18].…”

Section: Introductionmentioning

confidence: 99%

In vitro reconstruction of the biosynthetic pathway of peptidoglycan cytoplasmic precursor in Pseudomonas aeruginosa

Zoeiby¹,

Sanschagrin²,

Havugimana

et al. 2001

FEMS Microbiology Letters

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Bacterial peptidoglycan is the cell wall component responsible for maintaining cell integrity against osmotic pressure. Biosynthesis of the cytoplasmic precursor UDP-N-acetylmuramyl pentapeptide is catalyzed by the Mur enzymes. Genomic analysis of the three regions encoding Mur proteins was achieved. We have cloned and over-expressed the murA, -B, -D, -E and -F genes of Pseudomonas aeruginosa in pET expression system by adding a His-Tag to the C-termini of the proteins. Mur proteins were purified to homogeneity by a single chromatographic step on affinity nickel columns. Protein identities were verified through N-terminal sequencing. Enzyme activity was proved by the identification of the pathway's final product.

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Effect of analogues of diaminopimelic acid on the meso‐diaminopimelate‐adding enzyme from Escherichia coli

Cited by 30 publications

References 23 publications

Functional and Biochemical Analysis of theChlamydia trachomatisLigase MurE

Functional and Biochemical Analysis of theChlamydia trachomatisLigase MurE

The MurE Synthetase from Thermotoga maritima Is Endowed with an Unusual d-Lysine Adding Activity

In vitro reconstruction of the biosynthetic pathway of peptidoglycan cytoplasmic precursor in Pseudomonas aeruginosa

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