A census analysis of the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase and EPSP-associated domains

Glyphosate is one of the most used herbicides against weeds that targets the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). EPSPS is the central enzyme in the shikimate pathway to synthesize 3 essential amino acids in plants, fungi, and prokaryotes. Although this pathway is not found in animals, herbicide may affect the biodiversity of environmental and host-associated microorganisms. In this master thesis I will survey the distribution of the EPSPS enzyme in thousands of microorganisms and I will analyse the evolution of the multi domain structure of the EPSPS enzyme in fungi. Data was gathered from public databases of proteins (e.g., Pfam and COG). The analysis of the distribution of the EPSPS was performed using Excel functions and a bipartite network was analysed with the program Cytoscape. The Count program was used to assess evolutionary scenarios by Dollo’s maximum parsimony, and the phylogenetic trees were visualized with iTOL. The EPSPS enzyme is widely distributed in archaea, bacteria, plants, and fungi. The multi domain structure of the EPSPS in fungi is strongly associated with six other genes of the shikimate pathway. The most common multi domain structure is composed by a group of five enzymes (HQ synthase, EPSPS, SKI, DHquinase I and Shikimate DH), which I call in this thesis the “Major 5”. The EPSPS multi domain structure in fungi ranges between two to eight domains. The evolutionary analysis shows that the ancestral of fungi had a multi domain structure of six domains. Thus, there have been domain gains and losses throughout the evolution of the EPSPS in fungi. Further investigations are needed to determine the effect of the EPSPS-associated domains to glyphosate resistance. A scientific article that includes data from this master thesis is publicly available as a preprint at biorxiv and submitted to a peer-reviewed Nature Methods journal

Analysis of the Protein Domain and Domain Architecture Content in Fungi and Its Application in the Search of New Antifungal Targets

<div><p>Over the past several years fungal infections have shown an increasing incidence in the susceptible population, and caused high mortality rates. In parallel, multi-resistant fungi are emerging in human infections. Therefore, the identification of new potential antifungal targets is a priority. The first task of this study was to analyse the protein domain and domain architecture content of the 137 fungal proteomes (corresponding to 111 species) available in UniProtKB (UniProt KnowledgeBase) by January 2013. The resulting list of core and exclusive domain and domain architectures is provided in this paper. It delineates the different levels of fungal taxonomic classification: phylum, subphylum, order, genus and species. The analysis highlighted <i>Aspergillus</i> as the most diverse genus in terms of exclusive domain content. In addition, we also investigated which domains could be considered promiscuous in the different organisms. As an application of this analysis, we explored three different ways to detect potential targets for antifungal drugs. First, we compared the domain and domain architecture content of the human and fungal proteomes, and identified those domains and domain architectures only present in fungi. Secondly, we looked for information regarding fungal pathways in public repositories, where proteins containing promiscuous domains could be involved. Three pathways were identified as a result: lovastatin biosynthesis, xylan degradation and biosynthesis of siroheme. Finally, we classified a subset of the studied fungi in five groups depending on their occurrence in clinical samples. We then looked for exclusive domains in the groups that were more relevant clinically and determined which of them had the potential to bind small molecules. Overall, this study provides a comprehensive analysis of the available fungal proteomes and shows three approaches that can be used as a first step in the detection of new antifungal targets.</p></div