Synthesis of Malonyl-ACP

In order to study the specificity of KS-catalysed chain elongation a new assay was required. This was achieved by incubation of a pre-acylated recombinant KS domain with one equivalent of malonyl-ACP. In principle, the fi-keto elongated product would be formed on the PPant moiety of the ACP, and could be accurately detected by ESI-MS. Malonylation of the ACP PPant thiol was achieved by incubation of /toZo-PsyA-ACP3(A37, 38) with 50 equivalents of malonyl-CoA as detailed in Sect. 2.2.9.1. Reactions were monitored by ESI-MS for the presence 

Of the malonyl-ACP product (Fig. 6.2a). Generally, malonylation reactions were complete after 7 h at 25 °C, yielding -70-75 % malonyl-ACP. The PPant ejection assay was employed to check that the malonyl moiety was attached to the PPant thiol, resulting in predominantly an ion at m/z 347.2 corresponding to malonyl-PPant (Fig. 6.2b). Minor breakdown products of the activation process were also observed, including acetyl-PPant and PPant-SH. The former arising from decarboxylation of the malonyl unit, and the latter from loss of either acetyl or malonyl. 

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Scheme 2.4 Synthesis of malonyl-ACP from malonyl-CoA and holo-ACP...

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