Stack, Deirdre and Frizzell, Aisling and Tomkins, Karen and Doyle, Sean (2009) Solid Phase 4′-Phosphopantetheinylation: Fungal Thiolation Domains are Targets for Chemoenzymatic Modification. Bioconjugate Chemistry, 20 (8). pp. 1514-1522. ISSN 1043-1802

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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 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.

Item Type:	Article
Keywords:	Solid Phase 4′-Phosphopantetheinylation; Fungal Thiolation Domains; Chemoenzymatic Modification;
Academic Unit:	Faculty of Science and Engineering > Biology
Item ID:	7395
Identification Number:	https://doi.org/10.1021/bc900071j
Depositing User:	Dr. Sean Doyle
Date Deposited:	26 Aug 2016 14:46
Journal or Publication Title:	Bioconjugate Chemistry
Publisher:	American Chemical Society
Refereed:	Yes
Funders:	Higher Education Authority (HEA), Enterprise Ireland (EI)
URI:	http://pubs.acs.org
Use Licence:	This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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