Structural biology of endophyte non-ribosomal peptide synthetases

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dc.contributor.advisor Lott, Shaun en
dc.contributor.advisor Johnson, Richard en
dc.contributor.advisor Arcus, Vic en
dc.contributor.author Lee, Tet Verne en
dc.date.accessioned 2020-07-08T04:49:59Z en
dc.date.available 2020-07-08T04:49:59Z en
dc.date.issued 2008 en
dc.identifier.uri http://hdl.handle.net/2292/51977 en
dc.description Full text is available to authenticated members of The University of Auckland only. en
dc.description.abstract Infection of many pastoral grasses by mutualistic fungal endophytes has significant effects on the biology of these grasses. Thus this fungal-plant interaction is also of very significant economic importance. Many of the effects are mediated by an array of secondary metabolites produced by the fungi. These secondary metabolites are in many cases synthesised by a class of enzymes known as the nonribosomal peptide synthetases (NRPSs). NRPSs are large enzymes that are organised into modules made up of functional domains. NRPSs are found in many organisms and synthesise a number of medically-important peptides such as antibiotics and immunosuppressants. This research used structural bioinformatics and experimental structural biology to further our understanding of the structure and function of NRPSs. An in silico method of predicting the specificity of NRPSs was investigated. Homology models of the NRPS domains responsible for substrate recognition, the adenylation domains were built and ligand docking was used to dock a library of 110 model ligands into the active sites. The docking protocol was refined to ensure that the mainchain atoms of the amino acid ligands were docked correctly. Testing of the method with adenylation domains of known specificity revealed a need for further experimental structural data in order to accurately model the active sites particularly data for domains from fungal sources such as endophytes. NRPS domains were targeted for structure determination using a structural genomics approach. Twenty four domain fragments from four fungal endophyte NRPSs and two domains from an Escherichia coli NRPS were cloned and expressed. Both of the E. coli domains and two of the endophyte NRPSs were solubly expressed. The purified E. coli NRPS domains were used for preliminary studies into developing a nuclear magnetic resonance- based specificity assay. Fungal NRPS domains were subject to extensive screening and fine-tuning of expression constructs, lysis buffer conditions, purification approaches and crystallisation conditions. The structure of the third adenylation domain from the fungal NRPS, SidN, was solved by X-ray crystallography to 2.0 A resolution. SidN is involved in synthesising an extracellular Siderophore and has previously been found to be essential for the maintenance of the mutualistic character of the grass-endophyte relationship. The structure is made up of two highly-conserved subdomains. The orientation between the two subdomains was an 'open' conformation previously seen in distantly-related members of the protein superfamily but not seen in NRPS adenylation domains. This finding has implications for the mechanism of catalysis. The SidNA3 structure revealed a large substrate binding pocket lined by fifteen residues and ligand docking showed that the pocket would accommodate the putative substrate, Nδ-anhydromevalonyl-Nδ-hydroxyornithine. This is the first eukaryotic NRPS domain structure to be solved and the first of any protein from a fungal endophyte of grass. This research provides the tools for a comprehensive analysis of the specificity determinants of fungal NRPSs and the refinement of a general in silico substrate specificity prediction method. en
dc.publisher ResearchSpace@Auckland en
dc.relation.ispartof PhD Thesis - University of Auckland en
dc.relation.isreferencedby UoA99187948914002091 en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. en
dc.rights Restricted Item. Full text is available to authenticated members of The University of Auckland only. en
dc.rights.uri https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm en
dc.title Structural biology of endophyte non-ribosomal peptide synthetases en
dc.type Thesis en
thesis.degree.discipline Biological Sciences en
thesis.degree.grantor The University of Auckland en
thesis.degree.level Doctoral en
thesis.degree.name PhD en
dc.rights.holder Copyright: The author en


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