Substrate Specificity of Ketosynthase Domains Part I -Branched Acyl Chains

Substrate Specificity of Ketosynthase Domains Part I -Branched Acyl Chains 

Recent phylogenetic work has shown that KS domains from trans-AJ PKSs correlate, at the sequence level, with their predicted biosynthetic intermediates [1], The extent of this evolution-based specificity is believed to reach as far as the -position for each given biosynthetic intermediate, as detailed in section KS Specificity-Based Assignment of trans-AI PKSs . Compared to textbook colinearity rules, employed for ds-AT PKS systems, a KS specificity-based approach works remarkably well as a predictive method for assigning biosynthetic intermediates to their PKS, however it lacks functional testing. 

In order to probe the functional substrate specificity of KS domains from trans-AI PKSs, a mass spectrometry (MS)-based methodology was developed to test a selection of KS-domains. Previous approaches designed to examine KS specificity from cis-AT systems have focussed upon radioactivity assays and MS analysis of trypsin-digested domains [2, 3]. The assay reported in this chapter possesses significant advantages over previous methods, allowing rapid and direct analysis of intact KSs domain and ability to observe exact mass-shifts for the attachment of acyl chains. 

Ketosynthase Qade Predicted Intermediate SNAC Mimic 

N-acetyl cysteamine (SNAC) thioester substrate mimics were used to test the specificity of four KS domains with a range of predicted biosynthetic intermediates (Table 3.1). In the case of PsyD KS3° and BaeL KS5, the synthesised SNAC mimic are shorter in length than the predicted biosynthetic intermediate. However, the phylogenetic KS-based substrate prediction would suggest that the specificity does not extend past the P-position [1], and therefore SNAC substrate mimics synthesised to this length may be sufficient to reveal KS specificity. 

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