Structure of a Eukaryotic Nonribosomal Peptide Synthetase Adenylation Domain That Activates a Large Hydroxamate Amino Acid in Siderophore Biosynthesis

Abstract

Nonribosomal peptide synthetases (NRPSs) are large, multidomain proteins that are involved in the biosynthesis of an array of secondary metabolites. We report the structure of the third adenylation domain from the siderophore-synthesizing NRPS, SidN, from the endophytic fungus Neotyphodium lolii. This is the first structure of a eukaryotic NRPS domain, and it reveals a large binding pocket required to accommodate the unusual amino acid substrate,hydroxy-L-ornithine (cis-AMHO). The specific activation of cis-AMHO was confirmed biochemically, and an AMHO moiety was unambiguously identified as a component of the fungal siderophore using mass spectroscopy. The protein structure shows that the substrate binding pocket is defined by 17 amino acid residues, in contrast to both prokaryotic adenylation domains and to previous predictions based on modeling. Existing substrate prediction methods for NRPS adenylation domains fail for domains from eukaryotes due to the divergence of their signature sequences from those of prokaryotes. Thus, this new structure will provide a basis for improving prediction methods for eukaryotic NRPS enzymes that play important and diverse roles in the biology of fungi.

The siderophore ferricrocin produced by specific foliar endophytic fungi in vitro

“…Only one DSE strain of Phialocephala fortinii has been reported to produce ferrirubin (Bartholdy et al 2001) and orchid mycorrhizal strains produce ferrichrome or basidiochromes, as an exception (Haselwandter et al 2006;Haselwandter & Winkelmann 2007). Epichloë festucae, the foliar endophyte of perennial ryegrass, produces extracellular siderophore of a novel fusarinine type, and ferricrocin as the intracellular siderophore (Johnson 2008;Verne Lee et al 2010). According to our results, specific foliar endophytes of pine and Labrador tea produce ferricrocin in vitro, similar to most mycorrhizal and DSE fungi.…”

“…However, endophytes are microbes that live in the plant tissue without harming the host (Petrini 1986). A recent report describes the significance of siderophore production by the foliar endophyte Epichloë festucae for symbiosis with perennial ryegrass (Johnson 2008;Verne Lee et al 2010).…”

Identification of extracellular siderophores and a related peptide from the endophytic fungus Epichloë festucae in culture and endophyte-infected Lolium perenne

“…Biochemical studies of the third adenylation domain of the SidN NRPS enzyme complex from the related fungus N. lolii which produces 1 in culture (Koulman and Lane, unpublished data) established it could bind and activate cis -AMHO, but not any of the 20 proteogenic amino acids (Lee et al, 2010). However LC-MS analysis of culture supernatants of Δ sidN mutant strains of E. festucae established these accumulated trans -AMHO rather than cis -AMHO as the putative precursor for the siderophore (unpublished data).…”

Structural Biology of Nonribosomal Peptide Synthetases

“…Two structural studies on the TycC protein from tyrocidin synthesis, describing the PCP domain from the third module [9,6] and the PCP-condensation didomain crystal structure [44], were internal domain constructs. Additionally, an adenylation domain from SidN [63], a protein that is involved in the production of a fungal siderophore [64], was also excised from the middle of a larger NRPS protein. In this last example, the authors note that successful structure determination of SidN required optimization of the N- and C-termini and that changes of as few as 14 residues could impact protein solubility.…”