Mechanism-based crosslinking as a gauge for functional interaction of modular synthases.

Protein-protein interactions between domains within fatty acid and polyketide synthases are critical to catalysis, but their contributions remain incompletely characterized. A practical, quantitative system for establishing functional interactions between modifying enzymes and the acyl carrier protein that tethers the nascent polymer would offer a valuable tool for understanding and engineering these enzyme systems. Mechanism-based crosslinking of modular domains offers a potential diagnostic to highlight selective interactions between modular pairs. Here kinetic activity analysis and isothermal titration calorimetry are shown to correlate the efficiency of a ketosynthase-carrier protein crosslinking method to the binding affinity and transacylase activity that occurs in ketosynthase chain elongation.