Abstract

BackgroundMost filamentous ascomycete fungi produce high affinity iron chelators called siderophores, biosynthesized nonribosomally by multimodular adenylating enzymes called nonribosomal peptide synthetases (NRPSs). While genes encoding the majority of NRPSs are intermittently distributed across the fungal kingdom, those encoding ferrichrome synthetase NRPSs, responsible for biosynthesis of ferrichrome siderophores, are conserved, which offers an opportunity to trace their evolution and the genesis of their multimodular domain architecture. Furthermore, since the chemistry of many ferrichromes is known, the biochemical and structural 'rules' guiding NRPS substrate choice can be addressed using protein structural modeling and evolutionary approaches.ResultsA search of forty-nine complete fungal genome sequences revealed that, with the exception of Schizosaccharomyces pombe, none of the yeast, chytrid, or zygomycete genomes contained a candidate ferrichrome synthetase. In contrast, all filamentous ascomycetes queried contained at least one, while presence and numbers in basidiomycetes varied. Genes encoding ferrichrome synthetases were monophyletic when analyzed with other NRPSs. Phylogenetic analyses provided support for an ancestral duplication event resulting in two main lineages. They also supported the proposed hypothesis that ferrichrome synthetases derive from an ancestral hexamodular gene, likely created by tandem duplication of complete NRPS modules. Recurrent losses of individual domains or complete modules from this ancestral gene best explain the diversity of extant domain architectures observed. Key residues and regions in the adenylation domain pocket involved in substrate choice and for binding the amino and carboxy termini of the substrate were identified.ConclusionIron-chelating ferrichrome synthetases appear restricted to fission yeast, filamentous ascomycetes, and basidiomycetes and fall into two main lineages. Phylogenetic analyses suggest that loss of domains or modules led to evolution of iterative biosynthetic mechanisms that allow flexibility in biosynthesis of the ferrichrome product. The 10 amino acid NRPS code, proposed earlier, failed when we tried to infer substrate preference. Instead, our analyses point to several regions of the binding pocket important in substrate choice and suggest that two positions of the code are involved in substrate anchoring, not substrate choice.

Highlights

  • Most filamentous ascomycete fungi produce high affinity iron chelators called siderophores, biosynthesized nonribosomally by multimodular adenylating enzymes called nonribosomal peptide synthetases (NRPSs)

  • Genomes surveyed for ferrichrome-associated nonribosomal peptide synthetases Candidate homologs of C. heterostrophus NPS2 [3,18] and A. nidulans SidC [19] were identified through blastp and tblastn searches using individual A domains from both NPS2 and SidC proteins as a query set

  • For the five basidiomycete genomes examined, two known NRPSs were found in U. maydis, one undescribed ferrichrome synthetase was identified in C. cinerea while P. chrysosporium, L. bicolor, and C. neoformans lacked genes encoding these enzymes

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Summary

Introduction

Most filamentous ascomycete fungi produce high affinity iron chelators called siderophores, biosynthesized nonribosomally by multimodular adenylating enzymes called nonribosomal peptide synthetases (NRPSs). The observation that A-T-C modules from a single NRPS often group together as a monophyletic clade suggests tandem duplication of modules as a possible mechanism by which multimodular NRPSs arise [3]. It is clear, that other mechanisms such as recombination and gene conversion operate [4]. Ferrichrome synthetases, which biosynthesize ferrichromes, fungal hydroxamate siderophores that function primarily in intracellular iron storage, are among the most conserved NRPSs, offering an opportunity to trace the evolutionary history of the corresponding genes across fungi

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