Overview
- Nominated as an Outstanding Ph.D. thesis by the University of Nottingham
- Describes novel use of intact MS for the study of enzyme acylation and elongation
- Comprehensive and accessible review of trans-AT PKS enzymology
- Majority of thesis published in high-impact journals
- Includes supplementary material: sn.pub/extras
Part of the book series: Springer Theses (Springer Theses)
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Table of contents(6 chapters)
About this book
This thesis reports studies on the substrate specificity of crucial ketosynthase (KS) domains from trans-AT Polyketide Synthases (PKSs). Using a combination of electrospray ionisation-mass spectrometry (ESI-MS) and simple N-acetyl cysteamine (SNAC) substrate mimics, the specificity of a range of KS domains from the bacillaene and psymberin PKSs have been succsessfully studied with regard to the initial acylation step of KS-catalysis.
In addition, the ability to alter the substrate tolerance of KS domains by simple point mutations in the active site has been demonstrated. A series of acyl-ACPs have been synthesised using a novel methodology and employed to probe the substrate specificity of both KS domains and the previously uncharcterised acyl hydrolase domain, PedC.
KS-catalysed chain elongation reactions have also been conducted and monitored by ESI-MS/MS. All KS domains studied exhibited higher substrate specificity at the elongation step than in the preceeding acylation step. Furthermore, a mechanism of reversible acylation is proposed using the PsyA ACP1-KS1 di-domain. The findings in this thesis provide important insights into mechanisms of KS specificity and show that mutagenesis can be used to expand the repertoire of acceptable substrates for future PKS engineering.
Authors and Affiliations
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School of Chemistry,, University of Warwick,, Coventry,, United Kingdom
Matthew Jenner
About the author
Bibliographic Information
Book Title: Using Mass Spectrometry for Biochemical Studies on Enzymatic Domains from Polyketide Synthases
Authors: Matthew Jenner
Series Title: Springer Theses
DOI: https://doi.org/10.1007/978-3-319-32723-5
Publisher: Springer Cham
eBook Packages: Chemistry and Materials Science, Chemistry and Material Science (R0)
Copyright Information: Springer International Publishing Switzerland 2016
Hardcover ISBN: 978-3-319-32722-8Published: 09 May 2016
Softcover ISBN: 978-3-319-81355-4Published: 27 May 2018
eBook ISBN: 978-3-319-32723-5Published: 27 April 2016
Series ISSN: 2190-5053
Series E-ISSN: 2190-5061
Edition Number: 1
Number of Pages: XVIII, 176
Number of Illustrations: 37 b/w illustrations, 99 illustrations in colour
Topics: Mass Spectrometry, Enzymology, Biochemical Engineering, Medical Biochemistry