Substrate Specificity of BaeL KS5 M237A

As discussed previously, the Met to Ala mutation was predicted to provide additional space in the binding pocket to accommodate a -carbon branched substrate. The KS5(M237A) mutant was successfiilly constructed using standard molecular cloning techniqnes (detailed in Sect., 2.2.1.4), followed by expression and purification (Figs. 3.1 and 3.2). 

The results presented provide strong evidence that the nature of the X-Cys residue has the ability to dictate KS specificity with regard to P-branching. In order to investigate the potential role of this residue in KS domains from other clades, a sequence analysis was conducted using the 138 KS domains from the initial phylogenetic study, in addition to 12 subsequently identified KSs from the psymberin PKS [1, 6]. The sequence analysis focussed upon the nature of the X-Cys residue, and the most highly represented residues were reported (Table 3.5). The study 

Initial rate was calculated from a ln[KS]/[KSo] versus t plot, given to 2 significant figures N.D—No acylation was detected over the tme scale of the incubation (40 min) 

In contrast, the clade containing BaeL KS6 (I), harbours exclusively X = Ala and is likely to provide the necessary space in the binding pocket to accommodate a -methyl branch (Fig. 3.11). Clade XVI contains all KS domains positioned immediately downstream of an NRPS module, and therefore accept amino acid-containing acyl chains. In this instance, X = Asn and the role of this residue is investigated further in Chap. 4. 

In summary, using a novel MS-based assay the molecular basis of carbon ( -branch specificity in trans-AT PKSs KS domains has been identified. A key residue has been characterised in the active site of BaeL KS5 which sterically dictates the tolerance for -branched substrates. Examination of this residne in the sequences of 150 KS domains from trans-AT PKSs suggests that the presence of 

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