Thioesterase-mediated side chain transesterification generates potent Gq signaling inhibitor FR900359

Abstract

The potent and selective Gq protein inhibitor depsipeptide FR900359 (FR), originally discovered as the product of an uncultivable plant endosymbiont, is synthesized by a complex biosynthetic system comprising two nonribosomal peptide synthetase (NRPS) assembly lines. Here we characterize a cultivable bacterial FR producer, enabling detailed investigations into biosynthesis and attachment of the functionally important FR side chain. We reconstitute side chain assembly by the monomodular NRPS FrsA and the non-heme monooxygenase FrsH, and characterize intermolecular side chain transesterification to the final macrocyclic intermediate FR-Core, mediated by the FrsA thioesterase domain. We harness FrsA substrate promiscuity to generate FR analogs with altered side chains and demonstrate indispensability of the FR side chain for efficient Gq inhibition by comparative bioactivity, toxicity and docking studies. Finally, evolution of FR and side chain biosynthesis is discussed based on bioinformatics analyses. Side chain transesterification boosts potency and target affinity of selective Gq inhibitor natural products.

Fig. 1. FR biosynthesis.

a Organization, size and GC content of the frs BGCs from “Ca. B. crenata” and C. vaccinii (gray = NRPS, white = modifying enzyme). b Biosynthetic pathway of 1. First, the NRPS FrsDEFG together with FrsH forms a seven-membered linear peptide chain which is then hydrolyzed and cyclized by FrsG_TE to 2. Then, FrsA_TE catalyzes intermolecular transesterification of N-Pp-Hle synthesized by FrsA_CAT and FrsH (see Fig. 3a) onto 2 to yield the final product 1 (or 3). Pp = Propionyl, Ac = Acetyl, Me = Methyl, Hle = l-3-Hydroxyleucine, PLA = Phenyllactic acid, Dha = Dehydroalanine. Domains are abbreviated as: C = condensation A = adenylation, T = thiolation, E = epimerization, MT = methyl transferase. Modules are colored according to their activated amino acid precursors. c Nucleotide alignment of frsA and frsD. All predicted domains and identity are indicated. Prediction of domains was conducted with InterPro (EMBL).

Fig. 2. Characterization of C. vaccinii knock-out mutants.

a HPLC-MS with extracted ion chromatograms (EIC) of m/z: 1002.539 (1), m/z: 817.430 (2) and m/z: 344.101 (4, violacein) for butanolic extracts of C. vaccinii and the ΔvioA, ΔfrsA and ΔvioA/frsA deletion mutant strains. b Colony phenotype of wild type (WT) C. vaccinii changes, after deletion of vioA, from purple (production of 4) to white.

Fig. 3. In vitro assays with FrsA/B, FrsH, and FrsA_TE.

a Reaction scheme of N-Pp-Hle formation and intermolecular transesterification onto 2 to yield 1 Biosynthetic modifications are color-coded according to the respective catalyzing domain. b Structures of synthesized N-Pp-Hle (5) and its SNAC (6). c In vitro production of 5 (m/z: 202.108). (i) Synthetic 5 (1 µg/ml). (ii) Enzymatic assay with purified FrsA_CAT/FrsB, FrsH, incubated with l-Leu and propionyl-CoA, hydrolyzed with KOH. (iii) Negative control (NC) with heat-inactivated protein. d In vitro production of 1 (m/z: 1002.540). (i) 1 (10 µg/ml). (ii) Purified FrsA_TE incubated with 6 and 2. (iii) Purified FrsA/B incubated with 6 and 2. (iv) Purified FrsA/B, FrsH incubated with propionyl-CoA, l-Leu and 2. e In vitro production of FR congeners 3 (m/z: 988.530) and 7 (m/z: 1016.550). (i) 3 (10 µg/ml). (ii) Purified FrsA/B, FrsH incubated with l-Leu, acetyl-CoA and 2. (iii) Negative control. (iv) 7 (10 µg/ml). (v) Purified FrsA/B, FrsH incubated with l-Leu, butyryl-CoA and 2. (vi) Negative control. f In vitro FrsA/D combinations: (i) 3 (1 µg/ml). (ii) Production of 3 by FrsA_TE and FrsD, incubated with acetyl-CoA, l-Leu and 2. (iii) 1 (1 µg/ml). (iv) Production of 1 by FrsA_TE and FrsD, incubated with propionyl-CoA, l-Leu and 2.

Fig. 4. Evaluation of FR-Core and FR-5 compared to FR.

a Concentration-dependent inhibition of activated Gαq proteins by 1 and 2 as determined by label-free whole-cell DMR biosensing. DMR recordings are representative (mean + s.e.m.) of at least four independent biological replicates conducted in triplicate. b. Competition binding experiments of 1 and 2 versus the FR-derived radiotracer [³H]PSB-15900 at human platelet membrane preparation (50 µg protein per vial), incubated at 37 °C for 1 h. c Exposure of nymphs of a stink bug (Riptortus pedestris) to different concentrations of 1 (left) and 2 (right), survival rate was measured. d Docked poses of 1 (left, represented in sticks and colored in orange, the N-Pp-Hle group present only in 1 is colored in green) and 2 (represented in sticks and colored in blue) in the binding pocket of the Gαq protein shown as line representation. Some of the interactions common for 1 and 2 are indicated by red dotted lines, and the interactions specific for 1 are shown as green dotted lines. Oxygen atoms are colored in red, nitrogen atoms in blue and polar hydrogen atoms in white. e Concentration-dependent inhibition of activated Gαq proteins by 1 and 7 as determined by label-free whole-cell DMR biosensing (see a). Source data are provided as a Source Data file.

Fig. 5. Bioinformatic analyses of the frs BGC and selected domains.

a BiG-SCAPE analysis network of frs and 2716 NRPS BGCs, obtained from a global BiG-SLiCE query against all non-fragmented NRPS from the BiG-FAM database (distance of d  < 1400) at distance cutoff 0.5. The frs BGCs from C. vaccinii and “Ca. B. crenata” (red) do not cluster with any other BGC. b Alignment of frs and their 4 closest BGCs from the BiG-SCAPE analysis in Fig. 5a. Color intensity denotes the degree of homology to the respective protein of the frs BGC. Red: frsD (NRPS), blue: frsB (MbtH-like protein), yellow: frsC (dehydrogenase), green: frsH (hydroxylase). None of the listed BGCs contains homologues to frsC or frsH. Alignment adapted from Corason. c Phylogenetic tree of C_starter domains. Taxonomy of clades is indicated. The scale bar represents 20 substitutions per 100 amino acids. d Phylogenetic tree of NRPS TE domains. The observed mode of peptide release is indicated. The scale bar represents 50 substitutions per 100 amino acids.