Extender Unit Specificity of PedD

Due to the nature of the analysis it was possible to monitor the malonylation state of PedD at the same time as the ACP. Upon examination of the PedD peaks, it was found to be 50 % malonylated after 20 min. This observation suggested that an equilibrium was reached during the transfer assay, with back-transfer of the malonyl moeity onto the AT allowing the equilibrium to approach a 50 50 ratio. These results provide the intriguing possiblity that back-transfer of extender units (such as malonyl) may indeed happen in the native PKS system. 

To investigate this phenomenon, the reverse reaction was carried out by incubating malonyl-ACP with unmalonylated-AT and monitoring any back-transfer of the malonyl unit onto the AT. Interestingly, the back reaction was found to proceed to approximately the same extent as the forward reaction, with 55 % of the PedD population observed to be malonyled (Fig. 5.13). 

The data presented in Sects. 5.3.5-5.3.S indicate that PedC is a hydrolytic enzyme with the ability to cleave acyl intermediates from ACPs, and PedD is a malonyl-specific acyltransferase domain. Despite such different catalytic roles, at the sequence level the domains are extranely similar. In an attempt to rationalise the difference in catalytic activities of these domains, homology models of PedC and PedD were constructed as described in Sect. 2.2.15.2. 

The homology models were subsequently aligned with the crystal structure of FabD, the AT domain from the E.coli FAS, which was successfully crystallised with a malonate unit attached to the acTive site serine [29]. This step allowed the same 

In order to investigate whether this is a common feature of AH domains, a sequence alignment of all the AT and AH domains from Fig. 5.2 was performed. All domains predicted to be ATs (ATI clade) harbored an Arg residue at this position. However, all but one of the domains in the AT2 clade possessed a Gin residue in the equivalent position (Fig. 5.16). 

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