Characterization of fungal phosphopantetheinyl transferases involved in primary and secondary metabolism.

D. Keszenman-Pereyra1, N. George2, K. Johnsson2 and G. Turner1.

Author address: 

1Department of Molecular Biology and Biotechnology, Krebs Institute for Biomolecular Research, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK; 2Institute of Chemical Sciences and Engineering, Ecole Polytechnique Federale de

Abstract: 

Primary and secondary metabolic pathways in Aspergillus sp utilize 30-50 phosphopantetheinylated proteins with specialized carrier protein (CP) domains. The conversion of apo-CPs to holo-CPs is catalysed by 4'-phosphopantetheine transferases (PPTases). Blast searches of available filamentous fungal genome sequences revealed that each organism has putative genes encoding Fas2 integrated-, AcpS- (postulated to target mitochondrial Acp1) and NpgA- type PPTases. A. nidulans NpgA seems capable of activating CPs of non-ribosomal peptide synthetases, polyketide synthases and alpha aminoadipate reductase (homologue of yeast Lys2). To analyse the wide substrate specificity of NpgA, the ORF was amplified by PCR from A. nidulans genomic DNA and cloned in frame with N-terminal hexahistidine tag; the PPTase was overexpressed in Escherichia coli and purified by nickel affinity chromatography. The wide substrate specificity of NpgA has been confirmed in vitro by using a range of CP substrates and assays. Moreover, co-expression experiments in E. coli show that NpgA is relatively efficient for the activation of heterologous substrates. NpgA is the first reported example of a eukaryotic PPTase involved in both primary and secondary metabolism. Phylogenetic relationships of fungal PPTases and their substrates will be discussed.
2005

abstract No: 

534

Full conference title: 

23rd Fungal Genetics Conference
    • Fungal Genetics Conference 23rd (2002)