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

Abstract

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.

Thioesterase Domains of Fungal Nonreducing Polyketide Synthases Act as Decision Gates during Combinatorial Biosynthesis

“…First, overall product flux is determined by these TEs, similar to the situation in modular PKSs of bacteria where the turnover rate of TE-catalyzed product release regulates extension cycles in “congested” upstream modules. 45 Next, the TE domains of the nrPKSs investigated in this study also display context-dependent release mechanisms, yielding macrolactones, carboxylic acids and their esters, and pyrones. Thus, the TE decision gate joins previouslyidentified control points for polyketide assembly on nrPKSs: selection of an appropriate primer unit by the SAT; acyl chain length monitoring and kinetic control of chain extension vs. cyclization by the KS; and regiospecific cyclization by the PT domain.…”

FT-ICR-MS Characterization of Intermediates in the Biosynthesis of the α-Methylbutyrate Side Chain of Lovastatin by the 277 kDa Polyketide Synthase LovF

“…Most surprising was our inability to detect the acetoacetyl-intermediate, which was expected to form in the presence of holo -LovF and malonyl-CoA, a result of decarboxylation and condensation between a malonyl extender unit and the acetate that resulted from decarboxylation of the initial malonyl unit loaded to the ACP (30). Initially, holo -LovF was incubated with 1mM malonyl-CoA for 10 minutes prior to proteolysis.…”

Interrogation of Global Active Site Occupancy of a Fungal Iterative Polyketide Synthase Reveals Strategies for Maintaining Biosynthetic Fidelity

“…41 Similarly, investigations of the erythromycin modular type I PKS megasynthase (DEBS) have brought forth a similar “congestion model” where chain extension cycles are triggered by TE release from the last module. 42 …”