Using Mass Spectrometry For Biochemical Studies On Enzymatic Domains From Polyketide Synthases (Jenner, Matthew)

[Farid-Khan]

3319327224 Jenner, Matthew Springer 2016

Catergory: Nonfiction, Science & Nature, Science, Physics, Spectrum Analysis, Biological Sciences, Biochemistry

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.

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