The changing patterns of covalent active site occupancy during catalysis on a modular polyketide synthase multienzyme revealed by ion-trap mass spectrometry.

A catalytically competent, homodimeric diketide synthase comprising the first extension module of the erythromycin polyketide synthase was analysed using MS, after limited proteolysis to release functional domains, to determine the pattern of covalent attachment of substrates and intermediates to active sites during catalysis. Using the natural substrates, the acyltransferase and acylcarrier protein of the loading module were found to be heavily loaded with propionyl starter groups, while the ketosynthase was fully propionylated. The acylcarrier protein of the extension module was partly occupied by the product diketide, and the adjacent chain-releasing thioesterase domain was vacant, implying that the rate-limiting step is transfer of the diketide from the acylcarrier protein to the thioesterase domain. The data suggest an attractive model for preventing iterative chain extension by efficient repriming of the ketosynthase domain after condensation. Use of the alternative starter unit valeryl-CoA produced an altered pattern, in which a significant proportion of the extension acylcarrier protein was loaded with methylmalonate, not diketide, consistent with the condensation step having become an additional slow step. Strikingly, when NADPH was omitted, the extension acylcarrier protein contained methylmalonate and none of the expected keto diketide, in contrast to results obtained previously by mixing individual recombinant domains, showing the importance of also studying intact modules. The detailed patterns of loading of the extension acylcarrier protein (of which there are two in the homodimer) also provided the first evidence for simultaneous loading of both acylcarrier proteins and for the coordination of timing between the two active centres for chain extension.