Tom Bretschneider

Vinylogous chain branching catalysed by a dedicated polyketide synthase module

Bacteria use modular polyketide synthases (PKSs) to assemble complex polyketides, many of which are leads for the development of clinical drugs, in particular anti-infectives and anti-tumoral agents. Because these multifarious compounds are notoriously difficult to synthesize, they are usually produced by microbial fermentation. During the past two decades, an impressive body of knowledge on modular PKSs has been gathered that not only provides detailed insight into the biosynthetic pathways but also allows the rational engineering of enzymatic processing lines to yield structural analogues. Notably, a hallmark of all PKS modules studied so far is the head-to-tail fusion of acyl and malonyl building blocks, which leads to linear backbones. Yet, structural diversity is limited by this uniform assembly mode. Here we demonstrate a new type of PKS module from the endofungal bacterium Burkholderia rhizoxinica that catalyses a Michael-type acetyl addition to generate a branch in the carbon chain. In vitro reconstitution of the entire PKS module, X-ray structures of a ketosynthase-branching didomain and mutagenesis experiments revealed a crucial role of the ketosynthase domain in branching the carbon chain. We present a trapped intermediary state in which acyl carrier protein and ketosynthase are covalently linked by the branched polyketide and suggest a new mechanism for chain alkylation, which is functionally distinct from terpenoid-like β-branching. For the rice seedling blight toxin rhizoxin, one of the strongest known anti-mitotic agents, the non-canonical polyketide modification is indispensable for phytotoxic and anti-tumoral activities. We propose that the formation of related pharmacophoric groups follows the same general scheme and infer a unifying vinylogous branching reaction for PKS modules with a ketosynthase-branching–acyl-carrier-protein architecture. This study unveils the structure and function of a new PKS module that broadens the biosynthetic scope of polyketide biosynthesis and sets the stage for rationally creating structural diversity.

Phenalenone-type phytoalexins mediate resistance of banana plants (Musa spp.) to the burrowing nematode Radopholus similis

Significance The ongoing decline of banana yields caused by pathogens and the use of toxic chemicals to manage them has attracted considerable attention because of the importance of bananas as a major staple food for more than 400 million people. We demonstrate that secondary metabolites (phenylphenalenones) of Musa are the reason for differences in cultivar resistance, and detected the phenylphenalenone anigorufone in greater concentrations in lesions in roots of a nematode-resistant cultivar than in those of a susceptible one. An in vitro bioassay identified anigorufone as the most active nematostatic and nematocidal compound. We discovered that large lipid–anigorufone complex droplets are formed in the bodies of Radopholus similis exposed to anigorufone, resulting in the nematode being killed. The global yield of bananas—one of the most important food crops—is severely hampered by parasites, such as nematodes, which cause yield losses up to 75%. Plant–nematode interactions of two banana cultivars differing in susceptibility to Radopholus similis were investigated by combining the conventional and spatially resolved analytical techniques 1H NMR spectroscopy, matrix-free UV-laser desorption/ionization mass spectrometric imaging, and Raman microspectroscopy. This innovative combination of analytical techniques was applied to isolate, identify, and locate the banana-specific type of phytoalexins, phenylphenalenones, in the R. similis-caused lesions of the plants. The striking antinematode activity of the phenylphenalenone anigorufone, its ingestion by the nematode, and its subsequent localization in lipid droplets within the nematode is reported. The importance of varying local concentrations of these specialized metabolites in infected plant tissues, their involvement in the plant’s defense system, and derived strategies for improving banana resistance are highlighted.

A ketosynthase homolog uses malonyl units to form esters in cervimycin biosynthesis

Ketosynthases produce the carbon backbones of a vast number of biologically active polyketides by catalyzing Claisen condensations of activated acyl and malonyl building blocks. Here we report that a ketosynthase homolog from Streptomyces tendae, CerJ, unexpectedly forms malonyl esters during the biosynthesis of cervimycin, a glycoside antibiotic against methicillin-resistant Staphylococcus aureus (MRSA). Deletion of cerJ yielded a substantially more active cervimycin variant lacking the malonyl side chain, and in vitro biotransformations revealed that CerJ is capable of transferring malonyl, methylmalonyl and dimethylmalonyl units onto the glycoside. According to phylogenetic analyses and elucidation of the crystal structure, CerJ is functionally and structurally positioned between the ketosynthase catalyzing Claisen condensations and acyl-ACP shuttles, and it features a noncanonical catalytic triad. Site-directed mutagenesis and structures of CerJ in complex with substrates not only allowed us to establish a model for the reaction mechanism but also provided insights into the evolution of this important subclass of the thiolase superfamily.

Imaging Mass Spectrometry and Genome Mining Reveal Highly Antifungal Virulence Factor of Mushroom Soft Rot Pathogen

Caught in the act: imaging mass spectrometry of a button mushroom infected with the soft rot pathogen Janthinobacterium agaricidamnosum in conjunction with genome mining revealed jagaricin as a highly antifungal virulence factor that is not produced under standard cultivation conditions. The structure of jagaricin was rigorously elucidated by a combination of physicochemical analyses, chemical derivatization, and bioinformatics.

Synthetic Remodeling of the Chartreusin Pathway to Tune Antiproliferative and Antibacterial Activities

Natural products of the benzonaphthopyranone class, such as chartreusin, elsamicin A, gilvocarcin, and polycarcin, represent potent leads for urgently needed anticancer therapeutics and antibiotics. Since synthetic protocols for altering their architectures are limited, we harnessed enzymatic promiscuity to generate a focused library of chartreusin derivatives. Pathway engineering of the chartreusin polyketide synthase, mutational synthesis, and molecular modeling were employed to successfully tailor the structure of chartreusin. For the synthesis of the aglycones, improved synthetic avenues to substituted coumarin building blocks were established. Using an engineered mutant, in total 11 new chartreusin analogs (desmethyl, methyl, ethyl, vinyl, ethynyl, bromo, hydroxy, methoxy, and corresponding (1→2) abeo-chartreusins) were generated and fully characterized. Their biological evaluation revealed an unexpected impact of the ring substituents on antiproliferative and antibacterial activities. Irradiation of vinyl- and ethynyl-substituted derivatives with blue light resulted in an improved antiproliferative potency against a colorectal cancer cell line. In contrast, the replacement of a methyl group by hydrogen caused a drastically decreased cytotoxicity but markedly enhanced antimycobacterial activity. Furthermore, mutasynthesis of bromochartreusin led to the first crystal structure of a chartreusin derivative that is not modified in the glycoside residue. Beyond showcasing the possibility of converting diverse, fully synthetic polyphenolic aglycones into the corresponding glycosides in a whole-cell approach, this work identified new chartreusins with fine-tuned properties as promising candidates for further development as therapeutics.

Enzymatic Polyketide Chain Branching To Give Substituted Lactone, Lactam, and Glutarimide Heterocycles†

Polyketides typically result from head-to-tail condensation of acyl thioesters to produce highly functionalized linear chains. The biosynthesis of the phytotoxin rhizoxin, however, involves a polyketide synthase (PKS) module that introduces a δ-lactone chain branch through Michael addition of a malonyl extender to an α,β-unsaturated intermediate unit. To evaluate the scope of the branching module, polyketide mimics were synthesized and their biotransformation by the reconstituted PKS module from the Rhizopus symbiont Burkholderia rhizoxinica was monitored in vitro. The impact of the type and configuration of the δ-substituents was probed and it was found that amino-substituted surrogates yield the corresponding lactams. A carboxamide analogue was transformed into a glutarimide unit, which can be found in many natural products. Our findings illuminate the biosynthesis of glutarimide-bearing polyketides and also demonstrate the utility of this branching module for synthetic biology.

Rational Design of an Apoptosis-Inducing Photoreactive DNA Intercalator**

Herein we report the first successful tailoring of thepotent chartarin pharmacophore by merging the strengths ofmolecular modeling, biosynthesis, and chemical synthesis. Wedemonstrate a viable chemobiosynthetic route to a novelvinyl-substituted chartreusin analogue, which forms covalentlinkstoDNAuponmildphotoactivationwithvisiblelight andwhich has a markedly higher antitumoral potency than theparent compound.To rationally design chartreusin analogues with poten-tially improved potencies we modeled the structures ofchartreusin and ring-substituted analogues into DNA andtookintoaccountcurrentdataonthemultivalent,nonrandombinding properties. Since chartreusin preferentially binds tosequences containing CpG or TpG triplets,

Biosynthesis and Mass Spectrometric Imaging of Tolaasin, the Virulence Factor of Brown Blotch Mushroom Disease

The button mushroom (Agaricus bisporus) is one of the most commonly cultivated mushrooms and is consumed worldwide. Unfortunately, it is prone to a wide range of bacterial, fungal, and even viral infections. One of the most severe mushroom diseases is brown blotch disease caused by Pseudomonas tolaasii, as it accounts for large economic losses in mushroom farming. Once a mushroom is infected by the pathogenic bacterium, the disease rapidly spreads throughout the farm. Typical symptoms include dark brown discoloration and characteristic lesions of the basidiocarp, caused by a toxin secreted by the bacteria. 3] The virulence factor, tolaasin, is a tensionactive lipodepsipeptide that consists of 18 amino acids and a beta-hydroxy-octanoic acid chain at the N terminus. In addition to the main derivative, tolaasin I (1; Scheme 1), several congeners have been characterized. The lipopeptides efficiently disrupt the fungal cell membrane by forming transmembrane pores. 6] P. tolaasii is not host specific; it can also infect various other mushroom species, such as the oyster

A Ribonucleotide Reductase‐Like Electron Transfer System in the Nitroaryl‐Forming N‐Oxygenase AurF

Many natural products endowed with the rare nitro moiety exhibit important biological activities such as antibiotic, antifungal, insecticidal, or antitumoral. The perhaps best-known representatives are the antibiotic chloramphenicol, the antifungal agent pyrrolnitrin, and the antiproliferative polyketide aureothin (1). Two principal biosynthetic pathways, nitration and Noxygenation of amino groups, lead to natural nitro compounds. Surprisingly, up to now only two genuine arylamine N-oxygenases have been identified: PrnD from the pyrrolnitrin pathway in Pseudomonas fluorescens, and AurF, which generates the p-nitrobenzoate (PNBA) building block for aureothin biosynthesis in Streptomyces thioluteus. Due to its remarkable chemoand regioselectivity, AurF has a high potential as biocatalyst for technical applications. 5] Although this unusual biocatalyst has been unprecedented, recently homologues of aurF have been identified through genomic analyses in yetunknown biosynthetic polyketide synthase (PKS) and nonribosomal peptide synthase (NRPS) gene clusters. Through a number of in vivo and in vitro studies, we could show that Noxygenation of the amino substrates occurs stepwise and involves hydroxylamine and nitroso intermediates. Furthermore, we could prove the participation of manganese in the catalytic action of AurF and were able to present the first X-ray structure of an N-oxygenase. 9] Later, another group reported the crystal structure of an iron-containing variant of AurF. In both structures, the overall chain folds were similar and featured a binuclear metal cluster in the active center. Both iron and manganese variants exhibit in vivo and in vitro activity, which led to some debate on the native form of AurF, also considering mixed Fe/Mn nuclei. 12] Nevertheless, it has been demonstrated for two structurally related dioxygenases that oxygen activation can be achieved by either metal. What is quite striking is that the aureothin biosynthesis gene cluster does not contain any gene coding for components of an electron transfer chain, which usually consists of a ferredoxin, a ferredoxin reductase and NAD(P)H. However, AurF has been proven to be active in vivo in at least three different organisms: in its natural host Streptomyces thioluteus, as well as in the heterologous hosts Streptomyces lividans and E. coli. Moreover, the purified enzyme can be regenerated in vitro by using the peroxide shunt. Whereas the native interaction partners are still unknown, AurF could be reconstituted in vitro by using surrogate ferredoxin and ferredoxin reductase from Anabaena sp. PCC 7119 (D9 desaturase). However, to date it has remained elusive how electrons are shuttled to the metals in the active site. Here we provide the first insight into the electron transport system of this unusual nitro-group-forming enzyme (Scheme 1). Although there is no obvious sequence homology of AurF with known enzymes, by DALI analyses we found that the chain fold and biometal cluster match remarkably well with ribonucleotide reductases (RNR), methane monooxygenases/ hydroxylases, and acyl desaturases/fatty acid reductases. In all cases, the metal cluster is embedded in a large helix bundle and coordinated by two (D/E)EXXH motifs. Furthermore, con-