New approaches to track chemoenzymatic loading reactions of NRPS carrier protein domains using NMR.

Abstract

The atomic-level detail provided by nuclear magnetic resonance (NMR) allows for the monitoring of biochemical reactions as substrates and intermediates are converted by enzymes. NMR signals are sensitive to chemical environments and this feature allows for scrutiny of the integrity of reactions. Routinely, time-consuming diversions are necessary with traditional approaches to monitor biochemical transformations as many require additional sample manipulations or their readouts are limited. Here, we highlight the advantages of NMR to both monitor and rescue a complex chemoenzymatic reaction that modifies an isotopically labeled nonribosomal peptide synthetase (NRPS) carrier protein with an unlabeled phosphopantetheine moiety through an enzymatic cascade. We present protocols to monitor this reaction that implement various 1D NMR experiments tailored to the detection of specific steps in the enzymatic cascade and can be easily applied even to reactions using unlabeled domains. Additionally, to highlight the signals of the unlabeled modification once attached to the labeled biomolecule, we present a modified diffusion and isotope-filtered pulse sequence. Importantly, when chemoenzymatic reactions are slow or are stalled, the technique provides an atomic-level readout of reaction byproducts and limiting or damaged reagents, and thus allows for quick interventions to rescue them. The advantages of using NMR highlighted in this study more generally serve as a dogma to monitor and restore complex chemoenzymatic reactions.