The Phosphopantetheine Ejection Assay

Studies conducted by Kelleher and co-woikers in 2006 characterised an efficient gas-phase tandem MS elhnination reaction to identify phosphopantetheine-bound substrates on ACPs [15]. During collisional activation of holo-ACP domains, it was found that the phosphopantetheinyl moeity is ejected from the ACP, yielding a characteristic protonated imine ion at m/z 261. The mechanism of this elimination is believed to leave a phosphate anion attached to the serine of the ACP (apo-ACP + 80 Da) nunus one charge, as the carbonyl of the amide displaces the pantetheinyl unit, generating a five-membered tetrahydrofuranol ring with a protonated inune (Fig. 5.1). 

This methodology has allowed identification of biosynthetic intermediates during enzymatic synthesis, by effectively reducing large 100 kDa proteins to small molecule fragment ions. In this chapter, the phosphopantetheine ejection assay will simply be used to characterise the synthesis acyl-ACPs, however, in later chapters the assay will be employed for identification of other intermediates. 

AF4MSS6 LlYnO AT ER AM 2766 OzmM AT AT ER ABS904 74 Of mM AT AT ER CANB9639 KiCIATATER AAM12912Minp1IIATAT ER 

The ACP used in this study originated from the psymberin (psy) trans-AT PKS. Psymberin is an extremely rare and highly potent cytotoxin isolated from the marine sponge Psammocinia aff. Bulbosa [17], but produced by an as-yet uncul-turable symbiotic bacterium [18]. Since gene inactivation experiments for pathway 

To investigate MS-based strategies using ACP-bound intermediates, we focused on PsyA ACP3 as a model, which is situated at the C-terminus of the PsyA protein. The holo-ACP was prepared as described in the Sect. 2.2.1. ESI-MS measurement of the ACP revealed a mass of 10,991 0.43 Da, in good agreement with the theoretical mass of 10,990 Da (Fig. 5.3). Collision induced activation of the ACP yielded the ejected PPant moiety at m/z 261, demonstrating an unacylated holo-KCP [15]. 

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