Pacidamycins, a novel series of antibiotics with anti-Pseudomonas aeruginosa activity. II. Isolation and structural elucidation.

Chen, Randal H.; Buko, Alexander M.; Whittern, David N.; McAlpine, James B.

doi:10.7164/antibiotics.42.512

Cited by 90 publications

(64 citation statements)

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“…and related actinomycetes have been discovered that inhibit bacterial translocase I (MraY) involved in the biosynthesis of peptidoglycan cell wall, an essential component for the survival of all bacteria and a proven target for antibiotics (1). These nucleoside antibiotics have been classified into minimally four groups based on structural differences: peptidyl nucleosides represented by pacidamycin from Streptomyces coeruleorubidus (2) and mureidomycin from Streptomyces flavidovirens (3), lipodisaccharyl nucleosides represented by tunicamycins from Streptomyces lysosuperificus (4), lipopeptidyl nucleosides represented by A-90289 from Streptomyces sp. SANK 60405 (5) and caprazamycin from Streptomyces sp.…”

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Characterization of LipL as a Non-heme, Fe(II)-dependent α-Ketoglutarate:UMP Dioxygenase That Generates Uridine-5′-aldehyde during A-90289 Biosynthesis

Yang

Chi

Funabashi

et al. 2011

Journal of Biological Chemistry

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“…This orf encodes a protein with sequence similarity to proteins annotated as ␣-ketoglutarate:taurine dioxygenases (TauD), 2 which catalyze the conversion of taurine to aminoacetaldehyde and sulfite (supplemental Fig. S1) (16).…”

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Characterization of LipL as a Non-heme, Fe(II)-dependent α-Ketoglutarate:UMP Dioxygenase That Generates Uridine-5′-aldehyde during A-90289 Biosynthesis

Yang

Chi

Funabashi

et al. 2011

Journal of Biological Chemistry

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“…(Received for publication July 1 7, 2000) During the course of isolating gram quantities of pacidamycins from the fermentation of Streptomyces coeruleorubidus (NRRL 18730) for mode-of-action and chemical-modification studies, three new pacidamycins were isolated.1^Here we report the production, isolation, structure elucidation, and biological activities of pacidamycins D (1), 4N (2), and 5T (3). A fresh culture of strain NRRL18730 was inoculated into four 250-ml baffled shake flasks, each containing 50 ml of seed medium consisting of glucose monohydrate 1.0%, soluble starch 1.5%, yeast extract 0.5%, casitone 0.5%, CaCO3 0.1% (pH adjusted to 7.0 before sterilization).…”

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New Pacidamycins Produced by Streptomyces coeruleorubidus, NRRL 18370.

et al. 2000

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were isolated.1^Here we report the production, isolation, structure elucidation, and biological activities of pacidamycins D (1), 4N (2), and 5T (3). A fresh culture of strain NRRL18730 was inoculated into four 250-ml baffled shake flasks, each containing 50 ml of seed medium consisting of glucose monohydrate 1.0%, soluble starch 1.5%, yeast extract 0.5%, casitone 0.5%, CaCO3 0.1% (pH adjusted to 7.0 before sterilization). The flasks were incubated on a rotary shaker (250 rpm) at 28°C for 36 hours to give the stage-1 seed. The stage-1 seed (200ml) was inoculated into two 4000-ml baffled flasks, each containing 1000 ml of the same seed media. The flasks were incubated under the same condition as the above for 27 hours to give the stage-2 seed. The stage-2 seed (2000ml) was inoculated into 22 liters of the seed media in a 28-liter fermenter. The fermenter was maintained at 28°C, 300rpm agitation, 10 slpm aeration, and 4.0 psi backpressure for 26 hours to give the stage-3 seed. The stage-3 seed was inoculated into a 300-liter reactor, containing 230 liters of a production media consisting of 1% soytone, 1% soluble starch, 2% D-maltose and 5 ml/liter trace elements. The fermentation was allowed to proceed with 400rpm agitation, 100 slpm aeration, 5.0 psi backpressure, 29°C, and maintained at pH 6.5 with the addition of 6N sodium hydroxide or 30% phosphoric acid.The fermentation mash (220 liters) was harvested after 1 17 with 28% NH4OH and the major pacidamycin component in each pool was further purified by reversed phase column chromatography on Amberchrom®as described below for pacidamycin D. A 2.5-liter pool containing pacidamycin D was loaded on an Amberchrom® column (2.5cmXlOOcm, 490ml) pre-equilibrated in deionized water. Upon completion of loading, the column was washed successively with 2 bed volumes of water and 2 bed volumes of0.1 M NH4OAc(pH 7.8). It was then eluted with a gradient of 0% to 12% CH3CNin buffer over 100ml, and 12% CH3CNin buffer

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“…More than 10 related family members have been reported (1,2): Their common scaffold contains a central N β -methyl 2S,3S-diaminoburytic acid (DABA) moiety which is α-amino-capped by a ureido dipeptide (C-terminal), β-amino-capped by a single amino acid or a dipeptide (N-terminal), and carboxy-linked to a 3′-deoxy-4′,5′-enaminouridine ( Fig. 1).…”

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tRNA-dependent peptide bond formation by the transferase PacB in biosynthesis of the pacidamycin group of pentapeptidyl nucleoside antibiotics

Zhang

Ntai

Kelleher

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Pacidamycins are a family of uridyl tetra/pentapeptide antibiotics with antipseudomonal activities through inhibition of the translocase MraY in bacterial cell wall assembly. The biosynthetic gene cluster for pacidamycins has recently been identified through genome mining of the producer Streptomyces coeruleorubidus, and the highly dissociated nonribosomal peptide assembly line for the uridyl tetrapeptide scaffold of pacidamycin has been characterized. In this work a hypothetical protein PacB, conserved in known uridyl peptide antibiotics gene clusters, has been characterized by both genetic deletion and enzymatic analysis of the purified protein. PacB catalyzes the transfer of the alanyl residue from alanyl-tRNA to the N terminus of the tetrapeptide intermediate yielding a pentapeptide on the thio-templated nonribosomal peptide synthetase (NRPS) assembly line protein PacH. PacB thus represents a new group of tRNA-dependent peptide bond-forming enzymes in secondary metabolite biosynthesis in addition to the recently identified cyclodipeptide synthases. The characterization of PacB completes the assembly line reconstitution of pacidamycin pentapeptide antibiotic scaffolds, bridging the primary and secondary metabolic pathways by hijacking an aminoacyl-tRNA to the antibiotic biosynthetic pathway.aminoacyltransferase | uridyl pentapeptide | peptidyl carrier protein P acidamycins are a family of uridyl tetra/pentapeptide antibiotics produced by Streptomyces coeruleorubidus (1). More than 10 related family members have been reported (1, 2): Their common scaffold contains a central N β -methyl 2S,3S-diaminoburytic acid (DABA) moiety which is α-amino-capped by a ureido dipeptide (C-terminal), β-amino-capped by a single amino acid or a dipeptide (N-terminal), and carboxy-linked to a 3′-deoxy-4′,5′-enaminouridine ( Fig. 1). Pacidamycins were reported to exert antibiotic activities by virtue of functioning as a substrate analog of the UDP-MurNAc-pentapeptide of MraY in bacterial cell wall assembly of the pentapeptidyl-bactoprenol intermediate (3,4). Pacidamycins are of interest due to their unusual peptidyl-nucleoside structure features, and for the development of next generation antibacterial drugs inhibiting the clinically underexplored cell wall enzyme target MraY. The biosynthetic gene clusters for pacidamycins and closely related napsamycins were identified in parallel recently by three research groups via genome mining (5-7). Bioinformatic analysis revealed the building blocks in pacidamycins are assembled using a nonribosomal peptide synthetase (NRPS) thiotemplate logic, whereas the encoded NRPS modules and domains are highly dissociated and predicted to work with each other in trans. We further characterized the functions of the separate NRPS protein domains in vitro to delineate catalytic steps for activation and insertion of each building block in the pacidamycin scaffold (8, 9). We have shown that nine NRPS enzymes encoded by the producing organism S. coeruleorubidus, when heterologously expressed and purified ...

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Pacidamycins, a novel series of antibiotics with anti-Pseudomonas aeruginosa activity. II. Isolation and structural elucidation.

Cited by 90 publications

References 0 publications

Characterization of LipL as a Non-heme, Fe(II)-dependent α-Ketoglutarate:UMP Dioxygenase That Generates Uridine-5′-aldehyde during A-90289 Biosynthesis

Characterization of LipL as a Non-heme, Fe(II)-dependent α-Ketoglutarate:UMP Dioxygenase That Generates Uridine-5′-aldehyde during A-90289 Biosynthesis

New Pacidamycins Produced by Streptomyces coeruleorubidus, NRRL 18370.

tRNA-dependent peptide bond formation by the transferase PacB in biosynthesis of the pacidamycin group of pentapeptidyl nucleoside antibiotics

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