Expression and Characterisation of a Nonribosomal Peptide Synthetase from Aspergillus fumigatus.

Deirdre Stack, Kevin Kavanagh and Sean Doyle.

Author address: 

Department of Biology and National Institute of Cellular Biotechnology, National University of Ireland Maynooth, Co. Kildare, Ireland. Email: [email protected]

Abstract: 

Aspergillus fumigatus is a significant human pathogen. Organismal virulence has been proposed to be multi-factorial and mediated by a range of proteinaceous and non-proteinaceous moieties. Nonribosomal peptide synthesis (NRPS) is responsible for a significant proportion of toxin and siderophore production in the organism. Nonribosomal peptide synthetases are multi-modular enzymes that facilitate NRPS. Furthermore, it has been shown that 4’-phosphopantetheinylation is required for the activation of key enzymes involved in NRPS in both Aspergillus fumigatus and other species whereby a coenzyme A (CoA)- derived 4’phosphopantetheine group is transferred to thiolation (T) (or peptidyl carrier protein (PCP) domains) in NRPS enzymes. Here we report the identification of an atypical 25.5 kb NRP synthetase gene (pes3) with a non-linear modular arrangement. Phylogenetic analysis indicated that pes3 shows minimal relatedness to other NRPS. Quantitative RT-PCR analysis has confirmed that pes3 is differentially expressed in A. fumigatus. We have previously cloned and expressed a functional characterisation 4’-phosphopantetheinyl transferase from A. fumigatus [1] and now investigate if Pes3 is activated by the 4’-PPTase. Consequently, a 72 kDa fragment of A. fumigatus pes3, containing a putative thiolation domain, was cloned and expressed in an E. coli expression system and shown to be activated by 4’phosphopantetheinylation using a biotinylatedCoA co-factor analogue only in the presence of the 4’-PPTase. Overall, our data confirm that the NRPS, pes3, is expressed in A. fumigatus and that the cognate protein is activated, in vitro, by recombinant 4’-PPTase. Reference: 1. Neville, C., Kavanagh, K., Murphy, A. and Doyle S. (2005) A 4’-phosphopantetheinyl transferase mediates non-ribosomal peptide synthetase activation in Aspergillus fumigatus. ChemBiochem 6(4):679-685.
2006

abstract No: 

ABSTRACT 54

Full conference title: 

SGM Irish Branch Dublin meeting
    • Irish SGM