Fang, X. et al. The mechanism of action of ramoplanin and enduracidin. Mol. BioSyst. 2, 69-76

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02128, USA.
Molecular BioSystems (Impact Factor: 3.21). 02/2006; 2(1):69-76. DOI: 10.1039/b515328j
Source: PubMed


The lipoglycodepsipeptide antibiotic ramoplanin is proposed to inhibit bacterial cell wall biosynthesis by binding to intermediates along the pathway to mature peptidoglycan, which interferes with further enzymatic processing. Two sequential enzymatic steps can be blocked by ramoplanin, but there is no definitive information about whether one step is inhibited preferentially. Here we use inhibition kinetics and binding assays to assess whether ramoplanin and the related compound enduracidin have an intrinsic preference for one step over the other. Both ramoplanin and enduracidin preferentially inhibit the transglycosylation step of peptidoglycan biosynthesis compared with the MurG step. The basis for stronger inhibition is a greater affinity for the transglycosylase substrate Lipid II over the MurG substrate Lipid I. These results provide compelling evidence that ramoplanin's and enduracidin's primary cellular target is the transglycosylation step of peptidoglycan biosynthesis.

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    • "These peptides disrupt bacterial cell wall biosynthesis, but have a mechanism of action that is distinct from the b-lactams and vancomycin. Enduracidin and ramoplanin block the elongation step of peptidoglycan biosynthesis by binding to the transglycosylase substrate Lipid II (Cudic et al., 2002; Fang et al., 2006). This substrate-binding mechanism is analogous to the vancomycin mode of action, but these different peptide antibiotics recognize distinct regions of Lipid II. "
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    ABSTRACT: The biosynthetic gene cluster for the 17 aa peptide antibiotic enduracidin has been cloned and sequenced from Streptomyces fungicidicus ATCC 21013. The 84 kb gene cluster contains 25 ORFs and is located within a 116 kb genetic locus that was fully sequenced. Targeted disruption of non-ribosomal peptide synthetase (NRPS) genes in the cluster abolished enduracidin production and confirmed function. The cluster includes four genes, endA-D, encoding two-, seven-, eight- and one-module NRPSs, respectively, and includes unique modules for the incorporation of citrulline and enduracididine. The NRPS organization generally follows the collinearity principle, and starts with a condensation domain (C domain) similar to those found in other lipopeptide systems for the coupling of an acyl group to the starting amino acid. The sixth module of EndB, corresponding to Thr(8), is missing an adenylation domain (A domain) and this module is presumed to be loaded in trans by the single module protein EndD. The most striking feature of the NRPS organization is the lack of epimerization domains (E domains) in light of the fact that the product has seven d-amino acid residues. Sequence analysis reveals that C domains following modules corresponding to d-amino acids belong to a unique subset of C domains able to catalyse both epimerization and condensation reactions. Other genes directing lipid modification and activation, and formation of the non-proteinogenic amino acids 4-hydroxyphenylglycine and enduracididine are readily identified, as are genes possibly involved in regulation of antibiotic biosynthesis and export. These findings provide the basis to further genetically manipulate and improve lipodepsipeptide antibiotics via combinatorial and chemical methods.
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    ABSTRACT: Molecular genetic and enzymological techniques have been employed to study antibiotic biosynthesis. In this thesis, we studied the formation and modification of the nonproteinogenic amino acid enduracididine (End), which exists in two important antibiotics, mannopeptimycins (MPPs) and enduracidin. Sequence analysis of the MPP gene cluster revealed that the product of mppO belongs to His-3 variant of non-heme iron, [alpha]-ketoglutarate dependent oxygenase superfamily. The mppO gene was subcloned and heterologously expressed in E. coli. Enzyme activity assays showed that MppO stereospecifically catalyzes hydroxylation of the [beta]-carbon of L-End and results in the formation of 3S-hydroxy-L-End. MppO is the first known enzyme that catalyzes the [beta]-hydroxylation of a nonproteinogenic amino acid. The formation of enduracididine was also studied in the enduracidin biosynthesis pathway. Three genes in the end cluster, endP, endQ and endR are predicted to be involved in the formation of L-End. The gene products of endP and endQ are proposed to be pyridoxal phosphate (PLP)-dependent enzymes. These genes were subcloned and expressed in E. coli. A fragment containing the whole endPQR operon was introduced into S. lividans and S. fungicidicus. Two possible mechanisms of enduracididine formation were proposed with [beta]-OH-L-Arg or [gamma] -OH-L-Arg as precursor, respectively. Printout. Thesis (M.S.)--Oregon State University, 2006. Includes bibliographical references.
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    ABSTRACT: At the end of its tether: Solid-supported tethered lipid bilayers that present native versions of the peptidoglycan precursors lipid I and lipid II are used to compare the binding of vancomycin and ramoplanin. Through the use of E. coli inner membranes, the resulting “proteolipid layer” contains the full range of intrinsic and extrinsic proteins found in vivo, thus allowing the in vitro biosynthesis of a peptidoglycan cell wall. E=enzyme.
    No preview · Article · Mar 2006 · Angewandte Chemie International Edition
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