Biosynthesis of Phenylnannolone A, a Multidrug Resistance Reversal Agent from the Halotolerant Myxobacterium Nannocystis pusilla B150

Abstract

The myxobacterial strain Nannocystis pusilla B150 synthesizes the structurally new polyketides phenylnannolone A–C. Apart from some common volatiles and siderophores, these are the first natural products from the genus Nannocystis. Phenylnannolone A shows inhibitory activity towards the ABCB1 gene product P‐glycoprotein and reverses daunorubicin resistance in cancer cells. To decipher the biochemical reactions leading to the formation of phenylnannolone A, the putative biosynthetic genes were identified (phn1, phn2). Phn2 is a polyketide synthase (PKS) with an NRPS‐like loading module, and its domain order is consistent with the phenylnannolone A structure. The functionality and substrate selectivity of the loading module were determined by means of a γ‐18O4‐ATP pyrophosphate exchange and a phosphopantetheine ejection assay. A specific activation of cinnamic acid by the AMP‐ligase was detected. Phn1 is a putative butyryl‐CoA carboxylase (BCC), providing ethylmalonyl‐CoA for the formation of the ethyl‐substituted part of phenylnannolone A. Phn1 is the first BCC found in biosynthetic genes for an ethyl‐substituted natural compound. Biosynthesis of phenylnannolone A, putatively encoded by phn1 and phn2, thus utilizes the first biosynthetic machinery in which both a BCC and a PKS are involved.

Discovery, Structure Elucidation, and Biological Characterization of Nannocystin A, a Macrocyclic Myxobacterial Metabolite with Potent Antiproliferative Properties

“…[3] Thebest-studied compound class comprises the tubulin-stabilizing epothilons,which have yielded ac linically approved anticancer drug (Ixabepilone). [5] We were therefore pleased to detect as tructurally novel and highly bioactive metabolite in ac ulture broth of Nannocyctis sp.S T201196 (DSM18870). While the great majority of myxobacterial products has been isolated from Sorangium cellulosum, Myxococcus xanthus, and Chondromyces sp., [4] only few metabolites have been described from Nannocyctis sp., including relatively common volatiles and siderophores and the recently discovered phenylnannolones and pyrronazols.…”

“…While the great majority of myxobacterial products has been isolated from Sorangium cellulosum, Myxococcus xanthus, and Chondromyces sp., [4] only few metabolites have been described from Nannocyctis sp., including relatively common volatiles and siderophores and the recently discovered phenylnannolones and pyrronazols. [5] We were therefore pleased to detect as tructurally novel and highly bioactive metabolite in ac ulture broth of Nannocyctis sp.S T201196 (DSM18870). [6] In this report, the isolation of the compound, the elucidation of its macrocyclic scaffold, including the relative and absolute stereochemistry,the characterization of its potent anticancer properties,a nd initial structure-activity relationship (SAR) studies are described.…”

An Unusual Fatty Acyl:Adenylate Ligase (FAAL)–Acyl Carrier Protein (ACP) Didomain in Ambruticin Biosynthesis

“…proposed the biosynthesis of the divinylcyclopropane (DVC) fragment starting from 4 by a Favorskii rearrangement accompanied by a polyene shift to give carboxylic acid 6 . In this scenario, a ligation–oxidative decarboxylation cascade catalysed by AmbG and the flavin‐dependent monooxygenase AmbI converts 6 into 8 , which is then further processed by the PKS …”

“…Phylogenetic analysis and application of the Khurana algorithm for FAAL substrate specificity prediction suggest that AFD_AmbG (FAAL_AmbG in the following) is an atypical representative (Figures S8–S11) . Studying the 93 best results of a BLAST search for sequence homologues of FAAL_AmbG in more detail, we came across that 42 hits were N‐terminal FAAL domains from multimodular protein complexes, 15 were FAAL–carrier protein (CP) didomains, and 36 were lone‐standing FAALs (Table S1) . FAAL–ACP didomains are thus rather rare in PKSs, and ambG is the only example for which the genes are centrally coded within the PKS genes.…”

“…We then conducted in vitro testing of AmbG with a library of structurally diverse substrate surrogates. Some fatty acid and aromatic acid starter group transferring FAAL‐like enzymes in PKS, NRPS, and PKS/NRPS hybrid systems have been biochemically characterised so far, mostly with small collections of compounds closely related to the suspected natural substrate (Table S2). Specificity for one type of substrate was usually observed, often along with limited tolerance for different aliphatic chain lengths or varying substitution patterns on the aromatic core .…”