Characterization of the Formation of the Pyrrole Moiety during Clorobiocin and Coumermycin A<sub>1</sub> Biosynthesis

Garneau, Sylvie; Dorrestein, Pieter C.; Kelleher, Neil L.; Walsh, Christopher T.

doi:10.1021/bi0476329

Cited by 82 publications

(118 citation statements)

References 27 publications

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“…Of these, only ␣-ketopyrrole moieties are found in a wide variety of biologically active natural products. Feeding experiments have shown that the ␣-ketopyrrole of calcimycin is derived from L-proline (11,33). Biosynthesis of Table 3.…”

Section: Resultsmentioning

confidence: 99%

Characterization of the Biosynthesis Gene Cluster for the Pyrrole Polyether Antibiotic Calcimycin (A23187) in Streptomyces chartreusis NRRL 3882

Lin

et al. 2011

Antimicrob Agents Chemother

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The pyrrole polyether antibiotic calcimycin (A23187) is a rare ionophore that is specific for divalent cations. It is widely used as a biochemical and pharmacological tool because of its multiple, unique biological effects. Here we report on the cloning, sequencing, and mutational analysis of the 64-kb biosynthetic gene cluster from Streptomyces chartreusis NRRL 3882. Gene replacements confirmed the identity of the gene cluster, and in silico analysis of the DNA sequence revealed 27 potential genes, including 3 genes for the biosynthesis of the ␣-ketopyrrole moiety, 5 genes that encode modular type I polyketide synthases for the biosynthesis of the spiroketal ring, 4 genes for the biosynthesis of 3-hydroxyanthranilic acid, an N-methyltransferase tailoring gene, a resistance gene, a type II thioesterase gene, 3 regulatory genes, 4 genes with other functions, and 5 genes of unknown function. We propose a pathway for the biosynthesis of calcimycin and assign the genes to the biosynthesis steps. Our findings set the stage for producing much desired calcimycin derivatives using genetic modification instead of chemical synthesis.

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Section: Resultsmentioning

confidence: 99%

Characterization of the Biosynthesis Gene Cluster for the Pyrrole Polyether Antibiotic Calcimycin (A23187) in Streptomyces chartreusis NRRL 3882

Lin

et al. 2011

Antimicrob Agents Chemother

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“…In analogy to the requirement of CoA thioester-bound substrates for C ␣ -H acidification in fatty acid dehydrogenation (21), abstraction of the ␣-proton from ␦-chloro-(allo-)Ile-S-KtzC could be facilitated by the phosphopantetheinyl thioester, to be followed by a removal of ␤-hydride. Similar flavoprotein dehydrogenases catalyze ␣,␤-dehydrogenation of T domain-bound thioesters in the biosynthesis of pyrrole moieties in pyoluteorin and undecylprodigiosin (22,23). Analogous dehydrogenation of ␦-chloro-(allo-)Ile-S-KtzC would yield an enamine intermediate, where C ␤ could serve as an intramolecular nucleophile in cyclopropane formation.…”

Section: Resultsmentioning

confidence: 99%

Cloning and characterization of the biosynthetic gene cluster for kutznerides

Fujimori

Hrvatin²,

Neumann³

et al. 2007

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

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Kutznerides, actinomycete-derived cyclic depsipetides, consist of six nonproteinogenic residues, including a highly oxygenated tricyclic hexahydropyrroloindole, a chlorinated piperazic acid, 2-(1-methylcyclopropyl)-glycine, a ␤-branched-hydroxy acid, and 3-hydroxy glutamic acid, for which biosynthetic logic has not been elucidated. Herein we describe the biosynthetic gene cluster for the kutzneride family, identified by degenerate primer PCR for halogenating enzymes postulated to be involved in biosyntheses of these unusual monomers. The 56-kb gene cluster encodes a series of six nonribosomal peptide synthetase (NRPS) modules distributed over three proteins and a variety of tailoring enzymes, including both mononuclear nonheme iron and two flavindependent halogenases, and an array of oxygen transfer catalysts. The sequence and organization of NRPS genes support incorporation of the unusual monomer units into the densely functionalized scaffold of kutznerides. Our work provides insight into the formation of this intriguing class of compounds and provides a foundation for elucidating the timing and mechanisms of their biosynthesis.chlorination ͉ halogenases ͉ nonribosomal peptide biosynthesis K utznerides are antifungal and antimicrobial cyclic hexadepsipeptides isolated from the soil actinomycete Kutzneria sp. 744 (1). Structural elucidation of these metabolites revealed nine related compounds composed of five unusual nonproteinogenic amino acids and one hydroxy acid ( Fig. 1) but differing in the extent of substitution and stereochemistry of constituent residues (2). All kutznerides contain 2-(1-methylcyclopropyl)-D-glycine (MecPGly) connected to the ␣-hydroxyl moiety of either (S)-2-hydroxy-3-methylbutyric or (S)-2-hydroxy-3,3-dimethylbutyric acid. The hydroxy acid residue is followed by a piperazic acid moiety, found in four distinct forms: as piperazic acid in kutznerides 1, 3, 5, and 7; as dehydropiperazate in kutznerides 4 and 9; as ␥-chloro-piperazate in kutznerides 2 and 8; and as ␥-hydroxy-dehydropiperazate in kutzneride 6. Furthermore, kutznerides contain O-methyl-L-serine and either the threo or erythro isomer of 3-hydroxy-D-glutamate. Finally, an unusual tricyclic dihalogenated (2S,3aR,8aS)-6,7-dichloro-3a-hydroxy-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid (PIC) is conserved in all structurally characterized kutznerides. The structural subunits of kutznerides suggest unusual enzymatic mechanisms involved in their biosynthesis.Recently, our laboratory has elucidated the enzymatic logic of carbon-chlorine bond formation during the biosynthesis of several chlorinated secondary metabolites. Among these, dichlorination of the pyrrole moiety in the biosynthesis of pyoluteorin (3) and chlorination of tryptophan in rebeccamycin biosynthesis (4) are carried out by the flavin-dependent halogenases PltA and RebH, respectively. The work of van Pee and coworkers (5) on the pyrrolnitrin halogenase PrnA demonstrated the necessity of FADH 2 , chloride, and oxygen for catalytic activity of flavoprotein halogenase...

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“…Here we describe a mass spectrometry based method to identify substrates based on mass changes that take place during acylation of a phosphopantetheinyl functionality on the carrier domain(s) of an NRPS protein from very complex substrate reaction mixtures. Observing such acylations by mass spectrometry on carrier domains is now becoming routine (5,19,(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36).The general method to identify covalently loaded substrates or intermediates is shown in Figure 1. Overproduced protein that contains a carrier domain is purified and incubated with Sfp, a promiscuous phosphopantetheinyl transferase from B .subtilis, and CoA in order to generate the holo form of the carrier domain (37).…”

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confidence: 99%

“…Therefore we wanted to develop an alternative method for substrate screening to complement the more traditional radioactive assays (5,6). In NRPS and PKS systems, the substrates and intermediates are loaded onto and processed while attached to the pantetheinyl functionality on a carrier domain (7,8).…”

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Activity Screening of Carrier Domains within Nonribosomal Peptide Synthetases Using Complex Substrate Mixtures and Large Molecule Mass Spectrometry

et al. 2006

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For screening a pool of potential substrates that load carrier domains found in non-ribosomal peptide synthetases, large molecule mass spectrometry is shown to be an ideal, unbiased assay. Combining the high resolving power of Fourier-Transform Mass Spectrometry with the ability of adenylation domains to select their own substrates, the mass change that takes place upon formation of a covalent intermediate thus identifies the substrate. This assay has an advantage over traditional radiochemical assays in that many substrates, the substrate pool, can be screened simultaneously. Using proteins on the nikkomycin, clorobiocin, coumermycin A 1 , yersiniabactin, pyochelin and enterobactin biosynthetic pathways as proof-of-principle, preferred substrates are readily identified from substrate pools. Furthermore this assay can be used to provide insight into the timing of tailoring events of biosynthetic pathways as demonstrated using the bromination reaction found on the jamaicamide biosynthetic pathway. Finally, this assay can provide insight into the role and function of orphan gene clusters for which the encoded natural product is unknown. This is demonstrated by identifying the substrates for two NRPS modules from the genes pksN and pksJ, that are found on an orphan NRPS/PKS hybrid cluster from Bacillus subtilis. This new assay format is especially timely for activity screening in an era when new types of thiotemplate assembly lines that defy classification are being discovered at an accelerating rate. KeywordsElectrospray Fourier transform mass spectrometry; Non-ribosomal peptide synthetase; Polyketide synthase; Carrier domains; Post-translational modification Almost weekly, new non-ribosomal peptide synthetase (NRPS) gene clusters encoding for proteins that biosynthesize bioactive compounds are discovered (1-4). Since many of the compounds produced by non-ribosomal peptide synthetase (NRPS) as well as polyketide * To whom correspondence should be addressed, kelleher@scs.uiuc.edu, fax number 217 244-8068 and phone number 217 NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 October 9. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript synthase (PKS) paradigm have potent medicinal utility or are involved in virulence, and display unusual chemistry, they are of great academic and industrial interest. Therefore we wanted to develop an alternative method for substrate screening to complement the more traditional radioactive assays (5,6). In NRPS and PKS systems, the substrates and intermediates are loaded onto and processed while attached to the pantetheinyl functionality on a carrier domain (7,8).Examples of NRPS or hybrid NRPS/PKS natural products for which substrates load onto carrier domains are: the siderophore pyoverdine (9), a virulence factor excreted by pseudomonads, the antimicrobial agents penicillin (10), vancomycin (11) gramicidin (12) and the antitumor agent calicheamycin (13). With ∼300 genomes sequenced and available in the public ...

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Characterization of the Formation of the Pyrrole Moiety during Clorobiocin and Coumermycin A₁ Biosynthesis

Cited by 82 publications

References 27 publications

Characterization of the Biosynthesis Gene Cluster for the Pyrrole Polyether Antibiotic Calcimycin (A23187) in Streptomyces chartreusis NRRL 3882

Characterization of the Biosynthesis Gene Cluster for the Pyrrole Polyether Antibiotic Calcimycin (A23187) in Streptomyces chartreusis NRRL 3882

Cloning and characterization of the biosynthetic gene cluster for kutznerides

Activity Screening of Carrier Domains within Nonribosomal Peptide Synthetases Using Complex Substrate Mixtures and Large Molecule Mass Spectrometry

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Characterization of the Formation of the Pyrrole Moiety during Clorobiocin and Coumermycin A1 Biosynthesis

Cited by 82 publications

References 27 publications

Characterization of the Biosynthesis Gene Cluster for the Pyrrole Polyether Antibiotic Calcimycin (A23187) in Streptomyces chartreusis NRRL 3882

Characterization of the Biosynthesis Gene Cluster for the Pyrrole Polyether Antibiotic Calcimycin (A23187) in Streptomyces chartreusis NRRL 3882

Cloning and characterization of the biosynthetic gene cluster for kutznerides

Activity Screening of Carrier Domains within Nonribosomal Peptide Synthetases Using Complex Substrate Mixtures and Large Molecule Mass Spectrometry

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Characterization of the Formation of the Pyrrole Moiety during Clorobiocin and Coumermycin A₁ Biosynthesis