Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.719839
Title: Investigation of L-amino acid hydroxylases
Author: Smart, Tristan Jamie
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2015
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Abstract:
Ferrous-iron/2-oxoglutarate(2-OG)-dependent oxygenases have emerged as a widely distributed family of oxygenases. They have applications both in biocatalysis and as pharmaceutical/agrochemical targets. One of the first subfamilies of 2-OG oxygenases to be studied in detail were the prolyl hydroxylases. The work described in my thesis focused on substrate analogue studies on the proline hydroxylases and on a highly unusual type of 2-OG oxygenase that catalyses the production of ethylene from 2-OG. The work involved the development of assays, in particular mass spectrometry and nuclear magnetic resonance spectroscopy, to screen a panel of substrate analogues. The results identified the need for an a-amino acid for productive catalysis, and were consistent with previous studies showing that the proline hydroxylases can accept different ring sizes, and can catalyse production of dihydroxylated products. New substrates for the proline hydroxylases including bicyclic compounds, which undergo a bifurcating pathway to give singly hydroxylated products, were also identified. In a separate line of investigation, a novel ethylene forming enzyme was studied. Previous work had identified an arginine-oxidising 2-OG oxygenase from Pseudomonas spp. that also produced ethylene. This observation is interesting given the key role of ethylene, as produced by aminocyclopropanecarboxylate (ACC) oxidase, in plant biology. ACC oxidase is structurally related to the 2-OG oxygenases but does not employ 2-OG during catalysis. Methods for the production of the recombinant ethylene-forming enzyme from Pseudomonas spp were developed as were assays for monitoring catalysis. The results reveal that the enzyme produces ethylene in an arginine-dependent manner and coproduces pyrroline-5-carboxylic acid (?-P5C) (the cyclic form of glutamate-5-semialdehyde). Crystal structures were obtained for the ethylene forming enzyme in the presence of a 2-OG analogue with and without arginine. The combined biochemical and structural studies imply that the enzyme has bifurcating pathway, with ethylene production arising from 2-OG and requiring arginine, but which is not oxidised; alternatively arginine can be oxidised to give ?1-pyrroline-5-carboxylic acid (?-P5C) in a 'normal' 2-OG oxygenase type reaction with concomitant production of succinate. The results open up new possibilities for ethylene production in organisms ranging from bacteria to animals.
Supervisor: Schofield, Christopher Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.719839  DOI: Not available
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