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Solid phase 4'-phosphopantetheinylation: fungal thiolation domains are targets for chemoenzymatic modification.
- Source :
-
Bioconjugate chemistry [Bioconjug Chem] 2009 Aug 19; Vol. 20 (8), pp. 1514-22. Date of Electronic Publication: 2009 Jul 23. - Publication Year :
- 2009
-
Abstract
- 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 solution-phase 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, high-throughput method to determine PPTase activity.
- Subjects :
- Aspergillus fumigatus metabolism
Bacterial Proteins chemistry
Biocatalysis
Coenzyme A chemistry
Coenzyme A metabolism
Pantetheine chemistry
Pantetheine metabolism
Recombinant Proteins chemistry
Recombinant Proteins metabolism
Transferases (Other Substituted Phosphate Groups) chemistry
Aspergillus fumigatus enzymology
Bacterial Proteins metabolism
Pantetheine analogs & derivatives
Transferases (Other Substituted Phosphate Groups) metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1520-4812
- Volume :
- 20
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- Bioconjugate chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 19627078
- Full Text :
- https://doi.org/10.1021/bc900071j