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.
Worthington
Andrew S
AS
Department of Chemistry and Biochemistry, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA.
Porter
Douglas F
DF
Burkart
Michael D
MD
D000213:Acyl Carrier Protein / Q000378:metabolism*; D001419:Bacteria / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism*; D048630:Polyketide Synthases / Q000378:metabolism*; D011485:Protein Binding; D013816:Thermodynamics
D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D048630:Polyketide Synthases; C087193:polyketide beta-ketoacylsynthase