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Title: Identification and enzyme studies of rare amino acid biosynthesis from Streptomyces cattleya
Author: Chan, K. K. Jason
Awarding Body: University of St Andrews
Current Institution: University of St Andrews
Date of Award: 2013
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This thesis is focussed on the biosynthesis of three toxins: fluoroacetate, 4-fluoro-L-theronine and β-ethynyl-L-serine which are biosynthesised by the soil bacteria Streptomyces cattleya. The two fluorinated metabolites originate from a common biosynthetic pathway and the thesis describes studies carried out on an aldose-ketose isomerase enzyme of the pathway. The biosynthetic origin of β-ethynyl-L-serine is not known. A total synthesis of this acetylenic amino acid is descibed along with the development of a new analytical method for identifying the metabolite and for future isotope-labelling based biosynthetic studies. Chapter 1 presents the background of this research. It is focussed on the biosynthesis of fluoroacetate and 4-fluoro-L-threonine by S. cattleya and it also introduces alkyne-containing natural products and their biosynthesis. Chapter 2 describes the work carried out on crystallisation of the aldose-ketose isomerase of the fluorometabolite pathway in S. cattleya. Crystals of the isomerase were obtained and they were diffracted by X-ray, however a structure could not be solved. Chapter 3 contains site-directed mutagenesis studies of the isomerase from S. cattleya. Chapter 4 describes an enantioselective total synthesis of β-ethynyl-L-serine. A robust analytical technique based on derivatisation using 'Click' chemistry and LC-MS was developed for the detection of this amino acid directly from the fermentation broth. Chapter 5 details the experimental procedures for compounds synthesised in this thesis and the biological procedures for gene cloning and protein purification.
Supervisor: O'Hagan, David Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Streptomyces cattleya ; Fluorometabolite ; Organofluorine natural products ; Isomerase ; Fluorinase ; Protein crystallography ; Ethynylserine ; Rare amino acids ; Click chemistry ; Acetylenic amino acid ; QR88.3C5 ; Streptomyces--Metabolism ; Microbiological synthesis ; Toxins--Synthesis ; Amino acids--Synthesis ; Isomerases