Frueh, D. P. | Search bioRxiv

bioRxiv2022

NMR as a readout to monitor and restore the integrity of complex chemoenzymatic reactions

The non-invasive nature of NMR offers a means to monitor biochemical reactions in situ at the atomic-level. We harness this advantage to monitor a complex chemoenzymatic reaction that sequentially modifies reagents and loads the product on a nonribosomal peptide synthetase carrier protein. We present a protocol including a novel pulse sequence that permits to assess both the integrity of reagents and the completion of each step in the reaction, thus alleviating otherwise time-consuming and costly approaches to debug and repeat inefficient reactions. This study highlights the importance of NMR as a tool to establish reliable and reproducible experimental conditions in biochemical studies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/491371v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@11bc2e6org.highwire.dtl.DTLVardef@198ab6forg.highwire.dtl.DTLVardef@1f0747org.highwire.dtl.DTLVardef@116fcbf_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Global Dynamics as Communication Sensors in Peptide Synthetase Cyclization Domains.

Structural biology is the foundation for deriving molecular mechanisms, where snapshots of macromolecules and binding partners inform on mutations that test or modify function. However, frequently, the impact of mutations violates the underpinnings of structural models, and mechanisms become cryptic. This conundrum applies to multidomain enzymatic systems called nonribosomal peptide synthetases (NRPSs), which assemble simple substrates into complex metabolites often with pharmaceutical properties. Engineering NRPSs can generate new pharmaceuticals1-3 but a dynamic domain organization challenges rational design.4-8 Using nuclear magnetic resonance (NMR), we determined the solution structure of a 52 kDa cyclization domain and demonstrate that global intra-domain dynamics enable sensing of substrates tethered to partner domains and draw an allosteric response encompassing the enzymes buried active site and two binding sites 40 [A] apart. We show that a point-site mutation that impedes the domain dynamics globally hampers the allosteric response. We demonstrate this mechanism through NMR experiments that provide atomic-level read-outs of allosteric responses during biochemical transformations in situ. Our results establish global structural dynamics as sensors of molecular events that can remodel domain interactions and illustrate the need for integrating structural dynamics explicitly when deriving molecular mechanisms through mutagenesis and structural biology.

biophysics↗