Dichlorination of a pyrrolyl-S-carrier protein by FADH
            <sub>2</sub>
            -dependent halogenase PltA during pyoluteorin biosynthesis

Dorrestein, Pieter C.; Yeh, Ellen; Garneau‐Tsodikova, Sylvie; Kelleher, Neil L.; Walsh, Christopher T.

doi:10.1073/pnas.0506964102

Cited by 169 publications

(224 citation statements)

References 27 publications

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“…Using Electrospray Ionization Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry (ESI/FT-ICRMS), thiotemplate bound substrates and intermediates of NRPS and PKS systems have been visualized (9,12,16,18,19,23,24). The FT-ICRMS assay has recently been adapted into a tool for substrate screening, but the mass accuracy has been between 0.05 −0.5 Da and required, in many cases, digestion and active site mapping prior to the analysis (25).…”

Section: Resultsmentioning

confidence: 99%

“…The proteins SgcC2, SgcC1, SgcC4, Sfp, PigH, PigJ, PigG, PigI, PigA, PltL, SsuE, PltA, GrsA, MycA(construct A4N7), AcpK, PksN, PksL-2ACP, PksI, PksH, NikP1, EntE and EntB(ArCP) were obtained as described (9,12,16,18,19,23). The first di-domain of PksJ with an N-terminal histidine tag was overproduced in E. coli and also purified via nickel affinity chromatography (Straight, Dorrestein, Kelleher, Kolter unpublished).…”

Section: Methodsmentioning

confidence: 99%

“…We provide support for the robustness and generality of this assay via 33 examples as this approach was used to characterize the loading of substrates, intermediates and products of proteins involved in the biosynthesis of the antibiotic nikkomycin (8), the antifungal agent pyoluteorin (9,10), the antibiotic prodigiosin (11,12), the antibacterial gramicidin (13,14), the antibacterial agent mycosubtilin (15,16), and the siderophore enterobactin (17,18). Further, intermediates were detected via this gas phase elimination on four proteins from the orphan orphan pksX cluster of B. subtilis that produces an unknown antibiotic (19)(20)(21).…”

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confidence: 99%

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Facile Detection of Acyl and Peptidyl Intermediates on Thiotemplate Carrier Domains via Phosphopantetheinyl Elimination Reactions during Tandem Mass Spectrometry

et al. 2006

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With the emergence of drug resistance and the genomic revolution there has been a renewed interest in the genes that are responsible for the generation of bioactive natural products. Secondary metabolites of one major class are biosynthesized at one or more sites by ultra large enzymes that carry covalent intermediates on phosphopantetheine arms. Because such intermediates are difficult to characterize in vitro, we have developed a new approach for streamlined detection of substrates, intermediates and products attached to a phosphopantetheinyl arm of the carrier site. During vibrational activation of gas phase carrier domains, facile elimination occurs in benchtop and FourierTransform mass spectrometers alike. Phosphopantetheinyl ejections quickly reduce >100 kDa megaenzymes to <1000 Da ions for structural assignment of intermediates at <0.007 Da mass accuracy without proteolytic digestion. This "Top Down" approach quickly illuminated diverse acylintermediates on the carrier domains of the nonribosomal peptide synthetases (NRPSs) or polyketide synthases (PKSs) found in the biosynthetic pathways of prodigiosin, pyoluteorin, mycosubtilin, nikkomycin, enterobactin, gramicidin and several proteins from the orphan pksX gene cluster from Bacillus subtilis. By focusing on just those regions undergoing covalent chemistry, the method delivered clean proof for the reversible dehydration of hydroxymethylglutaryl-S-PksL via incorporation of 2 H or 18 O from the buffer. The facile nature of this revised assay will allow diverse laboratories to spearhead their NRPS/PKS projects with benchtop mass spectrometers.About 50% of today's drugs and 75% of today's antimicrobials are derived from secondary metabolites (1,2). Many of those secondary metabolites are of polyketide or nonribosomal peptide origin. With the emergence of resistance and the genomic revolution there is a "renaissance" ongoing in the discovery of bioactive natural products and the characterization of the genes responsible for their production (1). Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are large enzymes often >>100 kDa that biosynthesize their natural products (e.g., the antibiotics penicillin, vancomycin) via covalent intermediates on phosphopantetheine arms (3). Currently, even when NRPS and PKS proteins can be overproduced their direct interrogation by mass spectrometry (MS) is difficult and timeconsuming, with reports from relatively few laboratories appearing in the primary literature (4-6). Therefore it would be of great benefit for the NRPS and PKS community if new MSbased methods to characterize these proteins were developed and easier to implement. This paper describes such a method.The purpose of this paper is four-fold: 1) the paper introduces a new and efficient method that utilizes a gas phase elimination reaction that takes place during tandem mass spectrometry (MS/MS) to quickly characterize substrates, intermediates and products that are loaded onto the phosphopantetheinyl arm on carrier domains of NRPS...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Facile Detection of Acyl and Peptidyl Intermediates on Thiotemplate Carrier Domains via Phosphopantetheinyl Elimination Reactions during Tandem Mass Spectrometry

et al. 2006

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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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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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“…Therefore, we performed assays in the presence of additional sodium chloride, sodium bromide, and potassium iodide. As is typical for FDTH, 30,33,35 KrmI is able to utilize chloride and bromide but not iodide, with reduced activity when incorporating bromide (Figures 5 and 6). Figure 5.…”

Section: Halide and Substrate Scopementioning

confidence: 99%

An Unusual Flavin-Dependent Halogenase from the Metagenome of the Marine Sponge Theonella swinhoei WA

et al. 2017

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Dichlorination of a pyrrolyl-S-carrier protein by FADH ₂ -dependent halogenase PltA during pyoluteorin biosynthesis

Cited by 169 publications

References 27 publications

Facile Detection of Acyl and Peptidyl Intermediates on Thiotemplate Carrier Domains via Phosphopantetheinyl Elimination Reactions during Tandem Mass Spectrometry

Facile Detection of Acyl and Peptidyl Intermediates on Thiotemplate Carrier Domains via Phosphopantetheinyl Elimination Reactions during Tandem Mass Spectrometry

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

An Unusual Flavin-Dependent Halogenase from the Metagenome of the Marine Sponge Theonella swinhoei WA

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Dichlorination of a pyrrolyl-S-carrier protein by FADH 2 -dependent halogenase PltA during pyoluteorin biosynthesis

Cited by 169 publications

References 27 publications

Facile Detection of Acyl and Peptidyl Intermediates on Thiotemplate Carrier Domains via Phosphopantetheinyl Elimination Reactions during Tandem Mass Spectrometry

Facile Detection of Acyl and Peptidyl Intermediates on Thiotemplate Carrier Domains via Phosphopantetheinyl Elimination Reactions during Tandem Mass Spectrometry

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

An Unusual Flavin-Dependent Halogenase from the Metagenome of the Marine Sponge Theonella swinhoei WA

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Dichlorination of a pyrrolyl-S-carrier protein by FADH ₂ -dependent halogenase PltA during pyoluteorin biosynthesis