A 4'-phosphopantetheinyl transferase mediates non-ribosomal peptide synthetase activation in Aspergillus fumigatus.
Aspergillus fumigatus is a significant human pathogen. Non-ribosomal peptide (NRP) synthesis is thought to be responsible for a significant proportion 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-ribosomal peptide synthesis in other species. Here we report the cloning, recombinant expression and functional characterisation of a 4'-phosphopantetheinyl transferase from A. fumigatus and the identification of an atypical NRP synthetase (Afpes1), 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 alpha-aminoadipate reductase (Lys2p) from Candida albicans, a key enzyme involved in lysine biosynthesis.
Neville
Claire
C
National Institute for Cellular Biotechnology, Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland.
Murphy
Alan
A
Kavanagh
Kevin
K
Doyle
Sean
S
D000445:Aldehyde Oxidoreductases / Q000378:metabolism; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D002176:Candida albicans / Q000201:enzymology / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D004789:Enzyme Activation; D015870:Gene Expression / Q000235:genetics; D050647:L-Aminoadipate-Semialdehyde Dehydrogenase; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
D001426:Bacterial Proteins; D011994:Recombinant Proteins; C082662:phosphopantetheinyl transferase; D000445:Aldehyde Oxidoreductases; D050647:L-Aminoadipate-Semialdehyde Dehydrogenase; D017855:Transferases (Other Substituted Phosphate Groups); D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase