Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.603935
Title: The putative roles of thioesterase domains in polyketide biosynthesis
Author: Heathcote, M.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2000
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Abstract:
This study is concerned with the investigation of the substrate specificity and putative roles of both type I and type II thioesterase domains in polyketide biosynthesis. To ascertain the role of type II thioesterases, several TE II genes from various PKS sources were overexpressed in Escherichia coli. MonAIX TE II and tyl TE II were successfully overexpressed and purified to homogeneity in high yield using the pET expression system, thus providing a convenient source of enzyme for in vitro studies. A series of synthetic substrates were synthesised, as either N-acetylcysteamine thioesters, or p-nitrophenyl esters, to define the substrate specificity of the type II thioesterases from the tylosin- and monensin-producing PKS clusters. Analysis of the reaction kinetics was carried out using UV spectroscopy. These experiments showed that both type II thioesterases could hydrolyse a range of substrates in vitro. The trends of hydrolytic activity determined by the kinetic parameters kcar/KM for a series of simple fatty acyl chains reveals that propionate and butyrate are more effective substrates for tyl TE II than acetate and valerate. Substrates which modelled aberrant tylosin biosynthetic intermediates, arising from mistakes made during reductive processing, were poor substrates for tyl TE II. This suggests that the type II thioesterase associated with the tylosin PKS possesses an editing activity, so that decarboxylated extender units are removed from the PKS complex. Comparison of the activity of monAIX TE II with the same series of acyl chain analogues did not reveal any visible trend in reactivity. To investigate the mechanism of action of the type I thioesterase from the carboxyl terminus of the erythromycin-producing PKS, the gene encoding the DEBS 3 terminal acyl carrier protein (ACP) and TE domains was overexpressed in Escherichia coli.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.603935  DOI: Not available
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