Investigations into side chain assembly and attachment during biosynthesis of the G protein inhibitor FR900359

Abstract

The natural product FR900359 (FR) selectively inhibits Gaq proteins and thus intracellular signalling of many G protein-coupled receptors. This unique mechanism of action makes FR an indispensable pharmacological tool to study Gaq-related processes, as well as a promising drug candidate. FR is a complex cyclic depsipeptide with seven nonproteinogenic building blocks; it was isolated from the plant Ardisia crenata but is not produced by the latter. Instead, the endosymbiotic bacterium “<em>Candidatus Burkholderia crenata</em>” contains the biosynthetic gene cluster (BGC) frs, encoding two nonribosomal peptide synthetase (NRPS) systems. Recently, the soil bacterium <em>Chromobacterium vaccinii</em> was found to harbour a very similar frs BGC and to produce FR under laboratory conditions, facilitating <em>in vitro</em> and <em>in vivo</em> biosynthesis studies on FR. <br /> In this work, the successive assembly of the FR side chain N-propionylhydroxyleucine was achieved <em>in vitro</em> by utilising the purified monomodular NRPS FrsA, the MbtH-like chaperon protein FrsB and the non-heme diiron monooxygenase FrsH. The final step of FR biosynthesis is an unusual intermolecular transesterification reaction, connecting the side chain with a macrocyclic intermediate (FR-Core), that is assembled by the heptamodular NRPS FrsD-G. FR-Core was isolated from the deletion mutant <em>C. vaccinii &Delta;frsA</em>. It has been shown that the FrsA thioesterase domain catalyses this transesterification and the substrate promiscuity of the FrsA domains was utilised for the chemoenzymatic production of FR derivatives with altered side chains. A new and unnatural derivative, FR-5, was produced in an <em>in vitro</em> assay and then isolated from <em>C. vaccinii</em> after precursor-directed biosynthesis, induced by feeding of butyric acid. This new analogue contains <em>N</em>-butyrylhydroxyleucine instead of N-propionylhydroxyleucine as side chain. The Gaq protein inhibiting activity of this new compound was 7-fold decreased in comparison to FR, demonstrating that this position is unsuitable for further modifications. <br /> Comparative <em>in vivo</em> and <em>in vitro</em> studies on FR-Core and FR supported by <em>in silico</em> docking to Gaq demonstrate that the side chain of FR is crucial for its remarkable Gaq inhibition properties. An evolutionary scenario is presented, leading to this important biosynthetic modification. Phylogenetic analysis of the starter condensation and thioesterase domains of the two frs clusters revealed their closest relatives to be inside the BGC, and overall no closely related BGCs could be found in a global BGC search. So, our hypothesis involves duplication of the highly similar NRPS module FrsD and the incorporation and possible modification of the unusual thioesterase domain. <br /> Lastly, we started preparations for the structural investigation of the three-dimensional structure of the NRPS FrsA and its thioesterase domain using highly pure protein for first crystallisation and cryo-EM trials.