Purification and characterization of the D-alanyl-D-alanine-adding enzyme from Escherichia coli

Duncan, Kenneth; Heijenoort, Jean van; Walsh, Christopher T.

doi:10.1021/bi00461a023

Cited by 69 publications

(54 citation statements)

References 25 publications

(30 reference statements)

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“…m g -' protein and the turnover number to 931 min-'. A turnover number of 784 min-' was previously determined for the MurF synthetase [24]. Under the most optimal in vitro conditions considered here, the D-glutamate-adding activity could catalyse the formation in each cell of approximately 28 x 10' peptidoglycan units during a 30-min generation time (2000 x 931 x 30).…”

Section: Discussionmentioning

confidence: 91%

Over‐production, purification and properties of the uridine diphosphate N‐acetylmuramoyl‐l‐alanine: d‐glutamate ligase from Escherichia coli

Pratviel‐Sosa

Mengin‐Lecreulx

Heijenoort

1991

European Journal of Biochemistry

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The UDP-N-acetylmuramoyl-L-alanine: D-glutamate ligase of Esckevichiu coli was over-produced in strains that harbour recombinant plasmids bearing the murD gene under the control of the lac or PR promoter. Purification to homogeneity was achieved by a two-step procedure from a 181-fold over-producing strain. The N-terminal sequence of the purified protein was determined and correlated with the nucleotide sequence of the murD gene. The purified activity was highly dependent on the concentration of potassium phosphate and Mg2+. The enzyme also catalysed the reverse reaction. The K , values for UDP-N-acetylmuramoyl-L-alanine; D-glutamate and ATP/ Mg2+ were estimated at 7.5, 55 and 138 pM, respectively. Under the most optimal in v i m conditions determined, a turnover number of 931 min-' was estimated. When considering the plasmid-free parental strain, the copy number of the murD gene product was not more than 1000 . cell-'. . Owing to its high specificity and to its occurrence only in eubacteria, the D-glutamate-adding enzyme is a potential target for new antibacterial agents. It was initially partially purified from Staphylococcus aureus [3, 41. In Escherichiu coli, various studies of the D-glutamateadding enzyme were carried out with crude extracts [5 -81. However, recently it was partially purified (33-fold) and its specificity was studied with analogues of D-glutamate and its nucleotide substrate, UDP-MurNAc-L-Ala [9]. Furthermore, the murD gene coding for this ligase has been identified at 2min on the chromosome of E. coli, in a large cluster of genes over pbpB-envA that code for proteins involved in peptidogylcan biosynthesis or cell division [lo]. The nucleotide sequence of murD has been determined [Il, 121. It contains 1314 nucleotides which are translated into 438 amino acids, corresponding to a protein with a molecular mass of 46936 Da.In the present paper we report on the over-production, the purification to homogeneity and the study of several properties of the D-glutamate-adding enzyme from E. coli.

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Section: Discussionmentioning

confidence: 91%

Over‐production, purification and properties of the uridine diphosphate N‐acetylmuramoyl‐l‐alanine: d‐glutamate ligase from Escherichia coli

Pratviel‐Sosa

Mengin‐Lecreulx

Heijenoort

1991

European Journal of Biochemistry

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“…In E. coli, murein peptide ligase (Mpl) recycles the tri-, tetra-and pentapeptides by ligating them to UDP-MurNAc [179,180]. Another enzyme, MurF can also ligate dipeptide D-Ala-D-Ala (a product of ATP-dependent ligase Ddl), to a UDP-MurNAc tripeptide to form a UDP-MurNAc pentapeptide [181][182][183]. Homologues of Mpl (PA4020) and MurF (PA4416) and Ddl (PA4201/PA4410) are found in P. aeruginosa; however, their role in cell-wall recycling has not been elucidated.…”

Section: Convergence Of Recycling and Biosynthesismentioning

confidence: 99%

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa – their role in the development of resistance

Dhar

Kumari

Balasubramanian

et al. 2018

Journal of Medical Microbiology

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The bacterial cell-wall that forms a protective layer over the inner membrane is called the murein sacculus -a tightly crosslinked peptidoglycan mesh unique to bacteria. Cell-wall synthesis and recycling are critical cellular processes essential for cell growth, elongation and division. Both de novo synthesis and recycling involve an array of enzymes across all cellular compartments, namely the outer membrane, periplasm, inner membrane and cytoplasm. Due to the exclusivity of peptidoglycan in the bacterial cell-wall, these players are the target of choice for many antibacterial agents. Our current understanding of cell-wall biochemistry and biogenesis in Gram-negative organisms stems mostly from studies of Escherichia coli. An incomplete knowledge on these processes exists for the opportunistic Gram-negative pathogen, Pseudomonas aeruginosa. In this review, cell-wall synthesis and recycling in the various cellular compartments are compared and contrasted between E. coli and P. aeruginosa. Despite the fact that there is a remarkable similarity of these processes between the two bacterial species, crucial differences alter their resistance to b-lactams, fluoroquinolones and aminoglycosides. One of the common mediators underlying resistance is the amp system whose mechanism of action is closely associated with the cell-wall recycling pathway. The activation of amp genes results in expression of AmpC blactamase through its cognate regulator AmpR which further regulates multi-drug resistance. In addition, other cell-wall recycling enzymes also contribute to antibiotic resistance. This comprehensive summary of the information should spawn new ideas on how to effectively target cell-wall processes to combat the growing resistance to existing antibiotics.

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“…In cell-free MurF assay, each substrate was converted to the corresponding product, UDP-MurNAc-pentapeptide or UDP-MurNAc-dansylpentapeptide, which was confirmed by HPLC analysis. 30 This indicated that UDP-MurNAc-dansyltripeptide was used as the substrate of MurF, and suggested that MurF activity could be measured in a high-throughput mode by coupling translocase I.…”

Section: Resultsmentioning

confidence: 99%

“…HPLC detection of the MurF activity was carried out as described by Duncan et al 30 with slight modifications. The reaction mixture containing 100 mM TrisHCl (pH 8.6), 40 mM KCl, 10 mM MgCl 2 , 200mM UDP-MurNAc-tripeptide, 200 mM D-Ala-D-Ala, 200 mM ATP and 8% glycerol was added to 10 ml of MurF and incubated.…”

Section: Hplc Assaymentioning

confidence: 99%

A novel assay of bacterial peptidoglycan synthesis for natural product screening

et al. 2009

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Although a large number of microbial metabolites have been discovered as inhibitors of bacterial peptidoglycan biosynthesis, only a few inhibitors were reported for the pathway of UDP-MurNAc-pentapeptide formation, partly because of the lack of assays appropriate for natural product screening. Among the pathway enzymes, D-Ala racemase (Alr), D-Ala:D-Ala ligase (Ddl) and UDPMurNAc-tripeptide:D-Ala-D-Ala transferase (MurF) are particularly attractive as antibacterial targets, because these enzymes are essential for growth and utilize low-molecular-weight substrates. Using dansylated UDP-MurNAc-tripeptide and L-Ala as the substrates, we established a cell-free assay to measure the sequential reactions of Alr, Ddl and MurF coupled with translocase I. This assay is sensitive and robust enough to screen mixtures of microbial metabolites, and enables us to distinguish the inhibitors for D-Ala-D-Ala formation, MurF and translocase I. D-cycloserine, the D-Ala-D-Ala pathway inhibitor, was successfully detected by this assay (IC 50 ¼1.7 lg ml À1 ). In a large-scale screening of natural products, we have identified inhibitors for D-Ala-D-Ala synthesis pathway, MurF and translocase I.

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Purification and characterization of the D-alanyl-D-alanine-adding enzyme from Escherichia coli

Cited by 69 publications

References 25 publications

Over‐production, purification and properties of the uridine diphosphate N‐acetylmuramoyl‐l‐alanine: d‐glutamate ligase from Escherichia coli

Over‐production, purification and properties of the uridine diphosphate N‐acetylmuramoyl‐l‐alanine: d‐glutamate ligase from Escherichia coli

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa – their role in the development of resistance

A novel assay of bacterial peptidoglycan synthesis for natural product screening

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