Solid phase 4'-phosphopantetheinylation: fungal thiolation domains are targets for chemoenzymatic modification.
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.
Stack
Deirdre
D
National Institute for Cellular Biotechnology, Department of Biology, National University of Ireland Maynooth, Co. Kildare, Ireland.
Frizzell
Aisling
A
Tomkins
Karen
K
Doyle
Sean
S
D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D003065:Coenzyme A / Q000737:chemistry / Q000378:metabolism; D010204:Pantetheine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; C082662:phosphopantetheinyl transferase; D010204:Pantetheine; D017855:Transferases (Other Substituted Phosphate Groups); C003129:4'-phosphopantetheine; D003065:Coenzyme A