Peer-Reviewed Journal Details
Mandatory Fields
Neville, C;Murphy, A;Kavanagh, K;Doyle, S
2005
April
ChemBioChem
A 4 '-phosphopantetheinyl transferase mediates non-ribosomal peptide synthetase activation in Aspergillus fumigatus
Published
18 ()
Optional Fields
DIRECTED MUTATIONAL ANALYSIS PHOSPHOPANTETHEINYL TRANSFERASE SIDEROPHORE BIOSYNTHESIS INVASIVE ASPERGILLOSIS MOLECULAR-GENETICS CANDIDA-ALBICANS CLONING IDENTIFICATION NIDULANS SPECIFICITY
6
679
685
Aspergillus fumigatus is a significant human pathogen. Non-ribosomal peptide (NRP) synthesis is thought to be responsible for a significant proportien of toxin and siderophore production in the organism. Furthermore, it has been shown that 4'-phosphopantetheinylation is required for the activation of key enzymes involved in non-ribosomol peptide synthesis in other species. Here we report the cloning, recombinant expression and functional characterisation of a 4'-phosphopontetheinyl transferase from A. fumigatus and the identification of an atypical NRP synthetase (Afpes 1), spanning 14.3 kb. Phylogenetic analysis has shown that the NRP synthetase exhibits greatest identity to NRP synthetases from Metarhizium anisolpiae (PesA) and Alternaria brassicae (AbrePsy1). Northern hybridisation and RT-PCR analysis have confirmed that both genes are expressed in A. fumigatus. A 120 kDa fragment of the A. fumigatus NRP synthetase, containing a putative thiolation domain, was cloned and expressed in the baculovirus expression system. Detection of a 4'-phosphopantetheinylated peptide (SFSAMK) from this protein, by MALDI-TOF mass spectrometric analysis after coincubation of the 4'-phosphopantetheinyl transferase with the recombinant NRP synthetase fragment and acetyl CoA, confirms that it is competent to play a role in NRP synthetase activation in A. fumigatus. The 4'-phosphopantetheinyl transferase also activates, by 4'-phosphopantetheinylation, recombinant a-aminoadipate reductase (Lys2p) from Candida albicans, a key enzyme involved in lysine biosynthesis.
WEINHEIM
1439-4227
10.1002/cbic.200400147
Grant Details