Synthesis and activity of 5′-Uridinyl dipeptide analogues mimicking the amino terminal peptide chain of nucleoside antibiotic mureidomycin A

Howard, Nigel; Bugg, Timothy D. H.

doi:10.1016/s0968-0896(03)00270-0

Cited by 58 publications

(50 citation statements)

References 27 publications

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“…The consequences of mutating D115 and D116 in MraY are more deleterious than those observed upon mutation of D90 and D91 in WecA (4), implying some differences in the Mg 2ϩ binding sites of the two enzymes, which do have different affinities for MgCl 2 (optimum activity at 3 mM MgCl 2 for WecA [4] and at 25 mM for MraY [13]). Recent studies on the mechanism of action of the inhibitor mureidomycin A indicate that the amino terminus of the antibiotic binds in place of the magnesium cofactor (21,25); therefore, the characterization of this interaction may be of importance for the design of future translocase 1 inhibitors.…”

Section: Discussionmentioning

confidence: 99%

“…Further, relief by Mg 2ϩ of the inhibition of E. coli translocase 1 by nucleoside analogues of mureidomycin A suggests that the antibiotic binds (in part) to the same site occupied by the metal ion in the enzyme active site (21,25). Therefore, an understanding of the function of the active site of this enzyme is essential to the development of inhibitors of translocase 1.…”

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confidence: 99%

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Phospho- N -Acetyl-Muramyl-Pentapeptide Translocase from Escherichia coli : Catalytic Role of Conserved Aspartic Acid Residues

et al. 2004

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Phospho-N-acetyl-muramyl-pentapeptide translocase (translocase 1) catalyzes the first of a sequence of lipid-linked steps that ultimately assemble the peptidoglycan layer of the bacterial cell wall. This essential enzyme is the target of several natural product antibiotics and has recently been the focus of antimicrobial drug discovery programs. The catalytic mechanism of translocase 1 is believed to proceed via a covalent intermediate formed between phospho-N-acetyl-muramyl-pentapeptide and a nucleophilic amino acid residue. Amino acid sequence alignments of the translocase 1 family and members of the related transmembrane phosphosugar transferase superfamily revealed only three conserved residues that possess nucleophilic side chains: the aspartic acid residues D115, D116, and D267. Here we report the expression and partial purification of Escherichia coli translocase 1 as a C-terminal hexahistidine (C-His 6 ) fusion protein. Three enzymes with the site-directed mutations D115N, D116N, and D267N were constructed, expressed, and purified as C-His 6 fusions. Enzymatic analysis established that all three mutations eliminated translocase 1 activity, and this finding verified the essential role of these residues. By analogy with the structural environment of the double aspartate motif found in prenyl transferases, we propose a model whereby D115 and D116 chelate a magnesium ion that coordinates with the pyrophosphate bridge of the UDP-N-acetyl-muramyl-pentapeptide substrate and in which D267 therefore fulfills the role of the translocase 1 active-site nucleophile.Enzymes involved in the assembly of the peptidoglycan layer of bacterial cell walls represent important targets for antibacterial chemotherapy (15, 49). The study of this class of enzymes and the search for selective inhibitors are likely to lead to the development of new chemotherapeutic agents, which are urgently needed to combat antimicrobial drug resistance, the threat of which has recently been highlighted by the acquisition of resistance by methicillin-resistant Staphylococcus aureus to vancomycin (43).Peptidoglycan consists of a ␤-1,4-linked N-acetyl-glucosamine-N-acetyl-muramyl-pentapeptide (GlcNAc-MurNAc-pentapeptide) polymer, assembled from cytoplasmic precursors UDP-MurNAc-L-Ala-␥-D-Glu-X-D-Ala-D-Ala (UDPMurNAc-pentapeptide; X, L-Lys or meso-diaminopimelic acid [meso-DAP]) and 49

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Phospho- N -Acetyl-Muramyl-Pentapeptide Translocase from Escherichia coli : Catalytic Role of Conserved Aspartic Acid Residues

et al. 2004

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“…[13] Structureactivity studies with synthesised peptidyl nucleoside analogues revealed that the terminal amino group, the N-methyl-Daba and probably the uridine moiety are most important for the inhibition of translocase I. [14,15] Recently, the identification and analysis of the gene clusters for caprazamycins, [16,17] liposidomycins [18][19][20] and capuramycintype A-500359 [21] has given the first insights into the biosynthesis of the structurally unusual nucleoside antibiotics that target translocase I. However, little is known about the formation of the uridylpeptide-type translocase I inhibitors.…”

Section: Introductionmentioning

confidence: 99%

Identification of a Napsamycin Biosynthesis Gene Cluster by Genome Mining

et al. 2010

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Napsamycins are potent inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan biosynthesis, and are classified as uridylpeptide antibiotics. They comprise an N-methyl diaminobutyric acid, an ureido group, a methionine and two non-proteinogenic aromatic amino acid residues in a peptide backbone that is linked to a 5'-amino-3'-deoxyuridine by an unusual enamide bond. The napsamycin gene cluster was identified in Streptomyces sp. DSM5940 by using PCR probes from a putative uridylpeptide biosynthetic cluster found in S. roseosporus NRRL15998 by genome mining. Annotation revealed 29 hypothetical genes encoding for resistance, regulation and biosynthesis of the napsamycins. Analysis of the gene cluster indicated that the peptide core structure is assembled by a nonlinear non-ribosomal peptide synthetase (NRPS)-like mechanism that involves several discrete single or didomain proteins. Some genes could be assigned, for example, to the synthesis of the N-methyl diaminobutyric acid, to the generation of m-tyrosine and to the reduction of the uracil moiety. The heterologous expression of the gene cluster in Streptomyces coelicolor M1154 resulted in the production of napsamycins and mureidomycins as demonstrated by LC-ESI-MS and MS/MS analysis. The napsamycin gene cluster provides a molecular basis for the detailed study of the biosynthesis of this class of structurally unusual compounds.

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“…This enzyme, an integral membrane protein, is known to be the site of action of three nucleoside natural product antibiotics: tunicamycin, mureidomycin A and liposidomycin B (Brandish et al, 1996a, b). The inhibition kinetics for these inhibitors show that they compete for the UDPMurNAc-pentapeptide and undecaprenyl phosphate binding sites at the MraY active site, and in the case of mureidomycin A, compete for the Mg 2+ cofactor binding site (Howard & Bugg, 2003).…”

Section: Introductionmentioning

confidence: 99%

Interaction of the transmembrane domain of lysis protein E from bacteriophage ϕX174 with bacterial translocase MraY and peptidyl-prolyl isomerase SlyD

et al. 2006

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The molecular target for the bacteriolytic E protein from bacteriophage wX174, responsible for host cell lysis, is known to be the enzyme phospho-MurNAc-pentapeptide translocase (MraY), an integral membrane protein involved in bacterial cell wall peptidoglycan biosynthesis, with an essential role being played by peptidyl-prolyl isomerase SlyD. A synthetic 37 aa peptide E pep , containing the N-terminal transmembrane a-helix of E, was found to be bacteriolytic against Bacillus licheniformis, and inhibited membrane-bound MraY. The solution conformation of E pep was found by circular dichroism (CD) spectroscopy to be 100 % a-helical. No change in the CD spectrum was observed upon addition of purified Escherichia coli SlyD, implying that SlyD does not catalyse prolyl isomerization upon E. However, E pep was found to be a potent inhibitor of SlyD-catalysed peptidylprolyl isomerization (IC 50 0?15 mM), implying a strong interaction between E and SlyD. E pep was found to inhibit E. coli MraY activity when assayed in membranes (IC 50 0?8 mM); however, no inhibition of solubilized MraY was observed, unlike nucleoside natural product inhibitor tunicamycin. These results imply that the interaction of E with MraY is not at the MraY active site, and suggest that a protein-protein interaction is formed between E and MraY at a site within the transmembrane region.

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Synthesis and activity of 5′-Uridinyl dipeptide analogues mimicking the amino terminal peptide chain of nucleoside antibiotic mureidomycin A

Cited by 58 publications

References 27 publications

Phospho- N -Acetyl-Muramyl-Pentapeptide Translocase from Escherichia coli : Catalytic Role of Conserved Aspartic Acid Residues

Phospho- N -Acetyl-Muramyl-Pentapeptide Translocase from Escherichia coli : Catalytic Role of Conserved Aspartic Acid Residues

Identification of a Napsamycin Biosynthesis Gene Cluster by Genome Mining

Interaction of the transmembrane domain of lysis protein E from bacteriophage ϕX174 with bacterial translocase MraY and peptidyl-prolyl isomerase SlyD

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