By Matthew Jenner
This thesis studies reviews at the substrate specificity of an important ketosynthase (KS) domain names from trans-AT Polyketide Synthases (PKSs). utilizing a mixture of electrospray ionisation-mass spectrometry (ESI-MS) and easy N-acetyl cysteamine (SNAC) substrate mimics, the specificity of a number of KS domain names from the bacillaene and psymberin PKSs were succsessfully studied with reference to the preliminary acylation step of KS-catalysis.
In addition, the power to change the substrate tolerance of KS domain names by way of easy element mutations within the energetic website has been established. a chain of acyl-ACPs were synthesised utilizing a unique method and hired to probe the substrate specificity of either KS domain names and the formerly uncharcterised acyl hydrolase area, PedC.
KS-catalysed chain elongation reactions have additionally been performed and monitored via ESI-MS/MS. All KS domain names studied exhibited larger substrate specificity on the elongation step than within the preceeding acylation step. additionally, a mechanism of reversible acylation is proposed utilizing the PsyA ACP1-KS1 di-domain. The findings during this thesis offer vital insights into mechanisms of KS specificity and exhibit that mutagenesis can be utilized to extend the repertoire of appropriate substrates for destiny PKS engineering.