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Title: Inositol phosphorylceramide synthase of fungal sphingolipid biosynthesis : a potential target for selective agrochemical and therapeutic agents
Author: Breen, Rachel S.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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The membrane-bound enzyme, inositol phosphorylceramide synthase (IPC synthase), encoded by AUR1, has been identified in yeast and fungi. Since IPC synthase is unique to fungi, there is an opportunity for development of a species-specific enzyme inhibitor that might have low toxicity to the host for use as a fungicide. Michaelis-Menten kinetic parameters have been determined for S. cerevisiae IPC synthase using Triton X-100 solubilised microsomes (phosphatidylinositol KM 357µM and fluorescent ceramide, N-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-ceramide KM 5.22µM). The fluorescent ceramide was found to exhibit substrate inhibition at high concentration (KI 125.69µM). When the substrate inhibition parameter was considered the apparent KM of N-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-ceramide increased to 11.42µM. Both B. cinerea and S. cerevisiae Aur1p have been cloned, recombinantly overexpressed and purified using affinity tags. B. cinerea Aur1p was overexpressed in E. coli Top 10™ cells in inclusion bodies as an N-terminal Glutathione-S-transferase fusion. However, IPC synthase activity was low and expression in P. pastoris was investigated. A large number of constructs were prepared and successful intracellular overexpression was achieved for both B. cinerea and S. cerevisiae Aur1p in KM71H cells (MutS) as C-terminal c-myc His6 fusions. Although expression was achieved with both full length and N-terminally truncated forms, the full length proteins had greater activity. B. cinerea Aur1p was far more active when expressed in P. pastoris rather than E. coli. Native S. cerevisiae IPC synthase was found to be functionally glycosylated with high mannose content and the increased activity is probably due to the correct processing and post translational modifications occurring in the eukaryotic system. Native IPC synthase activity was demonstrated in P. pastoris and an isolation method based on affinity chromatography has been identified for native P. pastoris and B. cinerea Aur1p.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available