Using Mass Spectrometry For Biochemical Studies On Enzymatic Domains From Polyketide Synthases (springer Theses)
by Matthew Jenner /
2016 / English / PDF
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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.
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.
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.
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.
