Thioesterase-mediated Side Chain Transesterification Generates Potent Gq Signaling Inhibitor FR900359

This post relates to our paper on the biosynthesis of a complex, bioactive natural product that was published in Nature Communications here: https://doi.org/10.1038/s41467-020-20418-3

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G proteins play a crucial role in our bodies. They shuttle the signals, that the ubiquitous G protein-coupled receptors (GPCR) get from outside, to the inside of our cells and are thus the starting point of a large number of signaling cascades. In recent years, a small group of natural products gained importance in GPCR signaling research due to their effective mechanism of action-the ability to inhibit Gq, one of the major G proteins, with high potency and selectivity. This ability makes them valuable tools to study various Gq-related signaling pathways (there are >800 GPCR but only four G protein families) by enabling one to selectively switch them off (Figure 1). FR900359 (FR), the most potent Gq inhibitor, additionally shows therapeutic promise against asthma and uveal melanoma. Since the original FR producer is an uncultivable bacterial leaf endosymbiont, our efforts to investigate the biosynthesis of this complex nonribosomal peptide were, however, complicated.

 

Figure 1. Left: FR inhibits Gq, a major family of G proteins, with high potency and selectivity. Right: As there are >800 GPCRs, but only four major G protein families, inhibition of Gq results in blockage of many GPCR-related signaling cascades. Picture credit: Evi Kostenis

Our journey that finally resulted in the accompanied publication started with the search for alternative, cultivable producers of FR, which would make our lives easier in several ways. Through bioinformatics searches we identified a cultivable bacterium harboring the FR biosynthetic gene cluster (BGC), and were even more delighted when we found this bacterium to produce FR in the lab. We then focused on the side chain of FR that is tethered to its macrocyclic peptide core. We attempted to answer three main questions:

  1. What are the biochemical details of FR side chain biosynthesis and attachment? 2. What is the influence of the FR side chain on its remarkable bioactivity? 3. How did this modification evolve and how is the relation to other natural product BGCs?

To answer the first question, we assembled the FR side chain in vitro with purified biosynthetic enzymes and side chain mimics, which were synthesized in collaboration with our collaborator Michael Gütschow. By using our in vitro system, we then proved the function of a thioesterase domain which was hypothesized to catalyze the unusual intermolecular side chain attachment to the macrocyclic core peptide (Figure 2). Through variation of precursors, we thereby also generated FR analogues with altered side chains. 

Figure 2: Assembly of the FR side chain by the nonribosomal peptide synthetase FrsA, MbtH-like protein FrsB and monooxygenase FrsH. The FrsA thioesterase (TE) catalyzes the attachment of the side chain onto the macrocyclic biosynthetic intermediate FR-Core.

To investigate the second question, we teamed up with our colleagues from the labs of Evi Kostenis, Christa Müller and Peter Mergaert. Through a series of comparative experiments, we were able to show that the FR side chain is crucial for its extraordinary Gq inhibition potency. Lastly, we performed detailed bioinformatics analyses which showed us that the side chain biosynthesis gene very likely originates from duplications of sequences already present within the FR BGC. This suggests an extension of the FR BGC during evolution leading to the synthesis of more potent inhibitors, which we discuss in detail. A global comparison with 1.2 million other BGCs, enabled by the fantastic bioinformatic tools developed by Marnix Medema, revealed that there is no close relationship of the FR BGC to any other BGC currently in the databases, coining the FR biosynthetic machinery a rare and specialized system, that we will continue to explore.

Finally, I would particularly like to thank Paco Barona-Gomez and the other two reviewers of our manuscript for their very constructive reviews, which were instrumental in improving and shaping this interdisciplinary story about our favorite natural product.

Max Crüsemann

Dr., University of Bonn