A Highly Conserved Basidiomycete Peptide Synthetase Produces a Trimeric Hydroxamate Siderophore

The model white-rot basidiomycete, ( ) B, encodes putative natural product biosynthesis genes. Among them is the gene for the seven-domain nonribosomal peptide synthetase CsNPS2. It is a member of the as-yet-uncharacterized fungal type VI siderophore synthetase family, which is highly conserved and widely distributed among the basidiomycetes. These enzymes include only one adenylation (A) domain, i.e., one complete peptide synthetase module, and two thiolation/condensation (T-C) didomain partial modules which together constitute an AT C T C T C domain setup. The full-length CsNPS2 enzyme (274.5 kDa) was heterologously produced as a polyhistidine fusion in as a soluble and active protein. -acetyl- -hydroxy-l-ornithine (l-AHO) and - -anhydromevalonyl- -hydroxy-l-ornithine (l-AMHO) were accepted as the substrates, based on results of an substrate-dependent [ P]ATP-pyrophosphate radioisotope exchange assay. Full-length -CsNPS2 catalyzed amide bond formation between three l-AHO molecules to release the linear l-AHO trimer, called basidioferrin, as the product , which was verified by liquid chromatography-high-resolution electrospray ionization-mass spectrometry analysis. Phylogenetic analyses suggested that type VI family siderophore synthetases are widespread in mushrooms and evolved in a common ancestor of basidiomycetes. The basidiomycete nonribosomal peptide synthetase CsNPS2 represents a member of a widely distributed but previously uninvestigated class (type VI) of fungal siderophore synthetases. Genes orthologous to are highly conserved across various phylogenetic clades of the basidiomycetes. Hence, our work serves as a broadly applicable model for siderophore biosynthesis and iron metabolism in higher fungi. Also, our results on the amino acid substrate preference of CsNPS2 support a further understanding of the substrate selectivity of fungal adenylation domains. Methodologically, this report highlights the /SM-Xpress-based system as a suitable platform to heterologously express multimodular basidiomycete biosynthesis enzymes in the >250-kDa range in soluble and active form.