Manipulation of Carrier Proteins in Antibiotic Biosynthesis

Clair, James J. La; Foley, Timothy L.; Schegg, Tracy R; Regan, Conor M; Burkart, Michael D.

doi:10.1016/j.chembiol.2004.02.010

Cited by 154 publications

(131 citation statements)

References 24 publications

Supporting

Mentioning

128

Contrasting

Unclassified

Order By: Relevance

“…In this work, we describe the implementation of a cyanogen bromide digestion protocol and the use of several coenzyme A analogues with spectroscopic, ∆ mass, and affinity properties for mapping the modified active site peptides from the pyochelin synthetase biosynthetic enzymes, PchE (159 kDa) and PchF (198 kDa) ( Figures 6 and 7 of the Supporting Information) (20)(21)(22). These "reporter"-coenzyme A analogues were exclusively loaded onto the conserved active site serines within the thiolation domains of PchE and PchF using the indiscriminate 4′-phosphopantetheinyl transferase, Sfp (23).…”

mentioning

confidence: 99%

Chemoenzymatic Approaches for Streamlined Detection of Active Site Modifications on Thiotemplate Assembly Lines Using Mass Spectrometry

et al. 2005

View full text Add to dashboard Cite

For the direct interrogation of peptides harboring covalently modified serines in nonribosomal peptide synthetases, streamlined methodologies described here employ proteolysis and reporter-coenzyme A analogues of four types. The chromophoric and fluorescent coenzyme A analogues pyrene-maleimidyl-S-CoA and BODIPY-FL-N-(2-aminoethyl)maleimidyl-S-CoA were enzymatically loaded onto the active site serines harbored in the ArCP, PCP1, and PCP2 thiolation domains of PchE and PchF, the nonribosomal peptide synthetases responsible for the biosynthesis of the siderophore pyochelin. During the chromatographic separation of cyanogen bromide digests, observation of the absorbance (at 338 and 504 nm) or fluorescence (after irradiation at 365 nm) enabled the selective detection of peptides containing each active site serine. This resulted in quick detection of each active site peptide by Fourier transform mass spectrometry in the fully reconstituted pyochelin system. The loading of short acyl chain reporters in equimolar quantities permitted further insights into digestion heterogeneity and side reactions by virtue of a mass shift signature on each active site peptide. The chromatographic shift of the reporter-loaded peptides relative to peptides carrying on pathway intermediates was 2 min at 7 kDa, providing a general strategy for efficient localization of "carrier" peptides in complex digests of thiotemplate enzymes. Also, the use of the affinity reporter, biotin-maleimidyl-S-coenzyme A, permitted the isolation of intact synthetases at high purity via removal of contaminating Escherichia coli proteins.

show abstract

mentioning

confidence: 99%

Chemoenzymatic Approaches for Streamlined Detection of Active Site Modifications on Thiotemplate Assembly Lines Using Mass Spectrometry

et al. 2005

View full text Add to dashboard Cite

show abstract

“…fectively 90%+ efficiency of biotin-CoA conjugate synthesis and extends the utility of aldrithiol-4 and other reagents used for thiol group determination (18). Previously, identical biotinCoA analogues were synthesized, the unincorporated biotin reporter extracted via the use of scavenger resins, and the resultant biotin-CoA reporter subsequently used for the highsensitivity detection (100 pg) of the biotin labeled recombinant VibB carrier protein domain (6). However, we observed minimal background protein modification once DTT was added to biotinCoA conjugates, which implies that removal of unreacted MB may not always be necessary.…”

Section: E H S T D T S Ad N E Rmentioning

confidence: 99%

“…More recently, the application of protein mass spectrometry for the determination of apoenzyme 4′-phosphopantetheinylation has been reported (2,5). These conventional methods for assaying PPTase activity, however, can be costly and cumbersome and significantly limit high-throughput operations (6).…”

Section: Introductionmentioning

confidence: 99%

“…This labeling technique also has utility in the identification of carrier protein domains from lysates of native cultures, as a proportion of carrier proteins in the lysate may be present in an unmodified inactive state. Application of the technique to the 6-deoxyerythronolide B synthase (DEBS) pathway from Saccharapolyspora erythraea resulted in the identification of a 150 kDa protein indicative of native DEBS (6).…”

Section: Introductionmentioning

confidence: 99%

“…An elegant method of covalently labeling T/PCP domains of NRP synthetases, using biotinylated or fluorescently labeled CoA, and a recombinant Bacillus subtilis encoded PPTase, Sfp, in PPTase-mediated reactions has been described (6). These authors used Sfp to catalyze modification of carrier proteins with a single fluorescent or affinity reporter, providing a sensitive means of protein visualization, Western blot identification, and affinity purification.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Solid Phase 4′-Phosphopantetheinylation: Fungal Thiolation Domains are Targets for Chemoenzymatic Modification

et al. 2009

View full text Add to dashboard Cite

No data exist on the ability of thiolation domains from fungal non-ribosomal peptide synthetases to undergo 4′-phosphopantetheinylation, using either biotinylated or fluorescently labeled coenzyme A analogues, mediated by 4′-phosphopantetheinyl transferases (PPTase). Yet, this is a key requirement to confirm the amino acid recognition function, and coding potential, of either non-ribosomal peptide synthetases or recombinantly expressed regions of these enzymes (e.g., didomains or modules). Moreover, determination of 4′-phosphopantetheinylation activity remains cumbersome. Here, we demonstrate that a recombinant fungal PPTase catalyzes the solutionphase transfer of either biotin-or fluorescein-labeled 4′-phosphopantetheine region of coenzyme A to a fungal thiolation domain, which is either part of a non-ribosomal peptide synthetase didomain (72 kDa), derived from Aspergillus fumigatus, or fused to a non-native protein (glutathione s-transferase). Significantly, we demonstrate that this reaction can unexpectedly occur when the target protein (4.4 pmol) is immobilized on a solid surface. These findings (i) confirm that thiolation domains of fungal origin, in native or non-native configuration, can accept modified 4′-phosphopantetheine residues via PPTase-mediated labeling and (ii) illustrate a novel, highthroughput method to determine PPTase activity.

show abstract

CoA‐Dependent Enzymes, Chemistry of

Drueckhammer

2008

Wiley Encyclopedia of Chemical Biology

View full text Add to dashboard Cite

Coenzyme A (CoA) and its thioesters play a diverse array of roles in biologic systems. The enzymes that catalyze reactions of CoA are of interest for a variety of reasons, including, but not limited to, their potential as drug targets. The enzymology of CoA biosynthesis is now well understood, and the genes for all of the enzymes involved have been identified. Thioesters are inherently reactive toward acyl transfer reactions and toward reactions involving deprotonation of the α‐carbon, and these are the primary reactivity patterns in enzyme‐catalyzed reactions of CoA thioesters. CoA utilizing enzymes have been widely studied mechanistically and structurally. Analogs of the natural CoA thioester substrates have been widely used in these studies, including thioesters of unnatural or uncommon acyl groups as well as a large number of analogs in which the thioester is replaced with alternative functionality. Recent technical applications of CoA have included the tagging of carrier protein domains and carrier protein fusions with tagged phosphopantetheine derivatives transferred enzymatically from the corresponding tagged CoA derivatives using promiscuous phosphopantetheinyl transferases.

show abstract

Manipulation of Carrier Proteins in Antibiotic Biosynthesis

Cited by 154 publications

References 24 publications

Chemoenzymatic Approaches for Streamlined Detection of Active Site Modifications on Thiotemplate Assembly Lines Using Mass Spectrometry

Chemoenzymatic Approaches for Streamlined Detection of Active Site Modifications on Thiotemplate Assembly Lines Using Mass Spectrometry

Solid Phase 4′-Phosphopantetheinylation: Fungal Thiolation Domains are Targets for Chemoenzymatic Modification

CoA‐Dependent Enzymes, Chemistry of

Contact Info

Product

Resources

About